<16> AUTOMATED METHODS OF ANALYSIS

AUTOMATED METHODS OF ANALYSIS

Where a sufficiently large number of similar units are to be subjected routinely to the same type of examination, automated methods of analysis may be far more efficient and precise than manual methods. Such automated methods have been found especially useful in testing the content uniformity of tablets and capsules and in facilitating methods requiring precisely controlled experimental conditions. Many manufacturing establishments, as well as the laboratories of regulatory agencies, have found it convenient to utilize automated methods as alternatives to Pharmacopeial methods (see Procedures under Tests and Assays in the General Notices and Requirements). In addition, the detection system and calculation of results for automated methods are often computerized.

Before an automated method for testing an article is adopted as an alternative, it is advisable to ascertain that the results obtained by the automated method are equivalent in accuracy and precision to those obtained by the prescribed Pharmacopeial method, bearing in mind the further principle stated in the General Notices and Requirements that “where a difference appears, or in the event of dispute, only the result obtained by the procedure given in this Pharmacopeia is conclusive.”

It is necessary to monitor the performance of the automated analytical system continually by assaying standard preparations of known composition frequently interspersed among the test preparations. Where immiscible solvents are employed in the automated apparatus for rapid extractions, they are often separated for analysis before complete extraction is attained, and the chemical reactions utilized in automated methods rarely are stoichiometric. Both the accuracy and the precision of the determinations depend upon precise adjustment of the equipment, so maintained that all standard and test preparations are exposed to identical physical and chemical manipulations for identical time intervals. Excessive variability in the response of the standard preparations indicates that the analytical system is malfunctioning and that the test results are therefore invalid. However, where automated systems are shown to operate reliably, the precision of the automated method may surpass that of the manual procedure employing the same basic chemistry.

Many of the manual methods given in this Pharmacopeia can be adapted for use in automated equipment incorporating either discrete analyzers or continuous flow systems and operating under a variety of conditions. On the other hand, an analytical scheme devised for a particular automated system may not be readily transposable for use either in a manual procedure or in other types of automated equipment.

The apparatus required for manual methods is, in general, less complicated than the apparatus of automated systems, even those systems used for the direct automated measurement of a single analyte (i.e., the substance being determined or analyzed for) in a binary mixture. However, because of their versatility, automated systems designed for the rapid determination of a specified substance often can be readily modified by the addition of suitable modules and accessories to permit the determination of one or more additional substances in a dosage form. Such extended systems have been utilized, for example, in the automated analysis of articles containing both estrogens and progestogens.

The accompanying pertinent diagrams represent examples of automated methods. Diagrams for official methods are reproduced here rather than in the individual monographs. The descriptions of the procedural details in these methods exemplify the general approach in automated analysis applicable to dosage forms. It should be noted that the diagrams, with many minutiae, are an indispensable part of the directions for conducting the analysis.

DIAGRAMS

The diagrams shown below are arranged in alphabetic order by the name of the drug first mentioned, where the diagram is for a procedure for a specific article. Where there is no procedure in this chapter for a particular diagram, reference is to be made to the named monograph.

ANTIBIOTICS—HYDROXYLAMINE ASSAY

The following procedure is applicable for the assay of those Pharmacopeial antibiotics, such as cephalosporins and penicillins, that possess the beta-lactam structure.

Apparatus— Automatic analyzer consisting of (1) a liquid sampler, (2) a proportioning pump, (3) suitable spectrophotometers equipped with matched flow cells and analysis capability at 480 nm, (4) a means of recording spectrophotometric readings, and/or computer for data retrieval and calculation, and (5) a manifold consisting of the components illustrated in the accompanying pertinent diagram.

Diagram for Automated Hydroxylamine Assay for Antibiotics

Reagents—

Hydroxylamine Hydrochloride Solution— Dissolve 20 g of hydroxylamine hydrochloride in 5 mL of polyoxyethylene (23) lauryl ether solution (1 in 1000), and add water to make 1000 mL.

Acetate Buffer— Dissolve 173 g of sodium hydroxide and 20.6 g of sodium acetate in water to make 1000 mL. Dilute 75 mL of this solution with water to 500 mL, and mix.

Ferric Nitrate Solution— Suspend 233 g of ferric nitrate in about 600 mL of water, add 2.8 mL of sulfuric acid, stir until the ferric nitrate is dissolved, add 1 mL of polyoxyethylene (23) lauryl ether, dilute with water to 1000 mL, and mix.

USP Reference Standards

— Use the USP Reference Standard as directed in the individual monograph.

Standard Preparation— Unless otherwise directed in the individual monograph, dissolve an accurately weighed quantity of the USP Reference Standard in water, and quantitatively dilute with water to obtain a solution having a known concentration of about 1 mg per mL.

