IDENTIFICATION TESTS

The true identification of a drug may be accomplished in a number of ways, namely : determination of physical constants, chromatographic tests and finally the chemical tests. The physical constants essentially include the melting point, boiling point, refractive index, weight per millilitre, specific optical rotation, light absorption, viscosity, specific surface area, swelling power, infra-red absorption, and the like. The chromatographic tests include specific spot-tests by thin-layer chromatography (TLC) of pure drug or its presence in a multi-component system. However, the most specific and reliable are the chemical tests which may be categorized separately under tests for inorganic substances and organic substances. The former may be carried out by well defined general quantitative inorganic analysis and the latter by specific reactions of one or more of the functional moieties present in a drug molecule.

PHYSICAL CONSTANTS

A wide range of physical constants, for instance : melting point, boiling point, specific gravity, viscosity, refractive index, solubility, polymorphic forms vis-a-vis particle size, in addition to characteristic absorption features and optical rotation play a vital role in characterization of pharmaceutical chemicals and drug substances. These physical constants will be discussed briefly with typical examples as under :

1. Melting Point

It is an important criterion to know the purity of a substance ; however, it has a few limitations. The accuracy and precision of melting point is dependent on a number of factors such as—capillary size, sample size, initial temperature of heating-block and the rate of rise of temperature per unit time (minutes). Keeping in view the different manufacturing processes available for a particular drug the melting point has a definite range usually known as the melting range.

Thus the melting range takes care of the variance in manufacture together with the storage variance over a stipulated period of time.

2. Boiling Point

It is also an important parameter that establishes the purity of a substance. Depending on the various routes of synthesis available for a substance a boiling point range is usually given in different official compendia.

3. Refractive Index

It is invariably used as a standard for liquids belonging to the category of fixed oils and synthetic chemicals.

4. Weight Per Millilitre

Weight per millilitre is prevalent in the Pharmacopoeia of India for the control of liquid substances, whereas Relative Density (20°/20°) or Specific Gravity is mostly employed in the European Pharmacopoeia.

5. Specific Optical Rotation

As pharmacological activity is intimately related to molecular configuration, hence determination of specific rotation of pharmaceutical substances offer a vital means of ensuring their optical purity.

6. Light Absorption

The measurement of light absorption both in the visible and ultraviolet range is employed as an authentic means of identification of offcial pharmaceutical substances.

7. Viscosity

Viscosity measurements are employed as a method of identifing different grades of liquids.

8. Specific Surface Area

The surface area of powders is determined by subsieve-sizer which is designed for measurement of average particle sizes in the range of 0.2 to 50 microns. The relationship between average particle diameter and specific surface area (SSA) is given by the following expression :

where, SSA = Specific surface area in cm² per g of material

d = Average diameter in microns

p = True density of material from which the powder was made in g per cm³

9. Swelling Power

The swelling power of some pharmaceutical products are well defined.

Examples :

(i) Isphagula Husk : When 1 g, agitated gently and occasionally for four hours in a 25 ml stoppered measuring cylinder filled upto the 20 ml mark with water and allowed to stand for 1 hour, it occupies a volume of not less than 20 ml and sets to a jelly.

(ii) Heavy Kaolin : When 2 g is titurated with 2 ml of water the mixture does not flow.

10. Infrared Absorption

Measurement and subsequent comparison of the infrared spectrum (between 4000-667 cm^–1) of compounds with that of an authentic sample has recently become a versatile method for the identification of drugs having widely varying characteristics.

Examples : Infrared spectroscopy is employed to compare samples of chloramphenicol palmitate (biologically active form) recovered from chloramphenicol palmitate mixture vis-a-vis an artificially prepared mixture of authentic sample consisting 10 per cent of the ‘inactive polymorph’.

Infrared spectra of known and newly reported compounds are provided in the British Pharmacopoeia (1998) and also in ‘Sadtler Standard Spectra’ published by Sadtler Research Laboratories, Philadelphia

(USA) is available to check the authenticity of pure drug samples.

11. Miscellaneous Characteristics

A large number of miscellaneous characteristics are usually included in many official compendia to ascertain the purity, authenticity and identification of drugs—including : sulphated ash, loss on drying, clarity and colour of solution, presence of heavy metals and specific tests.

11.1. Sulphated Ash

Specifically for the synthetic organic compounds, the Pharmacopoeia prescribes values for sulphated ash. The sulphated ash is determined by a double ignition with concentrated sulphuric acid. Metals thus remain as sulphides that are usually stable to heat. The method is one of some precision, and provides results which are rather more reproducible than those obtained by simple ignition.

11.2. Loss on Drying

Loss on drying reflects the net weight of a pharmaceutical substance being dried at a specified tempera-ture either at an atmospheric or under reduced pressure for a stipulated duration with a specific quantity of the substance.

11.3. Clarity and Colour of Solution

When a pharmaceutical substance is made to dissolve at a known concentration in a specified solvent it gives rise to a clear solution that may be either clear or possess a definite colouration.

11.4. Heavy Metals

Various tests are prescribed in the offcial compendia to control heavy metal e.g., Ag⁺, Hg²⁺, Pb²⁺, Bi²⁺, Cu²⁺, As³⁺, , Sb³⁺ and Sn⁴⁺ contamination in organic pharmaceutical substances. Hence, a stringent limit is recommended for the presence of heavy metals in medicinal compounds.

11.5. Specific Tests

In fact, certain known impurities are present in a number of pharmaceutical substances. The presence of such impurities may be carried out by performing prescribed specific tests in various official compendia in order to ascertain their presence within the stipulated limits.

Reagents :

·              Dilute 1 ml N. HCl and 2.0 ml ferric ammonium sulphate soln. (10% w/v in H₂O) with suffcient water to produce 100 ml.

·              Dissolve 50 mg cadmium acetate in a mixture of 5 ml DW and 1 ml glacial acetic acid and dilute with ethyl methyl ketone to 50 ml. Immediately before use add and dissolve suffcient Ninhydrin to produce a soln. containing 0.2% w/v.

·              Dissolve 10.0 g sodium tungstate and 2.5 g sodium molybdate in 80.0 ml DW in a 250 ml flask; add 5.0 ml phosphoric acid (85-90% w/w) and 10.0 ml HCl (= 11.5 N), connect to a reflux condenser and heat for 10 Hrs. Cool, add 15.0 g lithium sulphate, 5.0 ml DW and 1 drop of bromine and allow to stand for 2 Hrs. Remove the excess bromine by boiling the mixture for 15 mts. without the condenser. Cool, filter and dilute with DW to produce 100 ml.

Caution :

(i) The prepared soln. should be stored below 4°C, and

(ii) The soln. should be used within 4 months after preparation till it retains its original golden yellow colour. It must be rejected if it has a trace of green colour.

LIMIT TESTS VIS-A-VIS QUANTITATIVE DETERMINATIONS

In general, limit tests are quantitative or semi-quantitative tests particularly put forward to identify and control invariably small quantities of impurity that are supposed to be present in a pharmaceutical substance. Obviously the amount of any single impurity present in an official substance is usually small, and therefore, the normal visible-reaction-response to any test for that impurity is also quite small. Hence, it is necessary and important to design the individual test in such a manner so as to avoid possible errors in the hands of various analysts. It may be achieved by taking into consideration the following three cardinal factors, namely :

(a) Specificity of the Tests : A test employed as a limit test should imply some sort of selective reaction with the trace impurity. It has been observed that a less specific test which limits a number of possible impurities rather instantly has a positive edge over the highly specific tests.

Exmaple : Contamination of Pb²⁺ and other heavy metal impurities in Alum is precipitated by thioacetamide as their respective sulphides at pH 3.5.

(b) Sensitivity : The extent of sensitivity stipulated in a limit test varies widely as per the standard laid down by a pharmacopoeia. The sensitivity is governed by a number of variable factors having a common objective to yield reproducible results, for instance :

(i) Gravimetric Analysis : The precipitation is guided by the concentration of the solute and of the precipitating reagent, reaction time, reaction temperature and the nature and amount of other substance(s) present in solution.

(ii) Colour Tests : The production of visible and distinct colouration may be achieved by ascertain-ing the requisite quantities of reagents and reactants, time period and above all the stability of the colour produced.

(c) Personal Errors : In fact, the personal errors must be avoided as far as possible as explained.

VARIOUS TYPES OF TESTS FOR QUANTITATIVE DETERMINATIONS

In actual practice, it has been observed that different official compendia describe a number of detailed types of tests with a view to obtain a constant and regular check that might be possible to maintain the desired degree of optimum purity both in the pure pharmaceutical substances and the respective dosage-forms made therefrom.

A number of such tests shall be discussed here briefly with specific examples wherever possible and necessary :

1. Limits of Insoluble Matter

The limits of insoluble matter present in pharmaceutical substances and stated in various official com-pendia are given below :

In the same vein, tests for clarity of solution offer another means of limiting insoluble parent drug sub-stances in their correspondingly more highly water-soluble derivatives.

2. Limits of Soluble Matter

In order to detect the presence of some very specific impurities normally present in the official substances the limits of soluble impurities have been laid down in different pharmacopoeias. Some typical examples are cited below :

3. Limits of Moisture, Volatile Matter and Residual Solvents

A good number of pharmaceutical substances usually absorb moisture on storage thereby causing deterioration. Such an anomaly can be safely restricted and limited by imposing an essential requirement for the loss in weight (Loss on Drying) when the pharmaceutical chemical is subjected to drying under specified conditions. The quantum of heat that may be applied to the substance varies widely as per the following norms :

(a) Nature of the substance

(b) Decomposition characterisics of the substance.

Various official compendia recommended different temperatures and duration of drying either at atmos-pheric or reduced pressure (vacuum). A few typical examples are stated below :

There are four types of hydrates which may be observed amongst the pharmaceutical chemicals, namely :

(a) Inorganic Salt Hydrates e.g., Magnesium Sulphate (MgSO₄.7H₂O) ; Sodium Sulphate (Na₂SO₄. 10 H₂O).

(b) Salts of Inorganic Cations and Organic Acids e.g., Calcium Lactate, Ferrous Gluconate.

(c) Organic Hyrates e.g., Caffeine Hydrate, Theophylline Hydrate.

(d) Organic Substances e.g., Acacia, Hydroxymethyl Cellulose.

These substance either lose all or part of their water of crystallization on drying which sometimes attains a considerable value as could be seen in the following data :

3.1. Aquametry

It refers to the determination of water content titrimetrically with Karl Fischer Reagent (KFR). This technique has been used exclusively for the determination of water content in a number of pharmaceutical substances listed below :

Since the introduction of Gas-Liquid-Chromatography (GLC) as an essential analytical tool, it has been judiciously exploited as an useful alternative means for not only determining water content in pharmaceutical chemicals but also limiting specific volatile substances present in them. It may be expatiated with the help of the following examples :

Examples :

(i)  For Determination of Water Content :

Gonadorelin : (Limit NMT : 7.0 % w/v)

Procedure : Carry out the method for gas chromatography employing the following solutions

Solution (1) : Dilute 50 μ l of anhydrous methanol (internal standard) with sufficient

anhydrous propan-2-ol to produce 100 ml.

Solution (2) : Dissolve 4 mg of the sample in 1 ml of anhydrous propan-2-ol.

Solution (3) : Dissolve 4 mg of the sample in 1 ml of solution (1) above.

Solution (4) : Add 10 μ l of water to 50 μ l of solution (1).

The chromatographic procedure may be carried out by employing :

(a) A stainless-steel column (1 m × 2 mm) packed with porous polymer beads e.g., Chromosorb 102 (60 to 80 mesh) and maintained at 120°C.

(b)  Helium as the carrier gas.

(c) A Thermal Conducting Detector (TCD) maintained at 150°C. From the chromatograms obtained and taking into account any water detectable in solution

(1), calculate the percentage w/w of water taking 0.9972 as its weight per ml at 20°C.

(ii)  For Limiting Specific Volatile Substance :

Orciprenaline Sulphate : (Limit of Water and Methanol : 6.0% w/w)

Procedure : Perform the method for gas-chromatography using the following three solutions in water containing :

Solution (1) : 0.50% v/v of MeOH and 0.50% v/v of EtOH (96% v/v)—as Internal Standard

Solution (2) : 10% w/v of the sample

Solution (3) : 10% w/v of the sample and 0.50% v/v of the internal standard.

The chromatographic procedure may be performed using a glass column (1.5 × 4 mm) packed with porous polymer beads (80 to 100 mesh) e.g., Porapack-Q and maintained at 140°C.

Calculate the percentage w/v of methanol taking 0.792 as its weight per ml at 20°C.

4. Limits of Non-Volatile Matter

Pharmaceutical chemicals belonging to the domain of inorganic as well as organic substances containing readily volatile matter for which the various official compendia prescribe limits of non-volatile matter. It is pertinent to mention here that the Pharmacopoeia usually makes a clear distinction between substances that are readily volatile and substances that are volatile upon strong ignition, for instance :

(a) Readily Volatile : e.g., Organic Substances—alcohol (95% v/v), isopropyl alcohol, chloroform, halothane, anaesthetic ether, chlorocresol and trichloroethylene ; and Inorganic substances—ammonia solution, hydrogen peroxide solution, water for injection.

(b) Volatile Upon Strong Ignition : e.g., hydrous wool fat (lanolin).

5. Limits of Residue on Ignition

In fact, the limits of residue on ignition are basically applicable to the following two categories of pharmaceutical substances, namely :

(a) Those which are completely volatile when ignited e.g., Hg.

(b) Those which undergo total decomposition thereby leaving a residue with a definite composition e.g., calamine—a basic zinc carbonate that gives rise to ZnO as the residue.

According to BP, 68.0 to 74.0% when ignited at a temperature not lower than 900°C until, after further ignition, two successive weighings do not differ, by more than 0.2% of the weight of the residue.

6. Limits of Loss on Ignition

Official compendia include the limits of ‘loss on ignition’ which is generally applied to relatively stable pharmaceutical substances that are likely to contain thermolabile impurities. A few typical examples are stated below :

7. Limits on Ash Value

The ash values usually represent the inorganic residue present in official herbal drugs and pharmaceuti-cal substances. These values are categorized into four heads, namely :

(a) Ash Value (Total Ash),

(b) Acid-Insoluble Ash,

(c) Sulphated Ash, and

(d) Water-Soluble Ash.

These values would be explained with the help of some typical examples stated below :

7.1. Ash Value (Total Ash)

Ash value normally designates the presence of inorganic salts e.g., calcium oxalate found naturally in the drug, as well as inorganic matter derived from external sources. The official ash values are of prime importance in examination of the purity of powdered drugs as enumerated below :

(i) To detect and check adulteration with exhausted drugs e.g., ginger.

(ii) To detect and check absence of other parts of the plant e.g., cardamom fruit.

(iii) To detect and check adulteration with material containing either starch or stone cells that would modify the ash values.

(iv) To ensure the absence of an abnormal proportion of extraneous mineral matter incorporated acciden-tally or due to follow up treatment or due to modus operandi at the time of collection e.g., soil, floor sweepings and sand.

The most common procedure recommended for crude drugs is described below :

Procedure : Incinerate 2 to 3 g of the ground drug in a tared platinum or silica dish at a temperature not exceeding 450°C until free from carbon. Cool and weigh. If a carbon-free ash cannot be obtained in this way, exhaust the charred mass with hot water (DW), collect the residue on an ashless filter paper, incinerate the residue and filter paper, add the filtrate, evaporate to dryness and ignite at a temperature not exceeding 450°C. Calculate the percentage of ash with reference to the air-dried drug.

7.2. Acid-Insoluble Ash

The method described above for ‘total ash’ present in crude drugs containing calcium oxalate has certain serious anomalies, namely :

·              Offers variable results upon ashing based on the conditions of ignition.

·              Does not detect soil present in the drug efficaciously.

·              The limits of excess of soil in the drug are not quite definite.

Hence, the treatment of the ‘total ash’ with acid virtually leaves silica exclusively and thus comparatively forms a better test to detect and limit excess of soil in the drug than does the ash.

The common procedure usually adopted for the determination of ‘acid insoluble ash’ is given below :

Procedure : Place the ash, as described earlier, in a crucible, add 15 ml DW and l0 ml hydrochloric acid ( ~– 11.5 N), cover with a watch-glass, boil for 10 minutes and allow to cool. Collect the insoluble matter on an ashless filtre paper, wash with hot DW until the filtrate is neutral, dry, ignite to dull redness, allow to cool in a desiccator and weigh. Repeat until the difference between two successive weighings is not more than l mg. Calculate the percentage of acid-insoluble ash with reference to the air-dried drug.

A few typical examples are listed below :

7.3. Sulphated Ash

The estimation of ‘sulphated ash’ is broadly employed in the case of :

(a) Unorganized drugs e.g., colophony, podophyllum resin, wool alcohols, wool fat and hydrous wool fat.

(b) Pharmaceutical substances contained with inorganic impurities e.g.,

Natural Origin : Spray-dried acacia, Frangula Bark, Activated Charcoal

Organic Substances : Cephalexin, Lignocaine hydrochloride, Griseofulvin, Diazoxide, Medazapam, Saccharin.

Inorganic Substances : Ammonium chloride, Hydroxy urea.

The general method for the determination of ‘sulphated ash’ is enumerated below :

Procedure : Heat a silica or platimum crucible to redness for 30 minutes, allow to cool in a desiccator and weigh. Place a suitable quantity of the substance being examined, accurately weighed in the crucible, add 2 ml of 1 M sulphuric acid and heat, first on a waterbath and then cautiously over a flame to about 600°C. Continue heating until all black particles have disappeared and then allow to cool. Add a few drops of 1 M sulphuric acid, heat to ignition as before and allow to cool. Add a few drops of a 16% solution of ammonium carbonate, evaporate to dryness and cautiously ignite. Cool, weigh, ignite for 15 minutes and repeat the procedure to constant weight.

Following are the examples to depict the ‘sulphated ash’ present in various official pharmaceutical chemicals :

7.4. Water-Soluble Ash

Water-soluble ash is specifically useful in detecting such samples which have been extracted with water.

A detailed procedure as per the official compendium is enumerated below :

Procedure : The ash as described earlier, is boiled for 5 minutes with 25 ml DW, collect the insoluble matter in a sintered-glass crucible or on an ashless filter paper, wash with hot DW and ignite for 15 minutes at a temperature not exceeding 450°. Subtract the weight of the residue thus obtained from the weight of the ash.The difference in weight represents the water-soluble ash. Now, calculate the percentage of water-soluble ash with reference to the air-dried drug.