Assay Preparation— Unless otherwise directed in the individual monograph, using the specimen under test, prepare as directed under Standard Preparation.

Procedure— With the sample line pumping water, the other lines pumping their respective reagents, and the spectrophotometer set at 480 nm, standardize the system until a steady absorbance baseline has been established. Transfer portions of the Standard Preparation and the Assay Preparation to sampler cups, and place in the sampler. Start the sampler, and conduct determinations of the Standard Preparation and the Assay Preparation typically at the rate of 40 per hour, using a ratio of about 2:1 for sample and wash time. Calculate the potency by the formula given in the individual monograph, in which C is the concentration, in mg per mL, of USP Reference Standard in the Standard Preparation; P is the potency, in µg per mg, of the USP Reference Standard; and AU and AS are the absorbances, corrected for the absorbances of the respective blanks, of the solutions from the Assay Preparation and the Standard Preparation, respectively.

ASSAY FOR ASCORBIC ACID

The following procedure is applicable for the assay of ascorbic acid in Pharmacopeial multivitamin-minerals combination products (solid and liquid dosage forms) that contain components that interfere in other methods of assay.

Apparatus— Automatic analyzer consisting of (1) a liquid sampler; (2) a proportioning pump; (3) a suitable fluorimeter equipped with a flow cell and filters: primary—335 nm, and secondary—426 nm; (4) a means of recording fluorimeter readings; and (5) a manifold consisting of the components illustrated in the accompanying pertinent diagram.

Diagram for Automated Ascorbic Acid

Reagents—

Extracting Solution— Dissolve 600 g of metaphosphoric acid in 1200 mL of water. Add 400 mL of glacial acetic acid, dilute with water to 2000 mL, and mix.

Dilute Extracting Solution— Dissolve 60 g of metaphosphoric acid in 1200 mL of water. Add 160 mL of glacial acetic acid, dilute with water to 2000 mL, and mix.

Surfactant Solution— Prepare a 30% solution of polyoxyethylene (23) lauryl ether by melting 150 g in a container on a steam bath and slowly adding approximately 250 mL of water with continuous stirring. Cool and dilute with water to make 500 mL.

Wash Solution— Add 1 mL of Surfactant Solution to 3000 mL of Dilute Extracting Solution, and mix.

Carbon Extraction Solution— Dissolve 60 g of metaphosphoric acid in 1200 mL of water. Add 160 mL of glacial acetic acid, and mix. Add 33 g of activated charcoal powder, mix, and dilute with water to 2000 mL. Continually mix the solution at a rate that maintains homogeneity.

Sodium Acetate Solution— Dissolve 500 g of sodium acetate trihydrate in water to make 1000 mL, mix, and filter.

Phenylenediamine Solution— Dissolve 200 mg of o-phenylenediamine dihydrochloride in water to make 1000 mL, and mix. Prepare fresh daily.

USP Reference Standards

— USP Ascorbic Acid RS.

Diagram for Automated Aspirin Determinative Step of the Dissolution Test for Aspirin, Alumina, and Magnesium Oxide Tablets

Standard Stock Solution— Dissolve an accurately weighed quantity of USP Ascorbic Acid RS in Dilute Extracting Solution to obtain a solution having a known concentration of about 0.1 mg per mL.

Standard Preparations— Transfer 10.0, 20.0, 30.0, 40.0, and 50.0 mL of Standard Stock Solution to separate 100-mL volumetric flasks, dilute the contents of each flask with Carbon Extracting Solution to volume, mix, and filter to obtain Standard Preparations A, B, C, D, and E having known concentrations of 10 µg, 20 µg, 30 µg, 40 µg, and 50 µg of USP Ascorbic Acid RS per mL, respectively.

Assay Preparation—

For Liquid Preparations— Transfer an accurately measured volume of the liquid preparation, equivalent to 150 mg of ascorbic acid, to a 100-mL volumetric flask. Add 10 mL of Extracting Solution and 6 mL of glacial acetic acid. Dilute with water to volume, and mix. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with Carbon Extracting Solution to volume, mix, and filter.

For Tablet Preparations— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 250 mg of ascorbic acid, to a 250-mL volumetric flask. Add 25 mL of Extracting Solution, 15 mL of glacial acetic acid, and about 100 mL of water, and swirl to mix. Heat for 15 minutes in a 70

water bath, swirling after about 7 minutes. Cool, and dilute with water to volume. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with Carbon Extracting Solution to volume, mix, and filter.

For Capsule Preparations— Empty the contents, if necessary by cutting open with a sharp blade, of not fewer than 20 Capsules in a suitable container, and mix thoroughly. Transfer a portion of the capsule contents, equivalent to about 250 mg of ascorbic acid, to a 250-mL volumetric flask, and proceed as directed for Tablets above, beginning with “Add 25 mL of .”