A typical example of an official drug is that of ‘Ginger’, the water-soluble ash of which is found to be not more than 6.0%.

LIMIT TESTS FOR METALLIC IMPURITIES

The official compendia lay a great deal of emphasis on the control of physiologically dangerous, cumulative poisonous and harmful impurities, such as lead, arsenic and iron present in a host of pharmaceutical chemicals. These impurities very often creep into the final product through a number of means stated below, namely :

(a) Through atmospheric pollution.

(b) Most frequently derived from the raw materials.

(c) From materials used in the process of manufacture.

(d) Due to solvent action on the metal of the plant in which the substance is prepared.

In short, all prescribed tests for impurities in the Pharmacopoeia usually fix certain limits of tolerance. For lead, arsenic and iron general quantitative or limit tests are precisely laid down which, with necessary variations and modification are rigidly applicable to pharmaceutical substances.

1. Limit Tests for Lead

Theory : The offcial test is based on the conversion of traces of lead salts present in the pharmaceutical substances to lead sulphide, which is obtained in colloidal form by the addition of sodium sulphide in an alkaline medium achieved by a fairly high concentration of ammonium acetate. The reaction may be expressed as follows :

PbCl₂ + Na₂S  → PbS _(Dec) + 2NaCl

The brown colour, caused due to colloidal lead sulphide in the test solution is compared with that produced from a known amount of lead.

Equipment : Nessler Cylinders (or Nessler Glasses) : According to the British Standard Specification No : 612, 966—a pair of cylinders made of the same glass and having the same diameter with a graduation mark at the same height from the base in both cylinders (Figure 1).

The final comparison is made by viewing down through the solution against a light background.

Materials Required :

(i) Lead Nitrate Stock Solution : Dissolve0.1598 g of lead nitrate in 100 ml DW to which has been  added 1 ml nitric acid, then dilute with water to 1 Litre.

Note : The solution must be prepared and stored in polyethylene or glass containers free from soluble lead salts.

(ii) Standard Lead Solution : On the day of use, dilute 10.0 ml of lead nitrate stock solution with DW to 100.0 ml. Each ml of standard lead solution contains the equivalent of 10 microgrammes of lead. A control comparison solution prepared with 2.0 ml of standard lead solution contains, when compared to a solution representing 1.0 g of the substance being tested, the equivalent of 20 parts per million of lead.

(iii) Standard Solution : Into a 50 ml Nessler Cylinder, pipette 2 ml of standard lead solution and dilute with DW to 25 ml. Adjust with dilute acetic acid Sp. (IP)* or dilute ammonia solution Sp. (IP) to a pH between 3.0 and 4.0, dilute with DW to about 35 ml and mix.

(iv) Test Solution : Into a 50 ml Nessler Cylinder, place 25 ml of the solution prepared for the test as directed in the individual monograh, dissolve and dilute with DW to 25 ml the specified quantity of the substance being tested. Adjust with dilute acetic acid Sp. (IP) or dilute ammonia solution Sp. to a pH between 3.0 and 4.0, dilute with DW to about 35 ml and mix.

Procedure : To each of the cylinders containing the standard solution and test solution respectively, add l0 ml of freshly prepared hydrogen sulphide solution, mix, dilute with water (DW) to 50 ml, allow to stand for 5 minutes and view downwards over a white surface, the colour produced in the test solution is not darker than that produced in the standard solution.

 A few typical examples from the official compendium are given below :

2. Limit Test for Arsenic

Theory : The official process is a development of the Gutzeit Test wherein all arsenic present is duly converted into arsine gas (AsH₃) by subjecting it to reduction with zinc and hydrochloric acid. Further, it depends upon the fact that when arsine comes into contact with dry paper permeated with mercuric (Hg²⁺) chloride it produces a yellow strain, the intensity of which is directly proportional to the quantity of arsenic present. The various chemical reactions involved may be expressed by the following equations :

The details of experimental procedure described in the Pharmacopoeia are actually based upon a paper by Hill and Collins**, but have been adequately modified from time to time in accordance with the accumu-lated and acquired experience. Explicitly, the expressions provided in the Pharmacopoeia for limits of arsenic exclusively refer to parts per million, calculated as As.

Materials Required : Arsenic limit test apparatus; HgCl₂—paper : smooth white filter paper (having thickness in mm of 400 paper = weight in g per Sq. M.), soaked in a saturated solution of HgCl₂, pressed to get rid of excess of soln. and dried at about 60°C in the dark ; lead acetate solution 10.0% w/v soln. of PbAc₂ in CO_2– free water ; KI (AsT), 1.0 g ; Zn (AsT) : l0.0 g ; Dilute Arsenic solution (AST); Standard stains, Test Solutions—are prepared according to the Indian Pharmacopoeia 1996.

Arsenic Limit Test Apparatus (Figure 2)

A wide-mouthed glass bottle capable of holding about 120 ml is fitted with a rubber bung through which passes a glass tube. The latter, made from ordinary glass tubing, has a total length of 200 mm and an internal diameter of exactly 6.5 mm (external diameter about 8 mm). It is drawn out at one end to a diameter of about 1 mm and a hole not less than 2 mm in diameter is blown in the side of the tube, near the constricted part. When the bung is inserted in the bottle containing 70 ml of liquid, the constricted end of the tube is kept above the surface of the liquid, and the hole in the side is below the bottom of the bung. The upper end of the tube is cut off square, and is either slightly rounded or ground smooth.

The rubber bungs (about 25 mm × 25 mm), each with a hole bored centrally and through exactly 6.5 mm  in diameter,are fitted with a rubber band or spring clip for holding them tightly in place.

Procedure : The glass tube is lightly packed with cotton wool, previously moistened with lead acetate solution and dried, so that the upper surface of the cotton wool is not less than 25 mm below the top of the tube. The upper end of the tube is then inserted into the narrow end of one of the pair of rubber bungs, to a depth of l0 mm (the tube must have a rounded-off end). A piece of mercuric chloride paper is placed flat on the top of the bung and the other bung placed over it and secured by means of the spring clip in such a manner that the holes of the two bungs meet to form a true tube 6.5 mm diameter interrupted by a diaphragm of mercuric chloride paper.

The test solution prepared as specified, is placed in the wide-mouthed bottle, 1 g of KI (AsT) and 10 g of Zn (AsT) are added, and the prepared glass tube is placed quickly in position. The reaction is allowed to proceed for 40 minutes. The yellow stain that is produced on the HgCl₂ paper if As is present is compared by daylight with the standard stains obtained by performing in an identical manner with known quantities of dilute arsenic solution (AsT). The comparison of the stains is made immediately at the completion of the test.

By matching the intensity and depth of colour with standard stains, the proportion of arsenic in the substance may be estimated. Thus, a stain equivalent to the 1 ml standard stain obtained by performing on l0 g of a substance implies that the proportion of As is 1 part per million.

Cautions : 

(i) HgCl₂ paper should be protected from sunlight during the test to avoid lighter or no

stain.

(ii) The standard and test stains must be compared immediately as they fade out on retaining.

(iii) The reaction may be expedited by the application of heat and 40°C is considered to be the most ideal temperature.

(iv) The tube should be washed with HCl (AsT), rinsed with DW, and dried between successive tests.

Special Techniques : The special techniques are usually applicable to a host of pharmaceutical sub-stances before the normal test can be performed. A few typical examples would be discussed briefly here, namely :

(i) Free Acids : They are first converted to their respective sodium salts with Na₂CO₃ and As³⁺ oxi-dised to As⁵⁺ by evaporating the solution with Br₂. The residue is ignited carefully until carbonised to destroy organic matter, while As is kept as non-volatile sodium arsenate. The resulting residue is dissolved in brominated HCl and the test carried out in the normal manner.

Examples : Aspirin, Saccharin, Sodium Salicylate, Sodium Aminosalicylate.

(ii) Substances Reacting Vigorously with HCl : The As is readily converted to AsCl₃ which being volatile in nature is also carried off along with relatively large volumes of CO₂ (generated by the substance and HCl).

Examples : Magnesium Carbonate, Light Magnesium Oxide, Calcium Hydroxide, Chalk, KOH, NaOH.

(iii) Insoluble Substances : These substances, as those that do not interfere with the solution of As and its subsequent reduction to AsH₃ (arsine). Such substances are suspended in water along with stannated-HCl, and the normal test is performed.

Examples : Magnesium Trisilicate, Bentonite, Barium Sulphate, Light and Heavy Kaolin.

(iv) Metals Interfering with Normal Reaction

(a) Iron : It gets deposited on the surface of Zn thereby depressing the intensity of reaction between Zn and HCl to produce H₂.

Remedy : The sample is dissolved in H₂O and stannated HCl to allow conversion of all As to As³⁺ and finally as AsCl₃. The latter being volatile in nature can be separated by distillation from remaining metallic salts and the distillate examined in the normal manner.

Example : Ferrous Sulphate.

(b) Antimony : Sb-compounds are also reduced simultaneously by Zn/HCl to yeild SbH₃ (stilbine) that reacts with HgCl₂ paper to give a stain. Therefore, the sample is first distilled with HCl to yield a distillate containing all the As as AsC₃ (volatile), but yields only a fraction of Sb as SbCl₃ (non-volatile). A repeated distillation obviously gets rid of even the last traces of Sb.

Examples : Antimony Potassiun Tartrate, Antimony Sodium Tartrate.

A few typical examples are cited below from the official compendium.

3. Limit Test for Iron

Theory : The limit test for Iron is based on the reaction between iron and thioglycollic acid in a medium buffered with ammonium citrate to give a purple colour, which is subsequently compared with the standard colour obtained with a known amount of iron (0.04 mg of Fe). Ferrous thioglycollate is a co-ordination compound that attributes the purple colour ; besides thioglycollic acid converts the entire Fe³⁺ into Fe²⁺. The reactions involved may be expressed as follows :

Materials Required

Nessler cylinder : 1 pair ; Ferric ammonium sulphate : 1.726 g ; Sulphuric acid (0. 1 N) : 10.0 ml ; Iron-free citric acid (20% w/v) : 2.0 ml ; Thioglycollic acid : 0.1 ml; Iron-free ammonia solution : 20 ml.

Standard Iron Solution : Weigh accurately 0.1726 g of ferric ammonium sulphate and dissolve in 10 ml of 0.1 N sulphuric acid and suffcient water to produce 1 Litre. Each ml of this solution contains 0.02 mg of Fe.

Standard Colour : Dilute 2.0 ml of standard iron solution with 40 ml DW in a Nessler cylinder. Add 2 ml of a 20% w/v solution of iron-free citric acid and 0.1 ml of thioglycollic acid, mix, make alkaline with iron-free ammonia solution, dilute to 50 ml with DW and allow to stand for 5 minutes.

Procedure : Dissolve the specified quantity of the substance being examined in 40 ml DW, and trans-fer to a Nessler cylinder. Add to it 2 ml iron-free citric acid solution and 0.1 ml thioglycollic acid, mix, make alkaline with iron-free ammonia solution, dilute to 50 ml with DW and allow to stand for 5 minutes. Any colour produced is not more intense than the standard colour.

Some examples of this test for pharmaceutical substances are listed below :

Limit Tests for Lead

Theory : The offcial test is based on the conversion of traces of lead salts present in the pharmaceutical substances to lead sulphide, which is obtained in colloidal form by the addition of sodium sulphide in an alkaline medium achieved by a fairly high concentration of ammonium acetate. The reaction may be expressed as follows :

PbCl₂ + Na₂S  → PbS _(Dec) + 2NaCl

The brown colour, caused due to colloidal lead sulphide in the test solution is compared with that produced from a known amount of lead.

Equipment : Nessler Cylinders (or Nessler Glasses) : According to the British Standard Specification No : 612, 966—a pair of cylinders made of the same glass and having the same diameter with a graduation mark at the same height from the base in both cylinders (Figure 1).

The final comparison is made by viewing down through the solution against a light background.

Materials Required :

(i) Lead Nitrate Stock Solution : Dissolve0.1598 g of lead nitrate in 100 ml DW to which has been  added 1 ml nitric acid, then dilute with water to 1 Litre.

Note : The solution must be prepared and stored in polyethylene or glass containers free from soluble lead salts.

(ii) Standard Lead Solution : On the day of use, dilute 10.0 ml of lead nitrate stock solution with DW to 100.0 ml. Each ml of standard lead solution contains the equivalent of 10 microgrammes of lead. A control comparison solution prepared with 2.0 ml of standard lead solution contains, when compared to a solution representing 1.0 g of the substance being tested, the equivalent of 20 parts per million of lead.

(iii) Standard Solution : Into a 50 ml Nessler Cylinder, pipette 2 ml of standard lead solution and dilute with DW to 25 ml. Adjust with dilute acetic acid Sp. (IP)* or dilute ammonia solution Sp. (IP) to a pH between 3.0 and 4.0, dilute with DW to about 35 ml and mix.

(iv) Test Solution : Into a 50 ml Nessler Cylinder, place 25 ml of the solution prepared for the test as directed in the individual monograh, dissolve and dilute with DW to 25 ml the specified quantity of the substance being tested. Adjust with dilute acetic acid Sp. (IP) or dilute ammonia solution Sp. to a pH between 3.0 and 4.0, dilute with DW to about 35 ml and mix.

Procedure : To each of the cylinders containing the standard solution and test solution respectively, add l0 ml of freshly prepared hydrogen sulphide solution, mix, dilute with water (DW) to 50 ml, allow to stand for 5 minutes and view downwards over a white surface, the colour produced in the test solution is not darker than that produced in the standard solution.

 A few typical examples from the official compendium are given below :

Limit Test for Arsenic

Theory : The official process is a development of the Gutzeit Test wherein all arsenic present is duly converted into arsine gas (AsH₃) by subjecting it to reduction with zinc and hydrochloric acid. Further, it depends upon the fact that when arsine comes into contact with dry paper permeated with mercuric (Hg²⁺) chloride it produces a yellow strain, the intensity of which is directly proportional to the quantity of arsenic present. The various chemical reactions involved may be expressed by the following equations :

The details of experimental procedure described in the Pharmacopoeia are actually based upon a paper by Hill and Collins**, but have been adequately modified from time to time in accordance with the accumu-lated and acquired experience. Explicitly, the expressions provided in the Pharmacopoeia for limits of arsenic exclusively refer to parts per million, calculated as As.

Materials Required : Arsenic limit test apparatus; HgCl₂—paper : smooth white filter paper (having thickness in mm of 400 paper = weight in g per Sq. M.), soaked in a saturated solution of HgCl₂, pressed to get rid of excess of soln. and dried at about 60°C in the dark ; lead acetate solution 10.0% w/v soln. of PbAc₂ in CO_2– free water ; KI (AsT), 1.0 g ; Zn (AsT) : l0.0 g ; Dilute Arsenic solution (AST); Standard stains, Test Solutions—are prepared according to the Indian Pharmacopoeia 1996.

Arsenic Limit Test Apparatus (Figure 2)

A wide-mouthed glass bottle capable of holding about 120 ml is fitted with a rubber bung through which passes a glass tube. The latter, made from ordinary glass tubing, has a total length of 200 mm and an internal diameter of exactly 6.5 mm (external diameter about 8 mm). It is drawn out at one end to a diameter of about 1 mm and a hole not less than 2 mm in diameter is blown in the side of the tube, near the constricted part. When the bung is inserted in the bottle containing 70 ml of liquid, the constricted end of the tube is kept above the surface of the liquid, and the hole in the side is below the bottom of the bung. The upper end of the tube is cut off square, and is either slightly rounded or ground smooth.

The rubber bungs (about 25 mm × 25 mm), each with a hole bored centrally and through exactly 6.5 mm  in diameter,are fitted with a rubber band or spring clip for holding them tightly in place.

Procedure : The glass tube is lightly packed with cotton wool, previously moistened with lead acetate solution and dried, so that the upper surface of the cotton wool is not less than 25 mm below the top of the tube. The upper end of the tube is then inserted into the narrow end of one of the pair of rubber bungs, to a depth of l0 mm (the tube must have a rounded-off end). A piece of mercuric chloride paper is placed flat on the top of the bung and the other bung placed over it and secured by means of the spring clip in such a manner that the holes of the two bungs meet to form a true tube 6.5 mm diameter interrupted by a diaphragm of mercuric chloride paper.

The test solution prepared as specified, is placed in the wide-mouthed bottle, 1 g of KI (AsT) and 10 g of Zn (AsT) are added, and the prepared glass tube is placed quickly in position. The reaction is allowed to proceed for 40 minutes. The yellow stain that is produced on the HgCl₂ paper if As is present is compared by daylight with the standard stains obtained by performing in an identical manner with known quantities of dilute arsenic solution (AsT). The comparison of the stains is made immediately at the completion of the test.

By matching the intensity and depth of colour with standard stains, the proportion of arsenic in the substance may be estimated. Thus, a stain equivalent to the 1 ml standard stain obtained by performing on l0 g of a substance implies that the proportion of As is 1 part per million.

Cautions : 

(i) HgCl₂ paper should be protected from sunlight during the test to avoid lighter or no

stain.

(ii) The standard and test stains must be compared immediately as they fade out on retaining.

(iii) The reaction may be expedited by the application of heat and 40°C is considered to be the most ideal temperature.

(iv) The tube should be washed with HCl (AsT), rinsed with DW, and dried between successive tests.

Special Techniques : The special techniques are usually applicable to a host of pharmaceutical sub-stances before the normal test can be performed. A few typical examples would be discussed briefly here, namely :

(i) Free Acids : They are first converted to their respective sodium salts with Na₂CO₃ and As³⁺ oxi-dised to As⁵⁺ by evaporating the solution with Br₂. The residue is ignited carefully until carbonised to destroy organic matter, while As is kept as non-volatile sodium arsenate. The resulting residue is dissolved in brominated HCl and the test carried out in the normal manner.

Examples : Aspirin, Saccharin, Sodium Salicylate, Sodium Aminosalicylate.

(ii) Substances Reacting Vigorously with HCl : The As is readily converted to AsCl₃ which being volatile in nature is also carried off along with relatively large volumes of CO₂ (generated by the substance and HCl).

Examples : Magnesium Carbonate, Light Magnesium Oxide, Calcium Hydroxide, Chalk, KOH, NaOH.

(iii) Insoluble Substances : These substances, as those that do not interfere with the solution of As and its subsequent reduction to AsH₃ (arsine). Such substances are suspended in water along with stannated-HCl, and the normal test is performed.