Procedure— With the sample line pumping the Wash Solution, the other lines pumping their respective reagents, and the fluorimeter equipped with proper filters, standardize the system by pumping until a steady baseline has been established. Transfer portions of the Standard Preparations and the Assay Preparation to sample cups, and place in the sampler. Start the sampler, and conduct determinations of each Standard Preparation and the Assay Preparation at the rate of 40 per hour, using a ratio of about 2:1 for sample and wash time. Derive a standard response line by plotting the respective Standard Preparation concentration (10.0, 20.0, 30.0, 40.0, and 50.0 µg per mL) versus transmittance. From the measured transmittance and the standard response line, determine the ascorbic acid concentration, C, in µg per mL, of the Assay Preparation. Calculate the quantity, in mg, of C6H8O6 in the portion of liquids, tablets, or capsule contents taken by the appropriate formula:

For Liquids: 5C/V in which V is the volume, in mL, of liquid preparation taken to prepare the Assay Preparation.

For Tablets or Capsules: 12.5C.

ASSAY FOR IODIDE

Apparatus— Automatic analyzer consisting of (1) a liquid sampler, (2) a proportioning pump, (3) a heating bath, (4) a suitable colorimeter equipped with a 2.0- × 50-mm flow cell and analysis capability at 420 nm, (5) a means of recording colorimetric readings, and (6) a manifold consisting of the components illustrated in the accompanying pertinent diagram.

Diagram for Automated Iodide Assay

Reagents—

Acetic Acid Carrier Solution— Transfer 3.0 mL of glacial acetic acid to a 2000-mL volumetric flask containing about 800 mL of water. Add 2 mL of polyoxyethylene (23) lauryl ether, and dilute with water to volume.

Arsenious Acid Solution— Transfer 19.6 g of arsenic trioxide and 14.0 g of sodium hydroxide to a 2000-mL volumetric flask. Add about 150 mL of water, and dissolve with stirring. Dilute with water to a volume of about 800 mL, and add 66 mL of sulfuric acid. Cool to room temperature. Transfer 50.0 g of sodium chloride to the solution, and mix to dissolve. Add 2 mL of Surfactant Solution, dilute with water to volume, mix, and filter.

Ceric Ammonium Sulfate Solution— Transfer 12.65 g of ceric ammonium sulfate to a 1000-mL volumetric flask. Add about 700 mL of water followed by 100 mL of sulfuric acid, swirling to mix. Heat to dissolve, and cool to room temperature. Add 1 mL of Surfactant Solution, dilute with water to volume, mix, and filter.

3% Acetic Acid Solution— Transfer 30 mL of glacial acetic acid to a 1000-mL volumetric flask containing about 300 mL of water. Dilute with water to volume, and mix.

Standard Preparations—

Standard Stock Solution— Transfer an accurately weighed quantity of 1.3080 g of potassium iodide, previously dried for 24 hours at 105

, to a 1000-mL volumetric flask. Dilute with water to volume, and mix to obtain a solution having an iodide concentration of 1000 µg per mL.

Intermediate Standard Solution— Quantitatively dilute a suitable volume of Standard Stock Solution with water to obtain a solution having an iodide concentration of 1 µg per mL.

Working Standard Preparations— Transfer 2.0, 4.0, 6.0, 8.0, and 10.0 mL of Intermediate Standard Solution to separate 100-mL volumetric flasks. Add 5 mL of 3% Acetic Acid Solution. Dilute the contents of each flask with water to volume, and mix to obtain Standard Preparations A, B, C, D, and E having known iodide concentrations of about 0.02 µg per mL, 0.04 µg per mL, 0.06 µg per mL, 0.08 µg per mL, and 0.1 µg per mL, respectively.

Assay Preparation—

For Liquid Preparations— Transfer an accurately measured volume of the liquid preparation, equivalent to 16 µg of iodide, to a 200-mL volumetric flask. Add 10 mL of 3% Acetic Acid Solution to dissolve, dilute with deionized water to volume, mix, and filter. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, add 5.0 mL of 3% Acetic Acid Solution, dilute with deionized water to volume, mix, and filter to obtain a solution having an iodide concentration of about 0.08 µg per mL.

For Tablet Preparations— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 250 µg of iodide, to a 250-mL volumetric flask. Add 100 mL of 1 N hydrochloric acid, and mix with the aid of sonication for 30 minutes. Dilute with water to volume, mix, and filter. Transfer 8.0 mL of the filtered solution to a 100-mL volumetric flask, add 5 mL of 3% Acetic Acid Solution, dilute with water to volume, and mix to obtain a solution having an iodide concentration of about 0.08 µg per mL.

For Capsule Preparations— Empty the contents, if necessary by cutting open with a sharp blade, of not fewer than 20 Capsules into a suitable container, and mix thoroughly. Transfer a portion of the capsule contents, equivalent to about 250 µg of iodide, to a 250-mL volumetric flask and proceed as directed for Tablets above, beginning with “Add 100 mL of .”

Procedure— With the sample line pumping the Acetic Acid Carrier Solution, the other lines pumping their respective reagents, and the colorimeter equipped with 420-nm filters, standardize the system until a steady baseline has been established. Transfer portions of the Standard Preparations and the Assay Preparation to the sampler cups, and place in the sampler. Start the sampler, and conduct determinations of each Standard Preparation and the Assay Preparation at the rate of 30 per hour, using a ratio of about 1:4 for sample and wash time. Derive a standard response line by plotting the respective Standard Preparation concentration (0.02, 0.04, 0.06, 0.08, and 0.10 µg per mL) versus absorbance. [note—This is an indirect absorbance relationship: the greater the iodide amount, the less the absorbance.] From the measured transmittance and the standard response line, determine the iodide concentration, C, in µg per mL, of the Assay Preparation. Calculate the quantity, in µg, of iodide in the portion of liquids, tablets, or capsules contents taken by the formula:

For Liquids: 2000C/V in which V is the volume, in mL, of the liquid preparation taken to prepare the Assay Preparation.

For Tablets and Capsules: 3125C.

CONTENT UNIFORMITY OF NITROGLYCERIN TABLETS

This is not to be considered as the official method. It is detailed here for further illustration of descriptions of automated methods.

Apparatus— Automatic analyzer consisting of (1) a liquid sampler, (2) a proportioning pump, (3) a heating bath, (4) a suitable spectrophotometer equipped with a 5-mm flow cell and analysis capability at 545 nm, (5) a means of recording spectrophotometric readings, and (6) a manifold consisting of the components illustrated in the accompanying pertinent diagram.

Diagram for Automated Assay for Nitroglycerin Tablets

Reagents—

1 Percent Strontium Hydroxide Solution— Dissolve 20.0 g of strontium hydroxide [Sr(OH)2·8H2O] in 1800 mL of carbon dioxide-free water, heating if necessary. Cool to room temperature, dilute with carbon dioxide-free water to 2000 mL, and mix. Allow to stand overnight, and filter. Store the clear solution in tightly closed containers, protected from carbon dioxide.

0.3 Percent Procaine Hydrochloride Solution— Dissolve 3.0 g of procaine hydrochloride in water to make 1000 mL.

0.1 Percent N-(1-Naphthyl)ethylenediamine Dihydrochloride Solution— Dissolve 1.0 g of N-(1-naphthyl)ethylenediamine dihydrochloride in water to make 1000 mL. Prepare fresh each week.

Standard Preparation— Dissolve an accurately weighed portion of 10% nitroglycerin-betalactose absorbate, previously standardized, in water, and dilute quantitatively and stepwise with water to obtain a solution having a known concentration of about 30 µg per mL.

Test Preparation— Dissolve 1 Nitroglycerin Tablet in water to obtain a solution having a concentration of about 30 µg of nitroglycerin per mL.

Procedure— With the sample line pumping water, the other lines pumping their respective reagents, and the spectrophotometer set at 545 nm, standardize the system by pumping until a steady absorbance baseline has been established. Transfer portions of the Standard Preparation and the Test Preparation to sampler cups, and place in the sampler. Start the sampler, and conduct determinations of the Standard Preparation and the Test Preparation at a rate of 30 per hour, using a ratio of 1:1 for sample and wash time. First, run two standards, discarding the first value, then continue the run using one standard after each five samples, recording the absorbance values. Calculate the quantity, in mg, of C3H5N3O9 in the Tablet taken by the formula:

(T / D)C(AU / AS)

in which T is the labeled quantity, in mg, of nitroglycerin in the Tablet; D is the concentration, in µg per mL, of nitroglycerin in the solution from the Tablet, based on the labeled quantity per Tablet and the extent of dilution; C is the concentration, in µg per mL, of nitroglycerin in the Standard Preparation; AU is the absorbance of the Test Preparation; and AS is the average of the absorbances of the two Standard Preparations that bracket the Test Preparation.

Diagram of Dissolution Test Method for Erythromycin Ethylsuccinate Tablets Labeled as Chewable

Diagram for Automated Drug Release and Content Uniformity Test for Propranolol Hydrochloride and Hydrochlorothiazide Extended-Release Capsules

Diagram for Automated Dissolution and Content Uniformity Test for Reserpine Tablets

Diagram for Automated Content Uniformity Test for Reserpine, Hydralazine Hydrochloride, and Hydrochlorothiazide Tablets

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
General Chapter	Gary E. Ritchie, M.Sc. Scientific Fellow 1-301-816-8353	(GC05) General Chapters 05
Reference Standards	Lili Wang, Technical Services Scientist 1-301-816-8129 RSTech@usp.org

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