Examples : Magnesium Trisilicate, Bentonite, Barium Sulphate, Light and Heavy Kaolin.

(iv) Metals Interfering with Normal Reaction

(a) Iron : It gets deposited on the surface of Zn thereby depressing the intensity of reaction between Zn and HCl to produce H₂.

Remedy : The sample is dissolved in H₂O and stannated HCl to allow conversion of all As to As³⁺ and finally as AsCl₃. The latter being volatile in nature can be separated by distillation from remaining metallic salts and the distillate examined in the normal manner.

Example : Ferrous Sulphate.

(b) Antimony : Sb-compounds are also reduced simultaneously by Zn/HCl to yeild SbH₃ (stilbine) that reacts with HgCl₂ paper to give a stain. Therefore, the sample is first distilled with HCl to yield a distillate containing all the As as AsC₃ (volatile), but yields only a fraction of Sb as SbCl₃ (non-volatile). A repeated distillation obviously gets rid of even the last traces of Sb.

Examples : Antimony Potassiun Tartrate, Antimony Sodium Tartrate.

A few typical examples are cited below from the official compendium.

Limit Test for Iron

Theory : The limit test for Iron is based on the reaction between iron and thioglycollic acid in a medium buffered with ammonium citrate to give a purple colour, which is subsequently compared with the standard colour obtained with a known amount of iron (0.04 mg of Fe). Ferrous thioglycollate is a co-ordination compound that attributes the purple colour ; besides thioglycollic acid converts the entire Fe³⁺ into Fe²⁺. The reactions involved may be expressed as follows :

Materials Required

Nessler cylinder : 1 pair ; Ferric ammonium sulphate : 1.726 g ; Sulphuric acid (0. 1 N) : 10.0 ml ; Iron-free citric acid (20% w/v) : 2.0 ml ; Thioglycollic acid : 0.1 ml; Iron-free ammonia solution : 20 ml.

Standard Iron Solution : Weigh accurately 0.1726 g of ferric ammonium sulphate and dissolve in 10 ml of 0.1 N sulphuric acid and suffcient water to produce 1 Litre. Each ml of this solution contains 0.02 mg of Fe.

Standard Colour : Dilute 2.0 ml of standard iron solution with 40 ml DW in a Nessler cylinder. Add 2 ml of a 20% w/v solution of iron-free citric acid and 0.1 ml of thioglycollic acid, mix, make alkaline with iron-free ammonia solution, dilute to 50 ml with DW and allow to stand for 5 minutes.

Procedure : Dissolve the specified quantity of the substance being examined in 40 ml DW, and trans-fer to a Nessler cylinder. Add to it 2 ml iron-free citric acid solution and 0.1 ml thioglycollic acid, mix, make alkaline with iron-free ammonia solution, dilute to 50 ml with DW and allow to stand for 5 minutes. Any colour produced is not more intense than the standard colour.

Some examples of this test for pharmaceutical substances are listed below :

LIMIT TEST’S FOR ACID RADICAL IMPURITIES

Acid radical impurities constitute a serious but unavoidable source of impurities in a large number of pharmaceutical chemicals. However, the two most commonly found acid radical impurities are chloride (Cl^– ) and sulphate (SO₄^2–) that evidently arise from the inevitable use of raw tap-water in various manufacturing operations. As these two acid radical impurities are found in abundance due to contamination, the Pharmaco-poeia categorically stipulates limit tests for them which after due minor modifications are applicable to a number of pharmaceutical substances.

In addition to the above two commonly found impurities, there are a number of other acid radical impurities which exist in pharmaceutical substances, namely : arsenate, carbonate, cyanide, nitrate, oxalate, phosphate and silicate.

All these acid radical impurities shall be discussed briefly as under :

1. Limit Test for Chlorides

The limit test for chlorides is based on its precipitation with silver nitrate in the presence of dilute HNO₃, and comparing the opalescence produced due to the formation of AgCl with a standard opalescence achieved with a known quantity of Cl^– ions.

The equation may be expressed as :

Materials Required : Nessler cylinder 1 pair ; dilute nitric acid (10% w/w of HNO₃) 10.0 ml ; silver nitrate solution (5.0% w/v in DW) 1.0 ml.

Standard Opalescence : Place 1.0 ml of a 0.05845% w/v solution of NaCI in 10 ml of dilute HNO₃ in a Nessler cylinder. Dilute to 50 ml with DW and add 1 ml of AgNO₃ solution. Stir immediately with a glass rod and allow to stand for 5 minutes.

Procedure : Dissolve the specified quantity for the substance in DW, or prepare a solution as directed in the text and transfer to a Nessler cylinder. Add 10 ml of dilute nitric acid, except when it is used in the preparation of the solution, dilute to 50 ml with DW, and add 1 ml of AgNO₃ solution. Stir immediately with a glass rod and allow to stand for 5 minutes. The opalescence produced is not greater than the standard opalescence, when viewed transversely.

A few typical examples of this test representing a wide spectrum of pharmaceutical substances are enumerated below :

2. Limit Test for Sulphates

Theory : The limit test for sulphates is based upon its precipitation as barium sulphate in the presence of barium chloride, hydrochloric acid and traces of barium sulphate. In this combination, hydrochloric acid exerts its common ion effect whereas traces of BaSO₄ aids in the rapid and complete precipitation by seeding. Thus, the opalescence caused by the sample is compared immediately with a standard turbidity produced with a known amount of the SO₄^2– ion.

The main objective of this test is to provide a rigid control of sulphate as an impurity present primarily in inorganic pharmaceutical substances.

Materials Required : Nessler cylinders 1 pair ; dilute hydrochloric acid (10% w/v of HCl) 2.0 ml.

Barium Sulphate Reagent : Mix 15 ml of 0.5 M barium chloride, 55 ml of DW, and 20 ml of sulphate free alcohol, add 5 ml of a 0.0181% w/v soln. potassium sulphate dilute to 100 ml with DW, and mix. It should always be prepared fresh.

0.5 M Barium Chloride : BaCl₂ dissolved in DW to contain in 1 Litre 122.1 g of BaCl₂. 2H₂O.

Standard Turbidity : Place 1.0 ml of a 0.1089% w/v soln. of K₂SO₄ and 2 ml of dilute HCl in a Nessler cylinder, dilute to 45 ml with DW, add 5 ml BaSO₄ reagent, stir immediately with a glass rod and allow to stand for 5 minutes.

Procedure : Dissolve the specified quantity of the substance in DW, transfer to a Nessler cylinder, and the preparation of the solution. Dilute to 45 ml with DW, add 5 ml barium sulphate reagent, stir immediately with a glass rod, and allow to stand for 5 minutes. The turbidity is not greater than the standard turbidity, when viewed transversely.

A few examples of this test consisting of a cross-section of pharmaceutical substances are stated below :

3. Limit Test for Arsenate

Acetarsol : An organic arsenic compound, being therapeutically active when administered orally, that might be of value in the treatment of spirochaetal or protozoal diseases, for instance : syphilis, yaws, relapsing fever, sleeping sickness and amoebic dysentry.

It is made from p-hydroxyphenylarsonic acid, which may be prepared either by straight forward meth-ods from phenol or from p-aminophenylarsonic acid. The resulting compound obtained from either of these reactions is nitrated, reduced and the base is finally acetylated to afford acetarsol.

Inorganic arsenates are found to be extremely toxic in nature and hence careful control is maintained by the addition of magnesium ammonio-sulphate solution to an aqueous solution of the sample, thereby producing an instant white precipitate.

4. Limit Test for Carbonate

Carbonate impurity in pharmaceutical chemicals usually arise from contamination with atmospheric CO₂.

Examples of a few official compounds subject to this test from the Pharmacopoeia are given below :

5. Limit Test for Cyanide

Cyanide present in Edetate Disodium is assayed by titration with AgNO₃ in neutral solution employing dimethylaminobenzylidenerhodamine as an adsorption indicator with a colour change from yellow to orange.

A few typical examples are illustrated below :

A. Edetate Disodium

Materials Required : Edetate disodium 30.0 g ; sodium hydroxide solution (20% w/v in DW) 35.0 ml ; dimethylaminobenzylidenerhodamine solution (0.02% w/v in acetone) 1.0 ml ; 0.01 N AgNO₃ solution (1.699 g in 1 litre of DW) 100 ml.

Procedure : Dissolve 30.0 g in a mixture of 100 ml DW and 35 ml NaOH solution, add 1 ml dimethylaminobenzylidenerhodamine and titrate with 0.01N silver nitrate until the colour of the solution changes from yellow to orange. Repeat the operation without the disodium edetate. The difference between the titrations is not more than 1.25 ml.

B. Iodine

Materials Required : Iodine 3.5 g ; zinc powder 10 g ; ferrous sulphate solution (2.0% w/v in boiled and cooled DW) 1.0 ml ; sodium hydroxide solution (20% w/v in DW) 1 ml ; hydrochloric acid (~ 11.5 N) 20 ml.

Procedure : Triturate 3.5 g thoroughly with 35 ml DW, filter and decolorise the filtrate by the addition of a little zinc powder. To 5.0 ml of the filtrate add a few drops of ferrous sulphate solution and 1 ml NaOH solution ; warm gently and acidify with HCl, no blue colour or green colour is produced.

C. Potassium Iodide

Materials Required : Potassium iodide 0.5 g ; ferrous sulphate solution (2.0% w/v in boiled and cooled DW) 1 drop ; NaOH solution (20% w/v in DW) 0.5 ml ; HCl 20.0 ml.

Procedure : Dissolve 0.5 g in 5 ml warm DW, add 1 drop of ferrous sulphate solution and 0.5 ml NaOH solution and acidify with HCl, no blue colour is produced.

6. Limit Test for Nitrate

Basic nitrate is usually found as an impurity in bismuth salts (e.g., bismuth subcarbonate), very often due to the mode of preparation from the metal via bismuth nitrate.

BP (1914) first described a limit test, based upon the production of coloured nitro-compounds by the interaction of traces of nitrates with phenol-2, 4-disulphonic acid, and the conversion of these subsequently into dark-yellow ammonium salts. However, this test has a serious disadvantage of correctly matching the yellow colours with great difficulty.

BP (1932) put forward a more reliable test for nitrate based upon the oxidation of indigocarmine to colourless substances by the action of traces of nitrates in presence of hot and fairly concentrated sulphuric acid, and the reaction may be expressed as follows :

The quantities as specifed in the Pharmacopoeia allow an official limit of nitrate equivalent to about 0.29% BiONO₃.

A few typical instances of pharmaceutical substances are enumerated below :

7. Limit Test for Oxalate

Oxalate is found to be a frequent impurity in pharmaceutical substances belonging to the category of either organic acids e.g., anhydrous citric acid, tartaric acid; or salts of organic acids e.g., ferrous gluconate, sodium citrate, potassium citrate and sodium cromoglycate. The presence of this impurity is due to the following two prime factors, namely :

(a) The use of oxalic acid to get rid of Ca²⁺ during various manufacturing processes.

(b) The use of oxalic acid in the isolation and purification of organic bases e.g., ephedrine (thereby resulting into the formation of well defined crystalline oxalates).

A few typical examples are cited below :

8. Limit Test for Phosphate

The limit test for phosphate is based upon the formation of a yellow colour reaction with molybdovanadic reagent (combination of ammonium vanadate and ammonium molybdate) in an acidic medium. However, the exact composition of the molybdovanadophosphoric acid complex is yet to be established.

Three typical examples of pharmaceutical substances are stated below :

Molybdovanadic Reagent : Suspend 4.0 g of finely powdered ammonium molybdate and 0.1 g of finely powdered ammonium metavanadate in 70 ml DW and grind until dissolved. Add 20 ml of HNO₃ and dilute to 100 ml with DW.

Phosphate Standard Solution (5 ppm PO₄) : Dilute 0.5 ml of a 0.143% w/v soln. of potassium dihydrogen orthophosphate (KH₂PO₄) to 100 ml with DW.

Sulphomolybdic Solution : Dissolve with heating, 25 ml ammonium molybdate in 200 ml DW. Separately, with care, add 280 ml H₂SO₄ to 500 ml DW. Cool and mix the two solutions and dilute to 1 Litre with DW.

Methylaminophenol-sulphite Solution : Dissolve 0.1 g of 4-methylaminophenol sulphate, 20 g sodium metabisulphite and 0.5 g anhydrous sodium sulphite in sufficient DW to produce 100 ml.

LIMIT TESTS FOR NON-METALLIC IMPURITIES

Non-metallic impurities, such as boron, free halogens (I₂, Br₂ and Cl₂) and selenium in pharmaceutical substances usually contribute untoward reactions, skin manifestations and are found to be toxic to healthy tissues.

A few typical examples are described below which essentially contains the above cited nonmetallic impurities :

1. Boron

A. Salbutamol Sulphate : Boron shows its presence in the above compound as a result of the use of sodium borohydride (NaBH₄) in the manufacturing process. The estimation depends upon the conversion of boron to borate and the organic matter is subsequently destroyed by ignition with anhydrous sodium carbonate. The quantity of boron is finally determined by colorimetric assay.

Materials Required : Salbutamol sulphate 50 mg ; solution of an equimolar mixture of anhydrous sodium carbonate and potassium carbonate (3% w/v in DW) 5.0 ml ; Solution of curcumin (0.125% w/v in glacial acetic acid) 3.0 ml ; mixture of H₂SO₄ and glacial CH₃COOH (5 ml : 5 ml) 3.0 ml ; ethanol (96%) 100 ml ; solution of boric acid (dissolve 5 g of boric acid in a mixture of 20 ml DW and 20 ml absolute ethanol and dilute to 250 ml with absolute ethanol) : 100 ml.

Procedure : To 50 mg of substance add 5 ml of a 3% w/v solution of an equimolar mixture of anhydrous Na₂CO₃ and K₂CO₃, evaporate to dryness on a water-bath and dry at 120°C. Ignite the residue rapidly until the organic matter has been destroyed, allow to cool and add 0.5 ml DW and 3 ml freshly prepared 0.125% w/v soln. of curcumin in glacial acetic acid. Warm gently to effect solution, allow to cool and add 3 ml of a mixture of H₂SO₄, with stirring, to 5 ml of glacial acetic acid. Mix and allow to stand for 30 minutes. Add sufficient ethanol (96%) to produce 100 ml, filter and measure the absorbance of the filtrate at the maximum of 555 nm. Calculate the content of boron from a reference curve prepared from the absorbance obtained by treating suit-able aliquots of a solution of boric acid in the same manner.

Prescribed Limits : Not more than 50 ppm.

2. Free Halogens

A few typical examples of pharmaceutical chemicals in which free halogens like Iodine, Bromine, Fluo-rine and Chlorine are present as non-metallic impurities are given below.

A. Clioquinol : (Free Iodine)

Materials Required : Clioquinol 1.0 g ; potassium iodide 1.0 g ; H₂SO₄ (1 M) 1.0 ml ; chloroform 2.0 ml ; sodium thiosulphate (0.005 M) 0.1 ml.

Procedure : Shake 1.0 g with a solution of 1 g potassium iodide in 20 ml DW for 30 seconds, allow to stand for 5 minutes and filter. To 10 ml of the filtrate add 1 ml 1 M H₂SO₄ and 2 ml chloroform and shake.

Prescribed Limits : Any colour in the chloroform layer is discharged on the addition of 0.1 ml of 0.005 M sodium thiosulphate.

B. Diethylpropion Hydrochloride : (Free Bromine)

Test : Place 0.05 ml of a 10% w/v solution on starch-iodide paper.

Prescribed Limit : No colour is produced.

C. Doxycycline Hydrochloride : (Free Fluorine)

Materials Required : Doxycyline Hydrochloride : 0.30 g ; oxygen-combustion flask ; 1 L capacity;

Nessler cylinder 100 ml ; zirconyl alizarin solution* : 5.0 ml ; fluoride standard solution (10 ppm F) (dilute 5.0 ml of a 0.0442 % w/v soln. of sodium fluoride, previously dried at 300°C for 12 hours, to 100 ml with DW) : 3.0 ml.

Procedure : Burn 0.30 g, in three equal portions, by the method for oxygen-flask combustion (BP), using a 1 Litre flask and a separate 20 ml portion of DW as the absorbing liquid for each combustion, shaking the flask vigorously for about 15 minutes and transferring to the same 100 ml Nessler cylinder. Add 5 ml of acid zirconyl alizarin solution to the combined liquids, adjust the volume to 100 ml with DW and allow to stand for 1 hour.

Prescribed Limit : The colour of the resulting solution is greater than that obtained by repeating the operation with no substance enclosed in the successive portions of filter paper burnt in the method for oxygen flask combustion, but adding 3.0 ml of fluoride standard solution (10 ppm F) to the combined absorption liquids before adding the acid zirconyl alizarin solution.

D. Chloroform : (Free Chlorine)

Materials Required : Chloroform 10.0 ml ; cadmium iodide solution (5.0% w/v in DW) 1.0 ml ; starch mucilage 0.1 ml.

Procedure : Shake 10 ml of chloroform with 20 ml of freshly boiled and cooled DW for 3 minutes and allow to separate. To the aqueous layer add 1 ml cadmium iodide soln. and 0.1 ml of 10 ml of starch mucilage.

Prescribed Limit : No blue colour is produced.

E. Tetrachloroethylene (Free Chlorine)

Perform the limit test as stated under chloroform. No blue colour is produced.

3. Selenium

A. Selenium Sulphide

Theory : Selenium is very toxic and its contamination is usually controlled by an absorptiometric method after destruction of the organic compound with fuming nitric acid. The latter converts selenium (Se) as selenous acid (H₂SeO₃), which on subsequent treatment with 3,3′-diaminobenzidine under controlled experimental pa-rameters, results into the formation of a highly coloured compound known as 3,4-diaminophenylpiazselenol. The latter is consequently extracted with toluene after making the aqueous solution alkaline, and the colour compared with a standard prepared likewise from a known amount of selenium. The various reactions involved may be expressed as follows :

Materials Required : Selenium sulphide : 10.0 g ; formic acid (2.5 M) : 2.0 ml ; 3,3′-diaminobenzedine tetrahydrochloride solution (0.5% w/v in DW) : 2.0 ml ; ammonia (5 M) : 20 ml ; selenium standard solution (1 ppm Se) (Dilute 2.5 ml of a 0.00654% w/v solution of selenous acid to 100 ml with DW) : 5.0 ml.

Procedure : To 10 g of selenium sulphide add 100 ml DW, mix well, allow to stand for 1 hour with frequent shaking and filter. To 10 ml of the filtrate, add 2 ml of 2.5 M formic acid, dilute to 50 ml with DW, adjust the pH to 2.0 to 3.0 with 2.5 M formic acid, add 2.0 ml of a 3,3′-diaminobenzidine tetrahydrochloride in DW, allow to stand for 45 minutes and adjust the pH to 6.0 to 7.0 with 5 M ammonia. Shake the solution for 1 minute with 10 ml of toluene and allow to separate. Measure the absorbance at 420 nm.

Prescribed Limit : The measured absorbance at 420 nm is not greater than that of a solution prepared by treating 5 ml of selenium standard solution (1 ppm Se) in the same manner (5 ppm, calculated as Se).

THEORY AND TECHNIQUE OF QUANTITATIVE ANALYSIS

INTRODUCTION

The ‘technique of quantitative analysis’ is broadly based on the following three major heads, namely :

(a) Technique of Volumetric Analysis,

(b) Technique of Gravimetric Analysis, and

(c) Biomedical Analytical Chemistry.

Volumetric analysis essentially comprises of the most precise and accurate measurement of interacting solutions or reagents. It makes use of a number of graduated apparatus, such as : graduated (volumetric) flasks, burettes, pipettes and measuring cylinder of different capacities (volumes).

However, it is pertinent to mention here that quite a few techniques related to measurement of pharmaceutical substances and reagents involved is more or less common to both gravimetric and volumetric analysis. Besides, in gravimetric analysis, some more additional techniques play a vital role, namely : precipitation, filtration, washing of the precipitate and ignition of the precipitate.

Biomedical analytical chemistry happens to be one of the latest disciplines which essentially embraces the principles and techniques of both analytical chemistry and biochemistry. It has often been known as ‘clinical chemistry’. This particular aspect of analytical chemistry has gained significant cognizance in the recent past by virtue of certain important techniques being included very much within its scope of analysis, namely : colorimetric assays, enzymic assays, radioimmunoassays and automated methods of clinical analysis.

It is, however, important to mention here that certain other routine procedures also carried out in a clinical laboratory fall beyond the scope of biomedical analytical chemistry, narnely : microbiological assays, heamatological assays, serum analysis, urine analysis and assays of other body fluids.

It will be very much within the scope of this to discuss briefly the various important details, with specific examples wherever necessary, of volumetric analysis, gravimetric analysis and biomedical analytical chemistry

VOLUMETRIC ANALYSIS

Volumetric analysis may be broadly defined as those analytical methods whereby the exact volume of a solution of known concentration actually consumed during the course of an analysis is considered as a measure of the amount of active constituent in a given sample under determination (assay).

1. THEORY

According to the official method of analysis, hydrochloric acid can be determined by first weighing a given sample accurately, and secondly, by adding carefully a solution of known strength of sodium hydroxide in the presence of an appropriate indicator unless and until the exact equivalent amounts of HCl and NaOH have undergone the following chemical reaction :

Analyte (or Active Constituent) is the chemical entity under assay e.g., HCl.

Titrant is the solution of known strength (or concentration) employed in the assay e.g., NaOH.

Titration is the process of adding and then actually measuring the volume of titrant consumed in the assay. This volume is usually measured by the help of a calibrated burette.

Indicator is a chemical substance sensitive enough to display an apparent change in colour very close to the point in the ongoing titration process at which equivalent quantities of analyte and titrant have almost virtually reacted with each other.

Equivalence Point (or Stoichiometric Point) is the point at which there appears an abrupt change in certain characteristic of the prevailing reaction mixture—a change that is either ascertained electrometrically or is visibly spotted by the use of indicators.

In usual practice, the volumetric titrations may be accomplished either by direct titration method e.g., assay of HCl employing NaOH as the titrant, or by residual titration method e.g., assay of ZnO in which case a known-excess-measured volume of standardised solution of H₂SO₄, more than the actual amount chemically equivalent to ZnO, is added to the sample ; thereupon, the H₂SO₄ which remain unreacted with ZnO is subsequently titrated (sometimes referred to as back titration or residual titration in the text) employing standardized NaOH solution.

Thus, we have :

Known amount of H₂SO₄ consumed ≡ Known amount of NaOH + Unknown amount of ZnO Most official compendia usually record the results of drug assays in terms of % w/v, % w/w and % v/v.

2. DEFINITIONS

In order to have a clear-cut understanding of the various calculations involving volumetric assays through-out this book one needs to gain an in-depth knowledge of the various terms related to ‘equivalents’. They are :

(a) Gram-equivalent Weight (GEW) : It is the weight in grams that is chemically equivalent to 1 gram-atom of hydrogen (1.0079 g).

It is also sometimes simply referred to as the ‘gram-equivalent’. However, GEW has two distinct definitions for neutralization as well as as oxidation-reduction reactions as stated below :

(i)          For Neutralization Reactions : GEW is defined as that weight of a substance in grams which contains, furnishes, reacts directly or indirectly and replaces 1 gram-atom or ion of hydrogen.

(ii) For Oxidation—Reduction Reactions

Explanation : A reaction usually takes place by the combination of oxidizing and reducing agents and this may be considered as the basis for the quantitative measurement of one of the reactants. For instance, FeSO₄ can be determined quantitatively by its reaction with ceric sulphate [Ce(SO₄)₂] as expressed by the following equation :

                        .......(a)

Equation (a) can be split into two half-equations as shown below thereby depicting the loss of elec-trons by the Fe²⁺ ion [Eq. (b)] and the gain of electrons by the Ce⁴⁺ [Eq. (c)] :

                      ...........(b)

                  .................(c)

From Eq. (a) it is evident that each molecule of FeSO₄, upon oxidation, happens to lose one electron. Hence, one mole of FeSO₄ loses 6.02 × 10 ²³ electrons which is equivalent to 1 Faraday or 96,500 C. Thus, in electrochemical determination of equivalence point the quantity of electricity is almost identical with that required to reduce 1 mole of Ce(SO₄)₂. It follows from here that 1 mole of FeSO₄ and 1 mole of Ce(SO₄)₂ are chemical equivalents. In other words, 1 g of H, acting as a reducing agent, loses electrons equivalent to 96,500 C.

(b) Equivalent Weight of a Reducing Agent is that weight which loses electrons equivalent to 96,500 C.

It may be calculated by dividing the gram-molecular weight by the number of electrons lost by each molecule, for instance :

hence, the equivalent weight of FeSO₄ oxidizing to Fe₂(SO₄)₃ comes out to be 151.919 [FeSO₄ :

molecular weight = 151.91] or 1 gram-molecular weight.

(c) Equivalent Weight of an Oxidizing Agent is that weight which gains electrons equivalent to 1 Faraday, or to the electrons gained by 1 gram-ion of H⁺ ions (2H⁺ + 2e → H₂ ).

It may be calculated by dividing the gram-molecular weight by the number of electrons gained by each molecule, for example :

(a)   Ce⁴⁺ + e   → Ce³⁺      (cerous ion)

hence, the equivalent weight of ceric sulphate is 1 gram-molecular weight 332.24 g [Ce(SO₄)₂ : molecular weight = 332.24]

(b) MnO4– + 5e  → Mn2+   (manganous ion)

hence, the equivalent weight of potassium permanganate is 1/5th gram-molecular weight 31.61 g.

(KMnO₄ : 1/5 × 158.05 = 31.61)

(c)   Cr₂O₇^2– + 6e —→  2Cr³⁺                    (chromous ion)

hence, the equivalent weight of potassium dichromate is l/6 gram-molecular weight 49.03 g. (K₂Cr₂O₇ : 1/6 × 294.18 = 49.03)

(d) I2 + 2e  → 2I–  (iodide ion)

hence, the equivalent weight of iodine is 1 gram-molecular weight 126.90 g. (I₂ : Molecular Weight = 126.90)

(e)    BrO₃^– + 6e   → Br^–                            (bromide ion)

hence, the equivalent weight of potassium bromate is 1/6 gram-molecular weight 27.83 g.

(KBrO₃ : 1/6 × 167.01 = 27.83)

(d) Gram-milliequivalent Weight (GmEW) is nothing but GEW/1000. This term is very much used in all types of volumetric calculations.

(e) Equivalent (equiv) is the number of gram-equivalents involved in a quantitative method.

(f) Milliequivalent (meq) is the number of gram-milliequivalents involved in a quantitative method.

However, meq is used more frequently than equiv in quantitative procedures.

(g) Standard Solution is a solution of known (pre-determined) normality or molarity.

(h) Normality (expression of concentration) is the number of equivalents of solute per litre (equiv/lire) or milliequivalents per ml. (meg/ml) solution.

(i) Molarity is the expression of the concentration of a solution in terms of moles per litre.

(j) Standardization is the actual determination of either the normality or the molarity of a solution.

(k) Primary Standard is the substance of known purity (‘AnalaR’-grade reagents) whose carefully weighed quantity helps in the standardization of an unknown solution (normality or molarity).

(l) Secondary Standard is another standard solution that is used for standardization of an unknown solution.

Example : An unknown solution of HCl may be standardized volumetrically in two ways, namely :

(i) by the help of ‘AnalaR’-grade Na₂CO₃ i.e., purity is known-‘Primary Standard’, and

(ii) by the help of another standard solution of NaOH—‘Secondary Standard’.

(m) Titer : is the weight of a substance chemically equivalent to 1 ml of a standard solution.

Example : 1 ml of 1 N HCl contains 0.03646 g (i.e., 0.001 equiv or 1 meq) of HCl and hence is chemically equivalent to 0.04000 g (i.e., 0.001 equiv or 1 meq) of NaOH.

Thus, most calculations in volumetric determinations (titrimetry) are enormously facilitated by using titer values.

For instance, in the offcial procedure for the assay of tartaric acid, it is stated that ‘Each millilitre of 1 N sodium hydroxide is equivalent to 75.04 mg of C₄H₆O₆’. The C₄H₆O₆ titer of 1 N sodium hydroxide is, therefore, 75.04 mg/ml, a value that may be calculated as follows :

An examination of the equation indicates that 1 mole or 150.09 g of 

is 2 equiv, and the equivalent weight of H₂C₄H₄O₆ is 75.04 g. Hence, each millilitre of 1 NaOH contains 0.001 equiv of NaOH and is equivalent to 0.001 equiv or 0.001 × 75.04 = 0.07504 g or 75.04 mg of H₆C₄O₆.

In the broader sense, volumetric apparatus may be divided into two categories, namely :

(a) To deliver a definite volume of liquid, and

(b) To contain a definite volume of liquid.

1. Volumetric Apparatus Meant to Deliver a Definite Volume of Liquid

The two specific volumetric apparatus meant to deliver a defnite volume of liquid are burettes and pipettes which will be discussed very briefly below :

1.1. Burettes

Various official compendia specifies a standard temperature (°C) for glass volumetric apparatus as mentioned hereunder :

Pharmacopoeia of India (IP) : 27°C ;

United States Pharmacopoeia (USP) and National Formulatory (NF) : 25°C ;

National Bureau of Standards (NBS) : 20°C.

A burette is a graduated glass tube of uniform bore throughout the entire length, used for the accurate delivery and measurement of variable volumes of liquids. Burettes are graduated into millilitres (ml) and 1/10 millilitres (0.1 ml) and are made of varying capacity ranging from 1 ml to 100 ml ; however, the most common size is the 50 ml burette that is used invariably and conveniently for most volumetric titrations. They are usually closed at the bottom either by a Teflon or glass stopcock to monitor and control the outflow of liquid.

Specifications : The design, construction and capacity of volumetric glassware must be in accordance with those laid down by the Indian Standards Institution (ISI). The tolerances on capacity for burettes, as speci-fied in the relevant Indian Standards Institution, specifications are given in Table 2.1.

British Standards Institution (B.S. 846 : 1962) has laid down specifications for burettes and these are produced to either Class ‘A’ or Class ‘B’ specifications. All Class ‘A’ and a few of Class ‘B’ burettes have graduations that extend right round the barrel (or stem) of the burette to minimise errors due to parallax while taking the exact burette reading. It may be noted that Class ‘B’ burettes are normally graduated on one side only. Permitted tolerances on capacity for burettes used in common practice are stated in Table 2.2.

In fact, the tolerance actually represents the maximum error allowed at any point and also the maximum difference allowed between the errors at any two points. For instance, a tolerance of ± 0.05 ml signifies that the burette may have an error at any point by ± 0.05 ml, provided that the difference between the errors at any two given points does not exceed 0.05 ml.

Burettes calibrated at 20°C and 25°C deliver different weights of water for each 10 ml, when weighed with standard brass weights in air at 50% relative humidity (RH) at standard atmospheric pressure, as given below :

Hence, the true volume for each 10 ml segment of the burette can be calculated from the weights obtained and recorded on a convenient chart.

Leakage : A burette must be tested for any sort of leakage before putting it into operation. Teflon stopcocks are usually adjusted by a knurled nut for perfect use. Glass stopcocks may require a small quantity of a special type of grease or lubricant to allow both ease of operation and to check leakage.

Outlet Tip : From a practical point of view the outlet tip of either types of burette, i.e., having Teflon or glass stopcocks, must be of such diameter and taper as to allow the delivery of a single drop whose volume is significantly less than that which can be held between any two finest graduations of the scale with which the burette is calibrated.

Use of the Burette : The following steps are usually observed while operating a burette, namely :

(i) Burette tap is neatly lubricated with a thin-film of grease,

(ii) Rinse the burette, before putting it into operation, at least twice with small volumes of the solution (titrant), say about 5.0 ml, carefully draining out the solution between the addition of each portion,

(iii) Pour the solution into the burette until the former is little above the zero mark,

(iv) Open the burette tap slowly to fill up the tip of the burette and to expel all air bubbles,

(v) With the zero at eye-level carefully, drain out the liquid until the lower part of the meniscus is either at level or just below the Zero mark,

(vi) Remove the drop on the tip of the burette by just touching rapidly against the inner-neck of a flask or a porcelain tile,

(vii) The Class ‘B’ burettes should be read at level so as to avoid errors due to parallax,

(viii) To assist easy and accurate observation of the meniscus (lower for colourless solutions and upper for coloured solutions) it is always advisable to hold a piece of white paper behind the burette at the appropriate level,

(ix) Burette readings may be recorded to the nearest 0.02 ml, and

(x) Once a titration is completed, 15 seconds duration should be allowed to elapse before the final read-ing is made, to allow for drainage.

1.2. Pipettes

The pipette is the second volumetric apparatus that is meant to deliver a definite volume of liquid. Pipettes are of two types, namely :

(i) Transfer Pipettes : They have only one specific mark engraved on them and are specifically employed to deliver (or transfer) a definite volume of liquid under certain specified conditions, and

(ii) Graduated Pipettes : They have graduated stems and are used to deliver different small volumes as needed. However, they are not normally used for measuring very exact volumes of liquids.

The tolerances on capacity for pipettes, as specified by the Indian Standards Institution (ISI), are stated in Tables 2.3 and 2.4.

The British Standards Institution (BSI) has laid down the permitted tolerances and delivery times for commonly used bulb transfer pipettes as shown in Table 2.5.

The USP specifies the following tolerances accepted by the National Bureau of Standards for transfer pipettes :

The salient features of single-graduation mark transfer pipettes are :

(a) Capacity, temperature at which it was graduated (Ex) and reference to delivery time in seconds is stated on the bulb e.g., BOROSIL 1552 25 secs ‘A’ Ex 20 ml 20°C BS 1583.

(b) Class ‘A’ pipettes do mention the delivery time,

(c) Drainage time is specified, though an additional waiting time of 3 seconds after apparent cessation of flow is still important.

Note : The stated times apply only for water and aqueous solutions.

Use of the Transfer Pipette : The following steps mentioned sequentially must be followed while making use of a transfer pipette :

·              Always rinse the pipette with DW before use and allow it to drain as completely as possible,

·              Droplets of water remaining in the tip must be removed by touching against filter paper; and at the same time wipe out the outer surface of the pipette to prevent dilution of the solution to be pipetted,

·              Rinse the pipette with 2 to 3 small portions (5 ml) of the solution and drain out the liquid completely,

·              Gently suck the liquid up into the pipette a little above the single graduation mark and quickly shut the upper end of the pipette with the tip of the index finger. Now, remove the pipette from the stock solution and carefully wipe out the outer surface of the stem free from any liquid adhering to it. Hold the pipette vertically and keeping the graduation mark at the eye-level, slowly release the pressure on the index finger until the bottom of the meniscus just coincides with the graduation mark. Maintain sufficient pressure on the index-fnger so as to check any escape of liquid from the pipette, and quickly get rid of the drop attached to the tip by gently touching against a porcelain tile. Put the pipette into the receiving container, and permit the liquid to drain out with the tip of the pipette touching the inside of the container at an angle of 60°, taking care that the tip must not be dipping into the deliv-ered liquid. After all the solution has drained out, hold the pipette in this position for at least 3 seconds (waiting time), and then remove the pipette.

Note :

·              The National Physical Laboratory (NPL) describes a method of reading meniscus in graduated glassware, viz., a dark horizontal line on a white background is placed 1 mm below the meniscus. A slight adjustment of the position of the dark line causes the meniscus to stand out sharply against the white background,

·              The small drop of liquid that remains in the tip of the emptied pipette is taken into account while doing the calibration, and hence, it must not be added to the delivered liquid by blowing down the pipette.

·              Liquids having more viscosity and much larger surface tension than water must be provided with adequate draining time e.g., strong solution of iodine.

·              Presently, many analysts make use of pipette filler for sucking in and draining out of liquids from the transfer pipettes for obvious reasons.

Automatic Pipettes (Transfer Pipettes) : Automatic pipettes are always preferred to ordinary transfer pipettes because of their ability to handle corrosive and toxic liquids in routine analytical laboratories, e.g., determination of Iodine Value in edible oils by iodine-monochloride (ICl) solution.

The automatic pipette (Figure 2.1) dispenses a stated volume of liquid when filled with liquid used in the assay from tip (B) to tip (C) and is allowed to drain out in the normal manner. D is connected to an aspirator which is placed above the pipette so as to enable the solution to flow under gravity.

Operation of the Automatic Pipette : The automatic pipette may be operated by observing the following steps in a sequential manner :

·              Turn the two-way tap clockwise to open so that the solution starts flowing into the pipette.

·              After about 5.0 ml has run into the pipette, turn A clockwise through 180°, so that solution now flows from the pipette to fill the delivery tube B.

·              As soon as B is full upto the tip, again turn A clockwise through 180°, so that the body of the pipette is filled completely to the top-tip.

·              Close the tap A by turning clockwise through 90° when the solution starts to overflow at C.

·              The pipette is now full from the lower-tip to the upper-tip and is ready for operation.

·              Remove the drop of solution from tip B, run out and drain for 15 seconds in the usual way.

2. Volumetric Apparatus Meant to Contain a Definite Volume of Liquid

The two particular volumetric apparatus meant to contain a definite volume of liquid are volumetric flasks (also known as measuring or graduated flasks) and measuring cylinders (also known as graduated cylin-ders) which will be discussed here briefly :

2.1. Volumetric Flasks (Syn. Measuring Flasks or Graduated Flasks)

Volumetric flasks are normally round or pear-shaped, flat-bottomed; having a long-neck, which possesses a single graduation mark round the neck.

Flasks bearing one graduation mark, are meant to contain specified volume of liquid at 20°C, when the lower part of the meniscus coincides with the mark and are known as volumetric flasks.

The long and narrow neck of uniform diameter affords as a measure of accurate adjustment, since the height of the liquid is sensitive enough to small variations of volume.

Units of Capacity

Litre—is defined as ‘the volume occupied by one kilogram of water at its temperature of maximum density (4°C) and subjected to normal atmospheric pressure’. The litre is considered as the standard unit of volume for all volumetric measurements.

The cubic centimetre is the volume occupied by a cube of which each side is 1 cm in length, and thus, 1 litre equals 1000.028 c.c. Therefore, it follows from here that the millilitre and cubic centimetre are not the same, though the difference is quite negligible. Hence, all volumetric apparatus is universally standardized in millilitres.

Standard Calibrations

The Indian Standard Institution (ISI) has laid down the tolerances on capacity of volumetric flasks (with different capacities) calibrated at 27°C as stated in Table 2.6.

The United States Pharmacopoeia (USP) requirements for volumetric flasks calibrated to contain the indicated volume at 25°C are given in Table 2.7.

The British Standards Institution (BSI) and the National Physical Loaboratory (NPL) have laid down the tolerances in the capacity of volumetric flasks (i.e., measuring flask) at 20°C by two sets of toler-ances viz., Grade ‘A’ and Grade ‘B’ respectively, evidently to indicate the class of accuracy to which the flask has been subjected to for graduation, followed by the manufacturer’s name and finally the BS standard number. However, the permitted tolerances for volumetric flasks commonly used in analytical laboratories are de-picted in Table 2.8.

Preparation of Standard Solutions

A reasonably well established analytical laboratory requires a number of standard solutions for its routine as well as specific assays. Therefore, it necessitates to know the intricacies of preparing the standard solutions as detailed in the following steps :

·              Transfer the requisite quantity of the accurately weighed pharmaceutical substances or solid quantitatively into a beaker and dissolve it in either distilled water (DW) or other specified solvent,

·              Pour the resulting solution quantitatively, into the funnel placed in the mouth of the volumetric flask with the help of a glass rod and a sharp jet of water from a wash-bottle by holding the beaker with the right hand and the guiding rod with the left hand,

·              Wash down the contents of the beaker through the funnel by means of the glass rod and the jet of DW. Repeat the process several times till the flask is 2/3rd full,

·              Remove the funnel, swirl the contents of the volumetric flask and make up the volume upto the mark,

·              Final adjustment of the volume must be made with the help of a teat pipette by adding DW/solvent dropwise. In doing so, adequate care should be taken to allow suffcient time for water/solvent to drain-down the inside of the neck of the flask, and

·              Finally shake the contents of the flask thoroughly for 2 to 3 minutes to obtain a perfect homogeneous solution.

Note :

(i) For precise work, the temperature of the solution must be adjusted to 20°C before making the volume upto the mark,

(ii) Standard solutions are usually stored in stock-bottles,

(iii) Ensure before any transfer is actually affected that the receiving vessel must be rinsed with at least 2 to 3 successive small quantities of the solution, and

(iv) When a standard solution is used a while after preparation, the contents of the stockbottle must be shaken thoroughly before any solution is withdrawn, thereby the condensed droplets of water collected on the inside neck of the container gets mixed with the main bulk of the solution.

2.2. Graduated Cylinders

The graduated cylinders are also referred to as the measuring cylinders among volumetric apparatus meant to contain a definite volume of liquid. Measuring cylinders are containers either unstoppered or stoppered having a wide range of capacities varying from 5 ml upto 2000 ml (2 Litres). In usual practice, the smaller cylinders upto 100 ml are normally graduated either in fractions of a millimitre or in millilitres. On the contrary, the large cylinders are graduated in units of 2, 5, 10, 20, or 50 ml, as per their specific size and volume. However, it is pertinent to mention here that measuring cylinders are used in a broader sense for measuring volumes of solution when only approximate volumes are needed.

GENERAL COSIDERATIONS

Volumetric apparatus invariably used in titrimetric assays, meant either to deliver a definite volume of liquid viz., burettes and pipettes, or to contain a definite volume of liquid viz., volumetric flasks and measuring cylinders, have essentially the following three cardinal general considerations, namely :

(a) Cleaning of volumetric apparatus,

(b) Calibration of volumetric apparatus, and

(c) Effect of temperature on volumetric measurement.

These three aspects will be discussed briefly hereunder :

1. Cleaning of Volumetric Apparatus

New as well as used volumetric apparatus, namely : burettes, pipettes, volumetric flasks and measuring cylinders etc., employed in carrying out most of the pharmacopoeial assays should be extremely clean. It is particularly of great importance where small volumes of liquids are measured.

A positive evidence for a dirty apparatus may be sought by observing the adherence of droplets to the walls of a burette or pipette. However, in a clean volumetric apparatus, the liquid drains down quite uniformly thereby wetting the walls so that no droplets are visible to the naked eye.

A few very effective cleaning fluids that are used in good analytical laboratories are, namely :

(i) Chromic Acid Mixture,

(ii) Synthetic Detergent Solutions (or Alkaline Cleansing Agents), and

(iii) Teepol.

1.1. Chromic Acid Mixture

Materials Required : Sodium dichromate : 200 g ; Sulphuric acid : 1500 ml.

Procedure : Weigh 200 g sodium dichromate and transfer to a 2 Litre hard-boroslicate glass beaker. Dissolve it in 100 ml of water and cool in an ice-bath to about 10-15°C. Now, add to it 1500 ml of sulphuric acid (36 N) in small bits at intervals with constant stirring. Chromic acid mixture is extremely corrosive and hygroscopic and must be stored in closed glass-stoppered bottles.

Precautions :

(i) Chromate solution should be chilled before addition of H₂SO₄,

(ii) Safety goggles should be worn during the addition of the acid,

(iii) In case, a green colour develops, discharge the mixture into a sink with continuously flowing water,

(iv) Chromic acid must not be used for cleaning calibrated containers employed for optical measure-ments,

(v) Glass apparatus washed with chromic acid mixture must be subjected to adequate prolonged rinsing because glass (silicates and borosilicates) have a tendency to absorb the chromic acid,

(vi) Hot solutions should be avoided when cleaning accurately calibrated apparatus, due to the production of a permanent change in volume caused by heat known as thermal aftereffect,

(vii) All volumetric glasswares must be finally rinsed with purified water (distilled water) before use for analytical purposes.

1.2. Synthetic Detergent Solutions (or Alkaline Cleansing Agents)

Detergents are synthetic cleansing agents used with water. The most commonly used anionic surfactants containing carboxylate ions are known as soaps which are generally prepared by the saponification of natural fatty acid glycerides in alkaline solution. Usually a 2 to 5% (w/v) solution of a good detergent powder in water serves as a reasonably effective cleansing agent.

1.3. Teepol (or Gardinol Type Detergents)

It is a mixture of the sodium salts of sulphated fatty alcohols made by reducing the mixed fatty acids of coconut oil or cottonseed oil, and fish oils. Sometimes natural waxes such as spermaceti, wool fat and bees wax are sulphated directly.

A 1 to 3% (w/v) solution of Teepol in water may also serve as a good cleansing agent for the removal of stubborn deposits and stains present in glass apparatus.

TECHNIQUE OF VOLUMETRIC ANALYSIS

Following are the various steps that need to be observed carefully so as to achieve reasonably correct and reproducible results in the volumetric titrations :

1)          Conical flasks are considered to be the most suitable vessels meant for volumetric titrations because the mixing can be performed quite rapidly, easily and safely by gently swirling the contents of the flask during the titration,

2)          Beakers are not usually preferred, but in case they are to be used in volumetric analysis, following two provisions may have to be made for stirring :

·              use of a magnetic stirrer with a magnetic guide for the solution, and

·              use of a stirring rod,

3)          The titration container or vessel must always be kept polished so as to view the end point vividly,

4)          The solution under titration is normally viewed against a white background e.g., white tile or white paper,

5)          When the end point is being approached it is always advisible to have the drops of titrant split. It can be accomplished by opening the stopcock of the burette in such a manner that only a fraction of a drop flows out and remains adhered to the tip of the burette. Touch of the liquid against the inside of the flask and wash it down into the main bulk of the liquid with a fine jet of DW (from a wash-bottle),

6)          In a situation, where the colour-change at the end-point is rather gradual and not abrupt, it is always useful to have a comparison-solution readily available,

Example : Methyl orange offers a gradual end-point. Hence, two flasks containing the same volume of solution having approximately the same composition as the liquid being titrated may be prepared; first, slightly acidic—Red solution, second, slightly basic—Yellow solution.

7)          In fact, these carefully-prepared comparison solutions would ultimately help in deciding the colour change thereby confirming the actual end-point without any controversy, whatsoever,

8)          All titrations must be carried out in triplicate and the results of two concurrent readings (i.e., whose difference falls within 0.05 ml-based on a 20 ml titration) may be taken into consideration,

9)          Remainder solution in the burette, after titrations have been performed must be rejected and should not be put back to the stock-bottle for obvious reasons of contamination. The burette in operation is then washed thoroughly with DW and allowed to drain by placing it up-side down on a burette stand.

BIOMEDICAL ANALYTICAL CHEMISTRY

This particular aspect of analytical chemistry is the outcome of the unique amalgamation of the principles and techniques of analytical chemistry and biochemistry and was initially termed as ‘clinical chemistry’ but is more recently and more descriptively known as ‘biomedical analytical chemistry’.

Presently, both serum and urine assays are being used extensively in diagnostic medicine which evidently signifies that the pharmacist of today should be fully conversant with the ever-increasingly important techniques of biomedical analytical chemistry. It is, however, necessary to make a passing reference to microbiological assays and haematological assays, also being carried out in a clinical laboratory, though it should not be treated under this topic since these methods are outside the scope of biomedical analytical chemistry.

Classical example of SGOT-PAS episodes : Patients suffering from tuberculosis (TB) when diagnosed with para-aminosalicylic acid (PAS) invariably showed elevated serum levels of the intracellular enzyme serum-glutamic-oxaloacetic-transaminase (SGOT) which was initially considered and treated as a drug-induced hepatic toxicity. Later, an extensive and intensive studies revealed this to be an absolutely false diagnosis. In fact, the apparent enhanced SGOT levels were actually caused on account of the interference of PAS in the SGOT assay.

In the same vein, such analytical and biochemical interferences with respect to drug interference in various biomedical assays are being profusely cited in current scientific and research journals, such as the American Journal of Hospital Pharmacy and Clinical Chemistry.

It has been established beyond any doubt that analytical interferences can only take place when a drug or its resulting metabolite happens to interfere with the analytical method adopted for the assay.

In order to have a comprehensive account on the various aspects of ‘Biomedical Analytical. Chemistry’, we may have to study the following four methods of assay with specific emphasis on their principle and applications, namely :

(a) Colorimetric Assays,

(b) Enzymatic Assays,

(c) Radioimmunoassays, and

(d) Automated Methods of Clinical Analysis.

COLORIMETRIC ASSAYS

A. Theory : In fact, two fundamental laws actually govern the practice of colorimeteric assays of photometry.

First Law : Bougner’s (1729) or Lambert’s (1760) Law defines that—“when a beam of monochromatic light, previously rendered plane-parallel, enters an absorbing medium at right angles to the plane-parallel surfaces of the medium, the rate of decrease in radiant power with the length of light path through the absorbing medium `b’ is directly proportional to the radiant power of the beam, i.e., the light will be diminished in geometric (not arithmetic) or exponential progression”.

Alternatively, it may be explained that if a particular thickness absorbs half the light, the thickness which follows the first half and is equal to it will not absorb the entire second half, but instead only half of this half and will consequently reduce it to one-quarter. Thus, we have :

Upon integration and changing to logarithms of base 10, and substituting P = P₀ when b = 0, we may get :

2.303 log (P₀/P) = kb  ... (b)

In other words, the radiant power of the unabsorbed light decreases exponentially as the thickness of the absorbing medium increases arithmetically,

P = P₀ e^–kb = P₀ 10^{–0.43 kb}...(c)

Second Law : Bernard’s (1852) or Beer’s (1852) Law defines that—‘the radiant power of a beam of parallel monochromatic radiation decreases in a similar manner as the concentration of the light-absorbing constituent increases”. Thus we have :

2.303 log (P₀/P) = k′ C   ... (d)

where, C = concentration of substance, and

k′ = constant of proportionality.

Therefore, from Eq. (b) and Eq. (d), the two Laws may be combined and expressed with a single constant as follows :

log (P₀/P) = abc    ... (e)

or P = P₀ 10^–abc     ... (f)

where, a = absorptivity constant*.

[* and not to be tenned as absorbancy index, extinction coeffcient or specific extinction.]

In fact, the absorptivity constant ‘a’ is dependent upon the wavelength of the radiation as well as the nature of the absorbing material, whose concentration ‘C’ is usually expressed in grams per litre.

Molar Absorptivity (∈) : It is the product of the molecular weight of the substance and its absorptivity and is designated by the symbol ∈.

Beer’s Law (or Beer-Lambert’s Law) : The combined law is invariably referred to as ‘Beer’s Law’, while some texts refer to this as ‘Beer-Lambert’s Law’.

Eq. (f) is mostly expressed as shown below :

          A = abc       ...(g)

where, A = absorbance,

a = absorptivity,

b = optical path length, and

c = analyte concentration.

The term A^1%_1cm designates the absorbance of a 1 cm layer of solution that essentially contains 1% by weight of absorbing solute.

It is pertinent to mention here that most of the pure pharmaceutical substances (RS) do possess a definite characteristic absorbance (i.e., A^1%_1cn ) that forms the basis of their assay vis-a-vis the unknown sample.

Beer’s Plot : It is a plot of the absorbance value (along Y-axis) against a series of unknown solute concentrations in g/litre (along X-axis) thereby yielding a straight line passing through the origin.

Therefore, the solute-concentration present in an unknown solution can be estimated conveniently from the Beer’s Plot or sometimes referred to as the Standard Curve, merely by measuring the absorbance value of the solution and then finding the concentration value that corresponds to the measured absorbance value as is illustrated in the following Figure 2.2.

The colorimetric assay of sulphadiazine is based on the acid-catalysed equilibrium reaction that occurs between vanillin (an aldehyde) and sulphadiazine (an arylamine). The chemical species that forms as shown below is known as the Schiff’s Base and is yellow in colour.

Transmittance. The relationship between per cent transmittance and concentration is shown in Figure 2.3.

From Figure 2.3, it is quite evident that at lower concentrations the per cent trasmission is high and is vice varsa at higher concentrations.

However, a direct relationship between per cent transmittance and absorbance is illustrated in Figure 2.4.

B. Applications in Biomedical Analytical Chemistry Colorimetric assays have a wide spectrum of applications in biomedical analytical chemistry which may be categorized under the following four heads, namely :

(i) Colorimetric Assays of Biochemicals,

(ii) Colorimetric Assays Involving Complexation Reactions,

(iii) Colorimetric Assays Involving Redox Reactions, and

(iv) Colorimetric Assays of Enzyme Levels.

All these four categories of colorimetric assays shall be discussed briefly with appropriate examples, wherever necessary, to have an indepth knowledge and better understanding of the practical aspects.

1. Colorimetric Assays of Biochemicals

In this context, the discussion shall be restricted to the colorimetric assays of urea (BUN), bilirubin and cholesterol. However, the clinical significance of these substances and the extent to which they are present in biological fluids; besides the various drugs that usually interfere with their assay are also described adequately in the following pages :

1.1. Urea (BUN)

The extent of urea (BUN) present in biological fluids is normally determined in many Auto Analyzers by the following method :

The quantity of substance having an unknown structure is determined at 520 nm spectrophotometrically, while the normal BUN level is determined by averaging the BUN levels of a number of normal subjects.

Profile of BUN-levels

·              normal BUN level is 10-15 mg per 100 ml,

·              Enhanced BUN levels clearly signify a renal dysfunction, for instance urinary tract obstruction and nephritis i.e., inflammation of the kidney.

·              Increased incidence of BUN is also found in subjects suffering from diabetes, hepatic disorders and gastrointestinal disturbances,

·              Decreased BUN level is usually indicative of acute hepatic dysfunction and excessive dehydration,

·              A few important drugs, namely : thiazide diuretics (e.g., chlorothiazide, hydroflumethiazide, bendroflumethiazide, benzthiazide, cyclothiazide etc.), neomycin, tetracyclines, methyldopa etc., help in enhancing the BUN levels perhaps due to interference with normal renal function,

·              Phenothiazines (e.g., promethazine, chlorpromazine, ethopropazine etc.) on the contrary causes a significant decrease in BUN levels due to lowering of urea production from the liver, and

·              Substances that are inherently present in the serum and absorb at 520 nm shall interfere with these measurements, and therefore, necessary corrections for these materials have got to be made adequately.

1.2. Bilirubin

Bilirubin is diazotized with para-sulphonyl benzene diazonium compound and the absorbance of the resulting azobilirubin is measured at 600 nm to determine bilirubin level in the biological fluid e.g., blood serum. In usual practice, a serum blank is run simultaneously by reacting the serum with caffeine, sulphanilic acid and tartaric acid, and the absorbance of the blank is measured at 600 nm which is subsequently subtracted from the azobilirubin absorbance initially obtained before the bilirubin level is finally determined.

Profile of Bilirubin Levels

·              Normal bilirubin level ranges between 0-1.5 mg per 100 ml,

·              Enhanced bilirubin level may suggest drug toxicity, bile-tract obstruction, hepatitis and hepatic dysfunction,

·              As normal bilirubin level commences from zero, hence conditions responsible for its decrease are practically non-existent,

·              Increased bilirubin levels are caused due to the intake of large doses of such drugs as : chloroquine, vitamin K, sulpha-drugs, tetracyclines, paracetamol, nicotinic acid and monoamine oxidase inhibi-tors (e.g., iproniazid RP 1.0 ; nialamide RP 1.8 ; isocarboxazid RP 3.1 ; phenelzine RP 18 ; pheniprazine RP3l ; and tranylcypromine RP 45), where RP designates the ‘Relative Potency’ based on the tryptamine potentiation test. The elevated levels are due to hepatic injury, and

·              Drugs that interfere with the assay are, namely : (a) phenylazopyridine hydrochloride—a coloured drug, (b) azo-compound forming medicinals, and (c) degradation product of novobiocin.

1.3. Cholesterol

Cholesterol interacts with glacial acetic acid and acetic anhydride to result into the formation of a coloured product whose absorption is measured at 630 nm and this is found to be directly proportional to the level of cholesterol present in the serum. The reaction may be expressed as follows :

The above reactions is also referred to as the Libermann’s Reaction.

Profile of Cholesterol Levels

·              Normal total cholesterol level is 200 mg per 100 ml,

·              Increased cholesterol levels in serum are found in patients suffering from chronic hepatitis, atherosclerosis (deposit of fat in arteries of heart) and hypothyroidism,

·              Decreased cholesterol levels in serum is indicative of liver ailment and hyperthyroidism,

·              Corticosteroids (i.e., steroidal compounds) found in urine that possess biological properties resembling those of adrenal cortical extract, either in the increase or decrease of cholesterols levels,

·              Oestrogens, for instance : estrone, estriol, estradiol etc., are found to lower the cholesterol levels,

·              The broad-spectrum antibiotic chlorotetracycline and the aminoglycoside antibiotic kanamycin are observed to lower the cholesterol levels by forming salts with bile acids (e.g., cholic acid, deoxycholic acid and chenodeoxycholic acid) in the intestinal canal,

·              Likewise, the antoconvulsant phenytoin sodium and neomycin—an aminoglycoside antibiotic also decrease the cholesterol levels, and

·              Interestingly, penicillamine—a degradation product of penicillin and phenothiazines—the histamine H₁—receptor antagonists, such as : promethazine teoclate, methadilazine hydrochloride, trimeprazine tartrate are found to increase the cholesterol levels.

ENZYMATIC ASSAYS

A. Theory : 

All colorimetric enzymatic assays essentially involve the measurement of the activity of an ezyme under the following two circumstances, namely :

(a) When substrate is in large excess, and

(b) When enzyme concentration is in large excess.

A.1. Substrate Present in Large Excess : 

In reality, an enzyme reaction is nothing but a special kind of generalized reaction that may best be expressed as follows :

                     .....................(a)

Where, E = Enzyme,

            S = Substrate

          ES = Enzyme-substrate complex, and

          P = Product.

From Eq. (a), we have,

Rate of Product Formation = V_max [S]/K_m + [S]                                          ...(b)

Where, K_m = (k₂ + k₃) / k₁,

V_max = Max. rate of reaction

Assuming, [S] to be in large excess [S] >> K_m,

From Eq. (b) we have :

Rate of Reaction = V_max [S]/[S]

or    Rate of Reaction = V_max   ...(c)

Example : In order to measure the activity of an enzyme E, such as creatine phosphokinase (CPK), the concentration of the substrate S, for instance creatine, should be in large excesses so that the products measured shall be in the linear portion of the curve (Part ‘A’) in Figure 2.5.

Therefore, with a view to obtaining the best results, the two experimental parameters, namely : the temperature (constant-temperature-water-bath) and the time (phaser) should always be kept constant in order that the rate of reaction, as determined by the amount of product formed, specially designates the activity of the enzyme under assay, and devoid of the influence of any other variables on the reaction rate.

A.2. Enzyme Concentration in Large Excess

In order to analyze the quantity of substrate (S) present in a biological sample glucose oxidase is added in excess of the actual amount needed for the complete conversion of all the substrate to product ; and to achieve this object the reaction is allowed to run for a fairly long duration (i.e., to complete the reaction). It can be seen evidently in Part ‘B’ of Figure 2.5, wherein the sepecific reaction time the substrate (S) has been consumed completely and consequently, the concentration of the product achieves a maximum value.

1. Assay Methods

A few typical examples of colorimetric assay of enzyme levels will be discussed briefly hereunder :

1.1. Alkaline Phosphatase (AP)

Theory : 

Alkaline phosphatase is responsible for the cleavage of O-P bonds. It is found to be relatively non-specific and this characteristic permits the AP level to be assayed based on the fact that p-nitrophenylphosphate ion gets converted to p-nitrophenolate anion at pH 10.5; as expressed in the following reaction.

In actual practice, p-nitrophenylphosphate is present in large excesses, and the reaction is carried out at 38°C for 30 minutes. The resulting amount of p-nitrophenolate ion is estimated by the help of an usual standard curve employing known concentrations of p-nitrophenolate prepared from p-nitrophenol.

Bessey-Lowry Activity : 

One unit of activity may be defined as the amount of enzyme present in 1 millilitre of serum that liberates 1 μ mol of p-nitrophenol (0.1391 mg)* per hour at pH 10.5 after 30 minutes at 38°C.

Elimination of Interference due to Coloured Drugs

p-Nitrophenol is colourless, whereas the phenolate ion under basic conditions is yellow in appeanace. Therefore, the elimination of interference due to coloured drugs present in the serum is accomplished effectively by first, measuring the absorbance of the serum under basic conditions, and secondly, under acidic conditions. Thus we have :

A_{p-nitrophenolate} = A_basic – A_acidic

Profile of AP-levels

·              Normal AP-levels in adults range between 0.8 to 2.3 Bessey-Lowry units and in children between 2.8 to 6.7,

·              Increased AP-levels are observed in patients suffering from liver diseases, hyperparathyroidism and in rickets,

·              Decreased AP-levels could be seen in patients suffering from hypoparathyroidism and pernicious anaemia (i.e., an anaemia tending to be a fatal issue).

Interference due to Bilirubin

Bilirubin is eliminated by dializing the incubated p-nitrophenolate ion (at pH 10.5, and maintaining at 38°C for 30 minutes) into 2-amino-2-methyl-1-propanol, without carrying out the blank determination stated earlier.

There are a few medicinals that cause increased bilirubin levels which ultimately enhances AP-levels ; unless and until a corrective measure is taken in the respective procedure one may be left with false AP-level enhancement. Some typical examples are, namely : amitriptyline, chloropropamide, erythromycin, phenylbutazone, sulpha-drugs and tetracyclines.

Materials Required : 

0.01 M p-Nitrophenol (dissolve 140 mg of p-nitrophenol in 100 ml of DW) : 1.0 ml ; 0.02 N NaOH (dissolve 160 mg in 200 ml DW) : 200 ml ; 5 ml of alkaline-buffered substrate (l M p-nitrophenylphosphate) (dissolve 7.5 g glycine, 0.095 g anhydrous MgCl₂ and 85 ml of 1 N NaOH to 1 litre with DW ; and mixing with an equal volume of a solution prepared by dissolving 0. l0 g of p-nitro-phenylphosphate in 25 ml of water) ; temperature bath previously set at 38°C ; alkaline phosphatase for unknowns (commercial source) ; working standard [dilute 0.50 ml of a solution of p-nitrophenol (10.0 mol/ litre, 0.139 g/100 ml) to 100 ml with 0.02 N NaOH].

Procedure :

(1) First of all prepare a standard calibration curve as per Table 2.9.

(2) Plot a graph of absorbance A Vs units of alkaline phosphatase per millilitre.

(3) Proceed for the assay of AP in the serum sample sequentially as follows :

(i) Pipette 1.0 ml of alkaline—buffered substrate into each of two test tubes and keep in a water-bath preset at 38°C,

(ii) When both the test tubes have attained the temperature equlibrium, add 0.10 ml of serum and water to these tubes separately. The one with water serves as a reagent blank and is always needed per set of unknowns. Now, put the two tubes for incubation for exactly 30 minutes period,

(iii) Enzyme activity is arrested by adding 10.0 ml of 0.02 N NaOH to each tube. Remove them from the water-bath and mix the contents thoroughly,

(iv) Read out the absorbance of the unknown tube at 410 nm against the ‘reagent blank’ tube,

(v) Transfer the contents from the cuvets to the respective test-tubes and add 0.1 ml of HCl ( ₋~ 11.5 N to each tube and mix the contents carefully. This operation removes the colour developed due to p-nitrophenol,

(vi) Again read out the absorbance of the serum sample against the reagent blank tube at 410 nm. This gives the colour due to the serum itself,

(vii) Now, the corrected reading is achieved by subtracting the reading obtained in step (vi) from the reading in step (v). The alkaline-phosphatase activity of the serum as Bessey-Lowery units is obtained from the calibration-curve step (i). Under these experimental parameters, we have :

1 Bessey-Lowry Unit = 5 × 10 ^–8 mol of p-Nitrophenolate anion.

Thus, one unit of phosphatase activity liberated 1 μ mol of p-nitrophenol (l μ mol = 0.1391 mg) per hour per millilitre of serum under specified conditions.

Note : In case, a value more than 10 Bessey-Lowry Units is obtained, it is always advisable to repeat the process either with a smaller volume of serum or a shorter incubation period, and then finally adjust the calculations accordingly.

(4) Report the concentration of AP in units per millilitre.

1.2. Lactate Dehydrogenase (LDH)

 
Theory : 

The method of LDH assay is based on kinetic analysis. In a kinetic enzymatic assay a unit of enzyme activity is defined as ‘the quantity of enzyme that brings about a certain absorbance increase in 30 seconds or 1 minute at a fixed temperature (for instance 25 ± 0.2°C) ’.

The kinetic assay of LDH is based on the conversion of lactic acid to pyruvic acid, in the presence of nicotinamide adenine dinucleotide (NAD), and is closely monitored at intervals of 30 seconds or 1 minute by measuring the increase in absorbance at 340 nm. In this particular instance lactic acid available in an excess to ensure that the increase in pyruvic acid is linear with time, i.e., directly proportional to time. The reaction involved may be expressed as follows :

The liberated nicotinamide-adenine-dinucleotide hydrogenase (NADH) has an absorption maxima at 340 nm, whereas lactic acid. NAD⁺ and pyruvic acid do not absorb at all at this wavelenath.

Temperature Correction Factor : 

The rate of the above reaction is temperature dependent. Hence, if the temperature (experimental) is higher or lower than that used to define a unit of activity, a definite correction factor should be applied as per Table 2.10.

From Table 2.10 it may be observed that :

(a) At a temperature beyond 25°C (Tf = 1.0), the absorbance increases at a faster rate than at 25°C due to enhanced rate of reaction and enhanced formation of NADH, thereby lowering the correction factor from 1.0 e.g., 0.80 at 28°C,

(b) At a temperature lower than 25°C the rate of reaction is slower than at 25°C, thereby increasing the correction factor from 1.0 e.g., 1.24 at 24°C, and

(c) Rule of thumb suggests that for each 10°C rise in temperature the reaction rate is almost doubled and the correction factor is halved, for example : at 35°C the correction factor is 0.47 (or 1.0/2 −~ 0.47).

Profile of LDH-levels :

1)          Normal LDH levels are as follows : Absorbance Units per ml : 42 to 130, International Units per ml : 0.20 to 0.063

2)          LDH level in serum is found to be increased in 8 to 10 hours after a myocardial infarction (i.e., development or presence of an infarct in the heart) ; obviously the heart muscle is destroyed and consequently the enzymes leak into the serum,

3)          Increased LDH levels are found in patients suffering from diseases related to liver and renal func-tions, cancer and pulmonary infarction,

4)          Drugs like codeine and morphine help in enhancing LDH levels.

Materials Required : 

Dermatube LDH provided by Worthington Biochemical, USA.

Procedure : 

The following steps need to be followed in a sequential manner :

1)          Dissolve the contents of Dermatube LDH (containing NADH and lactic acid) with 2.8 ml of DW,

2)          Put this solution in a cuvette and then insert it in a colorimeter previously warmed up to 25°C. Set the wavelength at 340 nm. Carefully adjust the absorbance of this solution to 0.1 by making use of the proper variable control as explained earlier,

3)          Remove the cuvette and add to it 0.2 ml of serum. Mix the contents of the cuvette and replace it quickly in position. Carefully record the absorbance exactly at intervals of 30 seconds for 2 to 3 minutes. In case, the absorbance happens to rise very rapidly, repeat step 3 by diluting 0.1 ml of the serum to 0.2 ml with DW,

4)          From the foregoing measurement of absorbances calculate an average ∆A/min,

5)          Note the temperature at which the reaction is carried out accurately and then find out Tf from Table 2.10.

6)          Report the LDH concentration as follows :

RADIOIMMUNOASSAYS (RIAS)

An assay method based on immunological antibody-hapten (Ab-Ha) reaction that makes use of a radioactive tracer is usually known as radioimmunoassay. A hapten (or haptene) is a small molecule that represents the portion of an antigenic molecule or complex which determines its immunologic specificity, for instance : cortisol ; whereas an antibody is a relatively large protein that is specific for certain haptens. An antibody is generated by binding the hapten to a protein, resulting into the formation of an antigen that specifically stimulates the immune system to produce antibodies specific for the hapten.

The assays that utilize protein instead of antibody are normally termed as competitive protein bind-ing assays. As an antibody is also a protein, therefore, a radioimmunoassay may be looked upon as a type of competitive protein binding assay.

Theory : 

Generally, a radioimmunoassay makes use of a radioactive hapten and subsequently the percent of radioactivity bound to the antibody is measured. The radioactivity is determined by the help of a Geiger-Müller Counter or Geiger-Counter or G-M Tube and sometime by a Scintillation Counter.

First of all, a ‘Standard Curve’ or a ‘Calibration Curve’ is plotted between the reciprocal value (i.e., 1 × % ^–1 radioactivity bound to the antibody) versus the amount of standard for a series of unknowns. Thus, the amount of hapten present in the unknown sample is measured from the plotted calibration curve conveniently.

The radioimmunoassay is based on the evolved competition between the combination of radioactive (Ha⁺) and nonradioactive (Ha) hapten to the antibody as represented below :

Let us assume that the binding constants for Ha⁺ and Ha are equal ; now, for a fixed quantity of Ha⁺ but an increased concentration of Ha. The ultimate impact would be that lesser Ha⁺ shall be bound. In actual practice, however, the use of Tritium (H³) or Carbon-14 (C¹⁴), which helps to render the Ha radioactive, ulti-mately maintains the equality of these binding constants, namely : K_Ha⁺ and K_Ha . It also confirms that the chemical properties of both radioactive (Ha⁺) and nonradioactive (Ha) entities are more or less the same as far as the antibody is concerned.

Salient Features of Radioimmunoassays

·              They belong to a class of extremely sensitive methods of analysis,

·              Sample required for assay is usually very small e.g., 1 ml of serum,

·              Concentrations upto the nanogram range i.e., 10^–9 g can be measured accurately,

·              A large number of hormones and drugs which find their abundant usage in a bad way, namely :

·              cortisol (17-hydroxycorticosterone or hydrocortisone), insulin, morphine, barbiturates (sedatives), vitamin B₁₂, digoxin and human growth hormones, such as : somatotropin (elaborated in the placenta),

·              Incidence of interferences observed in the radioimmunoassays are fairly insignificant by virtue of the highly specific hapten-antibody complexation reaction, and

·              Exceptions do occur when two 5-substituted barbiturates present together cannot be assayed by this method, obviously due to interference.

1. Cortisol (In Plasma)

Theory : 

Cortisol (or hydrocortisone) was introduced in the year 1951, for the treatment of rheumatoid arthritis. It has a significant effect on protein metabolism. It also exerts widespread effects on carbohydrates, lipid and protein synthesis (or anabolism). The cardinal side effects such as excessive potassium excretion and sodium retention, enhanced gastric acidity, oedema, psychosis and negative nitogen balance are some of the exaggerated manifestations of the normal metabolite functions of cortisol.

Most importantly, the determination of cortisol levels is considered useful in the diagnosis and treatment of various ailments, namely : Addison’s Disease i.e., pernicious anaemia—a condition whereby the maturation of the red cells may not proceed beyond the stage of megaloblasts; Cushing’s Syndrome.

Adrenal Tumours : 

The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH₂Cl₂ (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H³ (i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H³) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve).

Materials Required : 

Schwartz-Mann-H³ Cortisol RIA-Kit ; liquid scintillation counter, centrifuge.

Procedure : 

The various steps to be followed sequentially for the assay of cortisol in plasma are as follows :

·              The cortisol is usually extracted from the samples drawn from the patients, as follows :

Place 100 μ l of plasma in each of two tubes and add 2.5 ml of methylene chloride. Shake the contents of the tube vigorously for 10 minutes and transfer 0.5 ml of clear extract (i.e., the lower layer) to another tube. Evaporate the methylene chloride either at 35°C in an oven or in a stream of N₂. The extract thus obtained is employed in the following step.

·              The following steps viz., Step 1 to Step 15, related to the procedure for the assay and the calibration curves must be performed simultaneously :

·              Results : Average the counts per minute in vials 3 and 4. This is the blank value. Now, subtract the blank from all other counts per minute to obtain the actual counts per minute and average the counts per minute for vials 1 to 2 to find the total count per minute. The percent bound may be calculated using the following expression :

Finally, plot the percent bound Vs nanograms (ng) per tube of cortisol standard either on linear or on semilog paper and make use of this Standard Curve to calculate the amount of cortisol present in the unkown samples.

AUTOMATED METHODS OF CLINICAL ANALYSIS

Theory : An ‘Autoanalyzer’ serves as the most versatile and important instrument in a well-equipped ‘clinical laboratory’ that caters for the rapid screening of serum levels for upto forty (40) important chemical substances in the field of diagnostic medicine. These autoanalyzers may be either ‘Single Channel’ i.e., per-forming only one determination on each sample or Multichannel’ i.e., carrying out several different determinations on each sample.

A few important substances that are routinely analyzed in a clinical laboratory with the aid of an ‘Autoanalyzer’ are, namely : serum-glutamic-oxaloacetic transaminase (SGOT) ; creatine-phophokinase (CPK); alkaline-phosphatase (AP) belonging to the class of enzymes ; and a host of biochemical substances, for instance : bilirubin, serum albumin, blood urea nitrogen (BUN), uric acid, creatinine, total protein, glucose, cholesterol, besides a few common inorganic ions, such as : Cl^–, Ca²⁺, K⁺, Na⁺.

The basic principles underlying both automated and unautomated methods of analysis are more or less the same. Out of the broad-spectrum of biological samples blood analysis is the most common one. There exists a number of parameters which may be assayed, and spectrophotometry is ideally suited for nearly all of them, a few typical examples are cited in Table 2.11.

Explanation : Glucose (having an aldehyde functional moiety) reduces Cu²⁺ to Cu₂O (i.e., Cu⁺) as per the following reaction :

As some other sugars are also present in blood sample, and besides the above reaction not being abso-lutely stoichiometric, it has become necessary in actual practice to establish an emperical calibration curve using known concentrations of glucose. The above reaction is allowed to proceed for exactly 8 minutes at 100°C. To the resulting solution phosphomolybdic acid is added, which is subsequently reduced by Cu₂O to give rise to an intensely coloured ‘molybdenum blue’ that is measured at 420 nm accurately.

Alternatively, glucose forms a specific complex with o-toluidine according to the following reaction that forms the basis of the colorimetric assay :

The diagnostic green colour is usually developed for exactly 10 minutes at 100°C and measured subse-quently at 635 nm.

1. Instrumentation

The schematic diagram of an Auto Analyser is shown in Figure 2.6. The major component parts com-prise of the various important sections namely : the preparation section, the reaction section and the analysis section which will be discussed briefly here.

1.1. Preparation Section

This particular section of the Auto Analyzer consists mainly of the sampler, proportioning pumps, and programmer. First, the sampler introduces a fixed quantity of serum sample into the ‘analysis train’, which varies from one instrument to another instrument supplied by different manufacturers. For instance, the SMA-12 Survey Auto Analyzer possesses 12 analysis trains or streams as illustrated in Figure 2.7.

The proportioning pump controls the rate of advancement, viz 10 inch/minute, of each sample through the analysis stream. Hence, a fixed length of tubing is equivalent to a fixed amount of time. Each analysis stream is made of transparent plastic flexible tubing, and each patient-sample is separated from one another by an air-bubble.

1.2. Reaction Section

The reaction section essentially comprises of the dialyzer, heat bath and phaser, and obviously the reaction takes place in this zone. Let us consider the following generalized reaction :

Where , [C]^c = Molar concentration of substance C raised to the cth power,

       A = Component in serum (e.g., cholesterol), and

       B = Reactant that reacts with A to give a coloured product.

Evidently, B is added always in excess to ensure :

(a)       rapid reaction, and

(b) complete reaction by forcing the reaction to the right in accordance to the Le Chatelier’s principle.

Now, the rate of forward reaction = k [A]^a [B]^b

Hence, the rate constant may be expressed as follows :

_{k  = Ae}–Ea/RT             ...............(c)

where , R = Gas constant ( 1.99 cal/K-mol),

T = Temperature, and

E_a = Activation energy of the reaction as depicted in Figure 2.8.

From Eq. (c) it may observed that as the temperature T is enhanced then the rate of reaction also enhances simultaneously because a higher value of T offers a smaller negative exponent of e or a larger number. Therefore, in actual experimental operations temperature is increased by the aid of a heat-bath so as to accelerate the reaction which in turn allows the reaction to attain equilibrium state as rapidly as possible.

Naturally at a very high temperature there is every possibility for decomposition of either the products or the reactants.

1.3. Analysis Section

The recent advancement in the field of computer technology and anlytical instrumentation it has become very easy and convenient to have the analyical data from a series of biological samples processed at high speed as digital readouts or on computerized recorders. Many hospitals round the globe make extensive use of advanced computer softwares for data processing as stated beiow :

·              Uptodate listing of various laboratory tests,

·              Listing of drugs and metabolites that cause interference both biochemically and analytically,

·              Storing of levels of biologically important compounds for various disease states, and

·              A tentative diagnosis for a patient based on his serum sample under investiation together with the drugs and dosages being administered and the levels of biologically important compounds.

Caution : Nevertheless, the concerned physician or pharmacist must exercise his or her own expertise and knowledge while prescribing drug(s) to a patient along with these computerized data informations.

Errors in Pharmaceutical divided into two different portions, namely : (a) Errors in Pharmaceutical Analysis, and (b) Statistical Validation, which will be discussed individually in the following sections :

ERRORS IN PHARMACEUTICAL ANALYSIS

INTRODUCTION

The skill, knowledge, expertise and above all the degree of confidence involved in the ultimate result of an analyst is solely governed by the extent of accuracy and precision achieved by the analytical procedure vis-a-vis the possible sources of error that may be incorporated inadvertently. In fact, the quantitative pharmaceutical analysis is not merely confined to just taking a random sample, performing a single assay quickly, and finally making a loud claim that the result so obtained cannot be challenged. Truly speaking an ideal analyst must have a total in-depth knowledge of the chemistry involved along with the pros and cons of interferences that may be caused due to the host of compounds, elements and ions besides adequate exposure and hands-on experience of the statistical distribution of values.

The terminology ‘error’ invariably refers to the difference in the numerical values between a measured value and the true value. It has become universally accepted in methods of comparison that the percentage composition of a ‘standard sample’ provided and certified by the National Institute of Standards and Technology (NIST) or the British Pharmacopoea Chemical Reference Substance (BPCRS) or the European Pharmacopoea Chemical Reference Substance (EPCRS) must be regarded and treated as absolutely correct, pure and authentic while evaluating a new analytical method. Consequently, the differences thus obtained between the standard values and those by the new analytical methods are then treated as ‘errors’ in the latest procedure.

CLASSIFICATION OF ERRORS

The numerous uncertainties usually encountered in a chemical analysis give rise to a host of ‘errors’ that may be broadly categorised into two heads, namely :

(i) Determinate (systematic) Errors, and

(ii) Indeterminate (random) Errors.

It is pertinent to mention here that it becomes rather difficult at times to place a particular ‘error’ into one of the above mentioned categories ; however, the classification may prove to be beneficial with regard to study of the various analytical errors that crop up in the course of routine analysis.

1. DETERMINATE (SYSTEMATIC) ERRORS

These are errors that possess a definite value together with a reasonable assignable cause; however, in principle these avoidable errors may be measured and accounted for coveniently. The most important errors belonging to this particular class are :

(a) Personal Errors : They are exclusively caused due to ‘personal equation’ of an analyst and have no bearing whatsoever either on the prescribed procedure or methodology involved.

(b) Instrumental Errors : These are invariably caused due to faulty and uncalibrated instruments, such as : pH meters, single pan electric balances, uv-spectrophotometers, potentiometers etc.

(c) Reagent Errors : The errors that are solely introduced by virtue of the individual reagents, for instance : impurities inherently present in reagents ; high temperature volatalization of platinum (Pt) ; unwanted introduction of ‘foreign substances’ caused by the action of reagents on either porcelain or glass apparatus.

(d) Constant Errors : They are observed to be rather independent of the magnitude of the measured amount ; and turn out to be relatively less significant as the magnitude enhances.

Example : Assuming a constant equivalence—point error of 0.10 ml is introduced in a series of titrations, hence for a specific titration needing only 10.0 ml of titrant shall represent a relative error of 1% and only 0.2% for a corresponding 50 ml of titrant consumed.

(e) Proportional Errors : The absolute value of this kind of error changes with the size of the sample in such a fashion that the relative error remains constant. It is usually incorporated by a material that directly interferes in an analytical procedure.

Example : Estimation of ‘chlorate’—an oxidant by iodometric determination. In this particular instance two things may happen, namely :

(i) Presence of ‘Bromate’—another oxidizing agent would give rise to positively higher results, and hence, it must be duly corrected for, and

(ii) Absolute error might increase while dealing with large samples, whereas the relative error would remain more or less constant if the sample is perfectly homogenous,

(f) Errors due to Methodology : Both improper (incorrect) sampling and incompleteness of a reaction often lead to serious errors. A few typical examples invariably encountered in titrimetric and gravimetric analysis are cited below :

(g) Additive Errors : It has been observed that the additive errors are independent of the quantum of the substances actually present in the assay.

Examples : (i) Errors caused due to weights, and

(ii) Loss in weight of a crucible in which a precipitate is incenerated.

Detection of this error is ascertained by taking samples of different weights.

2. INDETERMINATE (RANDOM) ERRORS

As the name suggests, indeterminate errors cannot be pin-pointed to any specific well-defined reasons. They are usually manifested due to the minute variations which take place inadvertently in several successive measurements performed by the same analyst, using utmost care, under almost identical experimental parameters. These errors are mostly random in nature and ultimately give rise to high as well as low results with equal probability. They can neither be corrected nor eliminated, and therefore, form the ‘ultimate limitation’ on the specific measurements. It has been observed that by performing repeated measurement of the same variable, the subsequent statistical treatment of the results would have a positive impact of ‘reducing their importance’ to a considerable extent.

Example : Figure 3.1, represents the absolute errors in nitrogen analysis by means of micro Kjeldahl’s Method*. Here, each vertical line labelled ( x’₁ – x_t) designates the absolute deviation of the mean of the set from the true value. In Figure 3.1, A represents ( x’₁ – x_t) the absolute error obtained by ‘analyst-1’ for the assay of benzyl-iso-thioureahydrochloride, whereas B represents ( x’₂ – x_t) the absolute error obtained by ‘analyst-2’ for the assay of the same compound.

Thus, it is evident from Figure 3.1, that the broad spread of individual errors centres around the mean values (x’_n – x ) thereby affording a direct indication of indeterminate type uncertainties. Hence, larger indeterminate errors seem to be linked with the performance of ‘analyst-2’ than with that of ‘analyst-1’.

Salient Features of Indeterminate Errors

The various salient features of indeterminate errors are enumerated below :

1)          Repeated mesurement of the same variable several times and subsequent refinement to the extent where it is simply a coincidence if the corresponding replicates eventually agree to the last digit,

2)          Both unpredictable and imperceptible factors are unavoidably incorporated in the results what generally appear to be ‘random fluctuations’ in the measured quantity,

3)          Recognition of specific definite variables which are beyond anyone’s control lying very close to the performance limit of an instrument, such as : temperature variations, noise as well as drift from an electronic circuit, and vibrations caused to a building by heavy vehicular-traffic,

4)          A variation that may be regarded as random by a slipshod analyst may at the same time prove to be quite evident and manageable by a careful observer, and

5)          The average of a number of fine observations having random scatter is definitely more accurate, precise and, hence, more cogent than coarse data that appear to agree perfectly.

3. ACCURACY

In connexion with the scientific data the two terminologies ‘accuracy’ and ‘precision’ are invariably practised synonymously, but there exists a clear distinction between them as discussed below :

In usual practice an accurate result is the one which matches very nearly with true value of a measured amount. The comparison is normally done with regard to the ‘error’; and the accuracy is inversely propor-tional to it i.e., the greater the accuracy, the smaller is the error. ‘Absolute error’ is the difference between the experimental value and the true value.

Example : An analyst determines a value of 70.55% cineole in a fresh sample of Eucalyptus Oil that actually contains 70.25%, the absolute error is given by :

70.55 – 70.25 = 0.30%

The error thus obtained is invariably stated with regard to the actual size of the measured quantity i.e., either in percent (%) or in parts per thousand (ppt). Therefore, the relative error is given by :

4. PRECISION

It may be defined as—‘the agreement amongst a cluster of experimental results ; however, it does not imply anything with respect to their relation to the ‘true value’ ’. Precision designates ‘reproducibility’ of a measurement, whereas accuracy the correctness of a measurement. Precision invariably forms an integral part of accuracy, but ironically a high degree of precision may not necessarily suggest accuracy.

Example : A sample of pure Peppermint Oil is known to contain 30.10 ± 0.03 per cent of Menthone. The results obtained by two Analysts-1 and 2 employing the same sample of peppermint oil and making use of the same analytical reagents and procedure are as stated below :

The arithmetic mean is 31.44% and the results vary between 31.40% to 31.46%

The ultimate results of the analysis put forward by the Analysts-1 and 2 may be summarized as under :

(i) The results achieved by Analyst-1 are fairly accurate i.e., in close proximity to the correct result ; however, the precision stands at an inferior level to the results obtained by Analyst-2. The results accomplished by Analyst-2 are indeed extremely precise but fail in accuracy,

(ii) The results of Analyst-1 lie on either sides of the average value as shown by two ‘cross-signs’ on each side which might have been caused due to ‘random errors’ discussed earlier. It is quite evi-dent that there exists a constant (determinate) error in the results obtained by the Analyst-2, and

(iii) In case, Analyst-3 had performed the estimations on the very same day in quick succession i.e., one after the other, this type of analysis could be termed as ‘repeatable analysis’. If the estimations had been carried out on two separate days altogether, thereby facing different laboratory conditions then the results so obtained would be known as ‘reproducible analysis’.

In short, there exists a marked and pronounced distinction between a within-run precision (i.e., repeatability) and an in-between-run precision (i.e., reproducibility).

5. MINIMISING SYSTEMATIC ERRORS

Systematic errors may be reduced substantially and significantly by adopting one of the following procedures rigidly, such as :

(i) Calibration of Instruments, Apparatus and Applying Necessary Corections

Most of the instruments, commonly used in an analytical laboratory, such as : UV-Spectrophoto-meter, IR-Spectrophotometer, single—pan electric balance, pH-meter, turbidimeter and nephelometer, polarimeter, refractometer and the like must be calibrated duly, before use so as to eliminate any possible errors. In the same manner all apparatus, namely : pipettes, burettes, volu-metric flasks, thermometers, weights etc., must be calibrated duly, and the necessary corrections incorporated to the original measurements

In some specific instances where an error just cannot be avoided it may be convenient to enforce an appropriate correction for the effect that it ultimately causes ; for instance : the inherent impu-rity present in a weighed precipitate can be estimated first and then deducted duly from its weight.

(ii) Performing a Parallel Control Determination

It essentially comprises of performing an altogether separate estimation under almost identical experimental parameters with a quantity of a standard substance that consists of exactly the same weight of the component as is present in the unknown sample. Thus, the weight of the component present in the unknown sample may be calculated with the help of the following expression :

where,             X = Weight of the component present in the Unknown Sample.

Note :             A good number of Standard Samples, including primary standards, such as : arsenic trioxide, benzoic acid, potassium hydrogen phthalate, sodium oxalate, are available as :

BPCRS = British Pharmacopoeia Chemical Reference Substance,

EPCRS = European Pharmacopoeia Chemical Reference Substance,

CRM = BCS—Certified Reference Materials,

ECRM = EURONORM—Certified Reference Materials.

(iii) Blank Determination :

In order to ascertain the effect of the impurities present in the reagents employed and reaction vessels used ; besides establishing exactly the extent to which an excess of standard solution required to locate the exact end-point under the prevailing experimental parameters of the unknown sample—a blank determination is an absolute necessity. It may be accomplished by performing a separate parallel estimation, without using the sample at all, and under identical experimental parmeters as employed in the actual analysis of the given sample.

Note : Always avoid using an appreciably large blank correction which gives rise to a vague and uncertain ‘exact value’ thereby minimising the precision of the analysis.

(iv) Cross-checking Results by Different Methods of Analysis

In certain specific cases tha accuracy of a result may be cross-checked by performing another analysis of the same substance by an altogether radically different method.

Examples :

(a) HCl-Solution : It may be assayed either by titration with a standard solution of a strong alkali (NaOH), or by precipitation and weighing as AgCl ; and

(b) Fe³⁺ : It may be assayed either by gravimetric method as Fe(III) hydroxide after getting rid of the interfering elements and igniting the precipitate to Fe(III) oxide, or by titrimetric method i.e., first reducing to the Fe(II) state and then titrating with a suitable oxidizing agent, for instance Ce(IV) sulphate, K₂Cr₂O₇. In short, the results thus obtained by the two fundamen-tally different techniques must be concordant thereby justifying and ascertaining the fact that the values obtained are fairly small limits of error.

(v) Method of Standard Addition

Here, a small known quantity of the component under estimation is added to the sample, which is subsequently subjected to analysis for the total amount of component present. The actual differ-ence in the quantity of components present in samples with or without the added component ultimately gives the recovery of the quantum added component. A good satisfactory recovery builds up the confidence in the accuracy of the method of analysis.

Note : The method of ‘standard addition’ is particularly useful to physicochemical techniques

of analysis, for instance : spectrophotometry, turbidimetry.

(vi) Method of Internal Standards

The specific method is of immense value both in chromatographic as well as spectroscopic determinations. Here, a fixed quantity of a reference substance (i.e., the ‘internal standard’) is added to a series of known concentrations of the material to be assayed.

A graph is plotted between the concentration values and the ratios obtained from the physical value (i.e., peak area of absorption) of the ‘internal standard’ and the series of known concentra-tions, thereby producing a straight line. Any unknown concentration may be determined effec-tively by adding the same amount of ‘internal standard’ and locating exactly where the ratio obtained falls on the concentration scale.

STATISTICAL VALIDATION

INTRODUCTION

After accomplishing the thorough investigation of various aspects of possible ‘determinate errors’ and having applied the relevant corrections, it has been observed that the data thus generated not only show fluctuations but also are found to be random in nature. The powerful and effective technique of statistics may render such results, which scatter in a random manner, into a better form that may be employed intelligently. Besides, the specific statistical treatment of the calibration data, aided by pre-programmable calculators and micro-computers, very often yields a fairly accurate and more presentable determination of the graphs between absorbance and concentration than those produced manually.

STATISTICAL VALIDATION

The statistical validation of analytical results will be discussed with regard to the following six as-pects individually, along with appropriate examples wherever possible, in the sections that follow :

(i) Statistical treatment of finite samples,

(ii) Distribution of random errors,

(iii) Significant errors,

(iv) Comparison of results,

(v) Method of least squares, and

(vi) Criteria for rejection of an observation.

STATISTICAL TREATMENT OF FINITE SAMPLES

The various techniques by which one may effectively treat the scientific data normally obtained in actual analytical procedures are enumerated below :

1. Mean

It is the average of a series of results. The mean of a finite number of measurements, x_l, x₂, x₃, x₄, ........, x_n, is commonly represented as x’. It may be calculated by taking the average of individual results as shown below :

It is evident that the mean of n results is  √n times more reliable than any one of the individual results. Therefore, there exists a diminishing return from accumulating more and more replicate meaurements. In other words, the mean of 9 results is 3 times as reliable as 1 result in measuring central tendency (i.e., the value about which the individual results tend to cluster) ; the mean of 16 results is 4 times as reliable etc.

2. Median

The median of an even number of results is nothing but the average of the ‘two middle values’ pro-vided the results are listed in order ; whereas for an odd number of results the median is the ‘middle value’ itself. However, the ‘mean’ and the ‘median’ are exactly identical in the case of a truly symmetrical distribu-tion. In short, median is an useful measure specifically when dealing with very small samples.

3. Average Deviation (or Mean Deviation)

It is the average of the differences between the individual results and the mean. It is regarded as a measure of variability. In the case of a small number of observations the average deviation is found to be not quite significant statistically. The average or mean distribution may be calculated by adopting the following steps, namely :

(i) To find the differences between individual results and the mean, without considering the +ve or –ve sign,

(ii) To add these individual deviations, and

(iii) To divide by the number of results (i.e., n).

Hence, an ‘average deviation’ may be expressed as :

4. Standard Deviation

It is the distance from the mean to the point of inflexion of the normal distribution curve. In compari-son to the average deviation the ‘standard deviation’ is usually considered to be much more useful and meaningful statistically. For a finite number of values it is normally symbolised as ‘S’, and may be expressed as follows :

In a situation, where ‘n’ is fairly large, say to the extent of 50 or more, it hardly matters whether the denominator in the above expression is either n – 1 or n; however, the former (i.e., n – 1) is strictly correct.

5. Coefficient of Variation (ν)

The coefficient of variation (ν) is simply the standard deviation(s) expressed as a percentage of the mean ( x’ ) as stated below :

6. Variance (s²)

The variance is the square of the standard deviation(s) i.e., s². However, the former is fundamentally more important in statistics than the latter, whereas the latter is employed more frequently in the treatment of chemical data.

7. Calculations of Fundamental Statistical Parameters

Example : The normality of a solution of sodium hydroxide as determined by an ‘analyst’ by FOUR different titrations are found to be 0.5038 ; 0.5049 ; 0.5042 ; and 0.5039. Calculate the mean, median, average deviation, standard deviation and coefficient of variation.

DISTRIBUTION OF RANDOM NUMBERS

Results obtained from a given set of measurements that scatter in a random manner are adequately treated by most logical methods of statistics.

In a situation whereby a large number of replicate readings, not less than 50, are observed of a titrimetric equivalence point (continuous variable), the results thus generated shall normally be distributed around the mean in a more or less symmetrical fashion. Thus, the mathematical model which not only fits into but also satisfies such a distribution of random errors is termed as the Normal or Gaussian distribution curve. It is a bell-shaped curve which is noted to be symmetrical about the mean as depicted in Figure 3.2.

The equation of the normal curve may be expressed as given below :

where, y = Relative frequency with which random sampling of the infinite population shall bring forth a specific value x,

 σ= Standard deviation, and

μ= Mean.

From the Normal distribution curve (Figure 3.2) it may be observed that 68.26% of results shall fall within one standard deviation on either side of the mean, 95.46% shall fall within two standard deviations, and 99.74% within three standard deviations.

SIGNIFICANT FIGURES

Generally, significant figures may be defined as—“All digits* that are certain plus one which contains some uncertainty are said to be significant figures”.

Examples :

(a) Burette Reading : Burettes are mostly graduated with the smallest graduation as 0.1 ml ; hence, while taking the burette reading the figures 6.3 ml can be read off with ample certainty. However, the second place of the decimal is normally estimated by arbitrarily sub-dividing the smallest division into 10 equal parts. Consequently, the final burette reading of 6.32 ml essentially contains three significant figures, of which two are certain, and one with some uncertainty.

(b) Measuring Weights : In the two measured quantities : 4.7350 g and 4.0082 g the zero is a significant figure ; whereas, in the quantity 0.0065 kg the zeros are not significant figures. Thus, in the latter instance the zeros only serve to locate the decimal point and, therefore, may be eliminated completely by proper choice of units, e.g., 6.5 g. Moreover, the first two numbers do have five significant figures, whilst 0.0065 only has two significant figures.

1. Computation Rules

The following computation rules are advocated to make sure that a calculated result, arrived at either by addition and subtraction or multiplication and division essentially contains only the number of ‘digits’ duly justified by the experimental data.

(a) Addition and Subtraction

In addition and subtraction, retain only as many decimal places as appear in the number that has the fewest decimals.

Example : Add algebraically the numbers given : 16.48 + 9.375 – 3.5450 + 118.9.

Following three steps are to be carried out sequentially :

(i) All numbers are required to be rounded up preliminarily to two decimal places,

(ii) Add the rounded numbers, and 

(iii) Final result is then rounded to one decimal place.

(b) Multiplication and Division

In multiplication or division, retain in each term one more signifcant figure than is contained in the term with the largest uncertainty. However, the percentage precision of product cannot be greater than the percentage precision of the least precise term entering the calculation. Hence, the multi-plication : 2.64 × 3.126 × 0.8524 × 32.9453 must be accomplished using the values

2.64 × 3.126 × 0.852 × 32.95

which is equal to 231.6796. Thus, the result obtained may be expressed to five significant figures as 231.68.

(c) Rounding Numbers

In rounding numbers, always drop the last digit in case it is less than 5, e.g., 8.62 will become 8.6. If the last digit is more than 5, always increase the preceeding digit by one i.e., 9.38 will become 9.4. In case, the digit to be dropped is 5, always round up the preceding digit to the nearest even number i.e., 8.75 will become 8.8 ; and 8.65 will become 8.6. Evidently, this method avoids a tendency to round up numbers in one direction only.

In rounding off quantities to the nearest correct number of significant figures, add one to the last figure retained provided the following figure is either 5 or over. Hence, the average of 0.6526, 0.6521, and 0.6524 is 0.6525 (0.65237).

(d) Always retain as many significant figures in a result as will yield only one uncertain figure.

Examples : (i) A volume read off from a burette reading that lies between 15.6 ml and 15.8 ml must be recorded as 15.7 ml, but not as 15.70 ml, because the latter would indicate that the reading lies between 15.69 and 15.71 ml.

(ii) A weight, to the nearest 0.1 mg, is recorded as 2.4500 g ; and it must not be written as either 2.450 g or 2.45 g, because in the latter instance an accuracy of a centigram is emphasized whereas in the former a milligram.

COMPARISON OF RESULTS

In a situation where the same sample has been analysed by two separate techniques altogether, each of them repeated several times, and that the mean values obtained are not the same ; statistically it may be possible to ascertain whether the analytical procedure adopted has been either accurate and precise or if it is superior to one of the two methods.

In fact there are two frequently employed methods that may be used to compare the results, namely :

(a) Student’s t-Test, and

(b) Variance-Ratio Test (or F-Test).

In order to perform these two tests one should have a clear understanding of the statistical term ‘the number of degrees of freedom’.

Degrees of Freedom : It is the number of individual observations which can be allowed to vary under conditions that the mean ( x ) and standard deviation(s), once determined, be held constant.

Thus, a sample having n values have n degrees of freedom, whereas the sum Σ(x – x’ )² is considered to have n – 1 degrees of freedom, because for any defined value of the mean, x’ , only n – 1 value can be assigned freely, as the nth is being defined from the other values automatically.

1. Student’s t-Test

It is usually employed for small samples only. It serves two main objectives, namely :

(i) It is employed to test the difference between the means of two sets of data x₁  and x₂ , and

(ii) It is used to compare the mean obtained from a sample having certain standard value and to express certain degree of confidence in the significance of the comparison.

The value of t may be obtained from the following expression :

                 ........................(i)

where,         x’  = Mean value,

μ = True value,

s = Standard deviation, and

n = Degree of Freedom.

Example : If x the mean of 12 determinations = 9.59, and µ the true value = 9.03, find out whether or not this result is significant provided the standard deviation(s) is 0.16.

From the above Eq. (i) we have :

Now, from the t-tables for (n – 1 i.e., 12 – 1 =) 11 degrees of freedom we have :

Evidently, the calculated value for t is 12.11 and the result is highly significant. Besides, the t-table also gives the information that the probability of obtaining the difference of 0.56 between the experimental and the true result comes out to be less than 1 in 100, which obviously is indicative of the fact that some kind of bias does exist in the laboratory method adopted.

2. Variance-Ratio Test (or F-Test)

A test that makes use of the ratio of the variances of two sets of results to determine if the standard deviations (s) are significantly different. Its application may also be extended to compare precisely the results obtained either from two different laboratories or from two different analytical procedures.

It is simply calculated from the following equation :

                                  ...................(ii)

where, s₁ and s₂ = Standard deviations of two sets of results.

Various steps invoived to determine F-Test are

(i) Find the ratio from Eq. (ii),

(ii) Place the larger s-value in the numerator so that F > 1,

(iii) Check for its significance against values in the F-table, and

(iv) If the F-value in the table is less than the calculated F-value, then the two standard deviations are significantly different ; otherwise, they are not.

Example : A sample of anhydrous sodium carbonate (Na₂CO₃) is analysed by two different methods which give the results for the percentage of Na₂CO₃ as follows :

Is there any significant difference between the precision of these two sets of results ?

Applying the variance-ratio or F-Test from Eq. (ii) we have :

From the standard table having F-values at the 95% probability level, under column n – 1 = 4 (since s₂ >s_l ) and row n – 1 = 5, find F = 6.39. Because, 6.39 > 1.69, the standard deviations are not significantly different [see step (iv) above].

METHOD OF LEAST SQUARES

A number of pharmaceutical analytical methods are solely based on instrumental measurements of an absolutely physical nature, such as : measuring peak areas with the help of a gas-chromatograph (GC), and measuring absorbance of a solution using a spectrophotometer (UV). In both these instances, the physical characteristics are directly proportional to the concentration of the analyte under examination. In usual prac-tice, a number of solutions having known concentrations is prepared and the response of the instrument is subsequently measured for each standard solution. Finally, a standard curve or calibration curve is plotted between the observed response Vs concentration, which invariably gives rise to straight line. It has been noticed, that the experimental points rarely fall exactly upon a straight line by virtue of the indeterminate errors caused by the instrument readings. At this juncture, an analyst is confronted with the tedious problem to obtain the ‘best’ straight line for the standard curve based on the observed points so that the error in estimating the concentration of the unknown sample is brought down to the least possible extent. At this stage, instead of deciding to draw the line merely on an analyst’s judgement, statistics comes to the rescue by providing a mathematical relationship whereby the analyst not only may calculate the slope objectively but also can obtain the ‘best’ straight line. The statistical process involved is termed as the method of least squares.

Example : The results obtained from the determination of concentration of the standard solutions and measurements of corresponding peak areas with a GC are recorded in Table 3.1 and plotted in Figure 3.3 ; where the former is represented along the x-axis and the latter along the y-axis. How to draw the’ ‘best’ straight line through all these points ?

Considering that the relationship between the concentration and the observed peak areas is a linear one, the equation for a straight line may be expressed as :

y = mx + b

where, m = Slope of the line, and

b = Intercept on the y-axis.

It may also be assumed that values of x are free of any error.

Presumably, the indeterminate errors caused by the instrument readings, y, are responsible for not allowing the ‘data points’ to fall exactly on the line. Therefore, the sum of the squares of the deviations obtained from the real instrument readings with respect to the correct values are minimized coinsiderably by adjusting adequately the values of the slope, m, and the intercept, b.

Table 3.1, comprises the values of x and y to enable plot of the graph in Figure 3.3, besides values of x², y² and xy and also the sums of all these terms.

Statistically, the slope (m) and intercept (b) of the straight line may be obtained by the help of the following equations :

Therefore, the equation of the line is

y = 1.99x + 1.02

Thus, the standard deviation of the y values, Sy, is given by :

The number of degrees of freedom in the above expression is n – 2, because two degrees have already been consumed while calculating the values of m and b earlier.

The standard deviation of the slope, Sm, is given by :

At this point, let us suppose that the ‘calibration curve’ is used to find out the concentration of the ‘unknown’. Assuming that three determinations have been carried out separately, thereby giving three y values of 5.85, 5.88, 5.91, or an average value, y_u , of 5.88. Thus, using the expression : y = mx + b, we have

The standard deviation (Su) in this result is obtained from the expression :

where, n_u = Number of determination of unknown,

n = Number of points in the calibration graph, and

y’ = Average of the y-values in the calibration graph (i.e., 34.94/5 = 6.99)

In case, the above statistical analysis has been based on a single determination, for instance : y = 5.88, the value of Su shall come out to be :

RECOMMENDATIONS FOR CRITERIA OF REJECTING AN OBSERVATION

An analyst, while carrying out a series of measurements, invariably comes across with ONE specific result in a set of replicates that obviously appears to be quite ‘out of place’ with the others, and at this juncture he should take an appropriate decision whether to discard (or expunge) this result from any further consideration. Thus, two situations often arise, namely :

(i) Number of replicates being small, and

(ii) Number of replicates being large.

A. Number of Replicates being Small

An analyst in the true sense encounters a serious problem when the number of replicates at his disposal is SMALL. Firstly, the divergent result shows a distinct and significant effect upon the mean value ( x’) ; and secondly, the prevailing scanty available data does not permit getting at the real statistical analysis of the status of the suspected result.

B. Number of Replicates being Large

In this instance, the analyst has the privilege of rejecting one value (i.e., the ‘out-of place’ value) as it is not an important one by virtue of the following two main reasons :

Firstly, a single value shall exert merely a small effect upon the mean value ( x ) ; and secondly, the treatment of data with the real statistical analysis would certainly reveal vividly the probability that the suspected ‘out of place’ result is a bonafide member of the same population as the others.

Blaedel et al.* (1951), Wilson** (1952) and Laitinen*** (1960) have put forward more broadly accepted and recommended criteria of rejecting an observation.

1. Rules Based on the Average Deviation

Both ‘2.5d’ and ‘4d’ rules are quite familiar to analysts. They may be applied in a sequential manner as follows :

(i) Calculate the mean ( x’ ) and average deviation ( d’ ) of the ‘good’ results,

(ii) Determine the deviation of the ‘suspected’ result from the mean of the ‘good’ results,

(iii) In case, the deviation of the suspected result was found to be either 2.5 times the average deviation of the good results (i.e., ‘2.5d’ rule) or 4 times the average deviation of the good results (i.e.‘4d’ rule) the suspected result was rejected out right ; otherwise the result was duly retained.

Note : The ‘limit for rejection’ seems to be too low for both the said rules.

2. Rules Based on the Range

The Q test, suggested by Dean and Dixon**** (1951) is statistically correct and valid, and it may be applied easily as stated below :

(i) Calculate the range of the results,

(ii) Determine the difference between the suspected result and its closest neighbour,

(iii) Divide the difference obtained in (ii) above by the range from (i) to arrive at the rejection Quotient Q,

(iv) Finally, consult a table of Q-values. In case, the computed value of Q is found to be greater than the value given in the table, the result in question can be rejected outright with 90% confidence that it was perhaps subject to some factor or the other which never affected the other results.

Table 3.2, records some of the Q-values as given below :

Example : Five determinations of the ampicillin content in capsules of a marketed product gave the following results : 0.248, 0.245, 0.265, 0.249 and 0.250 mg per capsule. Apply the Q-test to find out if the 0.265 value can be rejected.

The value of Q is :

The value in Table 3.2, at n = 5 is Q = 0.64. Because, the determined value 0.75 > 0.64, according to ‘rule based on the range’ the result i.e., 0.265 can be rejected.

Note : The Q-test administers excellent justification for the outright rejection of abnormally erroneous values ; however, it fails to eliminate the problem with less deviant suspicious values.

SAMPLING STATISTICS

The errors that are solely attributed to sampling, specifically in the instance of heterogeneous solids, usually give rise to the most important source of uncertainty in carrying out analysis of pharma-ceutical substances.

Thus, the overall standard deviation, s_T, i.e., the total error is given by the following expression :

              ......................(i)

where, S_S = Standard deviation of the sampling procedure (i.e., the sampling error), and S_A = Standard deviation of the analytical procedures (i.e., the analytical error).

The Eq. (i) may also be expressed as :

where  V_S = Variance due to sampling, and

V_A = Variance due to analytical method(s).

The individual determination of V_S and V_A may be accomplished by using the method described under variance.

Example : If the sampling error is ± 2.8% and the analytical errors by two different analysts come out to be ± 0.9% and ± 0.1% respectively, we may have :

From Eqs. (a) and (b) it is quite evident that the actual contribution of the analytical error viz., ± 0.9% and ± 0.1%, to the total error (S _T) is more or less insignificant.

Note : Youden* (1967) suggested that once the analytical error is reduced to 1/3rd of the sampling error, further reduction of the former is not required anymore.

In order to have a meaningful ‘sampling plan’ the following points should be taken into consideration**, namely :

·              Number of samples to be taken :

·              Size of the sample, and

·              Should separate samples be analysed or should a sample made up of two or more increments (i.e., composite sample) be prepared.

Unknown Bulk Material : 

A container-load of Paracetamol (10 MT) arrives at a raw-material stores and the composition of the bulk material is unknown, it will be a sensible and logical practice to carry out first and foremost a preliminary investigation by collecting a large number of samples and assaying the analyte of interest.

Thus, the confidence limits are given by the following expression :

           ............................(c)

where,      µ = Estimate of the true mean,

 x’ = Mean of the analytical results,

t = Parameter depending upon the number of degrees of freedom (ν) and the confidence level required,

S_S = Standard deviation of individual sample, and

n = Number of samples taken.

Example : The estimate in variability of Paracetamol in a consignment of 10 MT, based on 20 determinations, was found to be ± 1.4%. How many samples must be taken to give (at 95% confidence level) a sampling error of less than 0.5% paracetamol ?

The 0.5% value, in reality, represents the difference between the sample mean x and the actual value µ. If this value is designated by E, then Eq. (c) may be expressed as :

From the tables [Percentage Points of the t-Distribution] the value of t for (n – 1 ), 19 degrees of freedom at 95% confidence level is 2.09.

Conclusion : 

From this test it has been established that at least 34 samples are required if the specifi-cations provided in the above cited example are to be fulfilled adequately.

Sample Size : 

Another major problem associated with the sampling process is that of the sample size.

In fact, the sample size withdrawn from a heterogeneous material is solely guided by two factors, namely :