Limit tests

The limit test is a quality control test in pharmaceutical industries. A limit test is a semiquantitative test to determine whether the concentration of a particular impurity in a sample is within a predetermined limit or exceeded. Pharmacopoeia allows a certain limit of a particular impurity in the pharmaceuticals. This limit is expressed as a percentage of pharmacopoeial substances in micrograms per unit dose.

If the concentration of a particular impurity in the sample is below the limit then the sample is considered compliant with the pharmacopeia standards. It the concentration of a particular impurity exceeds the limit then API is considered noncompliant with the pharmacopeia standards.

Importance of limit test

It is very difficult to manufacture 100% pure pharmacopoeial substances. The absolute pure product shall be very costly. Pharmacopoeia allows some limit of impurity to make pharmacopoeial substances cost-effective and also safe for consumption. The primary purpose of a limit test is to ensure the safety, efficacy, and quality of the pharmaceutical substances and formulations. Limit test helps to protect patient health and well-being. Limit test compliance maintains product consistency and ensures compliance with regulatory standards.

Limit test importance in pharmaceutical industries can be explained by the below points

• 1. Quality assurance: Limit tests determine the maximum allowable concentration of impurities in the pharmaceuticals. This ensures the product meets the standards of pharmacopeia and regulatory bodies.

• 2. Safety: Impurities above fixed limits harm the patients. Limit tests highlight the potentially harmful pharmaceuticals. Compliance with limit tests ensures pharmaceuticals are safe for consumption.

• 3. Efficacy: Impurities in pharmaceuticals affect the efficiency of pharmaceutical formulation. Limit tests ensure that pharmaceutical formulations produce estimated effects.

• 4. Regulatory compliance: Regulatory authorities are strict in maintaining pharmaceutical formulation quality. Limit tests help pharmaceutical manufacturers to demonstrate compliance with regulatory standards.

• 5. Batch-to-batch consistency: Pharmaceutical formulations are manufactured in batches. The limit test ensures that each batch complies with the regulatory requirements and that quality with purity is the same in each batch.

• 6. Consumer confidence: Regulatory compliance of pharmaceutical formulations built confidence among consumers regarding effectiveness of the product.

• 7. Cost savings: Limit test compliance of raw materials helps to prevent noncompliance of pharmaceutical formulation with regulatory requirements. This saves both time and money for the pharmaceutical manufacturers.

Principle and procedures of Limit tests for chlorides, sulfates, iron, heavy metals, and arsenic.

Nessler cylinders: Nesseler cylinders are a pair of two glass cylinders having uniform diameter and height. They are designed to allow visual comparison of test and standard sample color, opalescence, or turbidity.

Key Features

• 1. Flat bottom: Ensures a stable base for accurate measurements.

• 2. Identical: Uniform diameter and length

• 3. Graduated scale: Markings for precise volume measurements.

• 4. Transparent material: Allows for clear observation of the solution's color.

The limit test ensures the presence of impurities within the limit in raw materials, pharmaceuticals, or pharmacopoeial substances. The standard solution and test solution of the sample are prepared in nessler’s cylinder.

Limit tests for chloride

• Principle: The limit test for chloride in the sample is based on the reaction between chloride ion (cl-) and silver ion (Ag-). This reaction forms a white precipitate of insoluble silver chloride (AgCl). Two reagents soluble chloride and silver nitrates are mixed in the presence of nitric acid that forms silver chloride (AgCl) precipitate. A very small amount of silver chloride (AgCl) is precipitated. Thus it develops opalescence. The opalescence in the test Nessler cylinder and standard Nessler cylinder are compared against a uniform illuminated background.

  • AgNO3 (Soluble) + Cl- = AgCl (Precipitate) + NO3+

  • AgNO3 (Soluble) + Cl- (Soluble) → AgCl (precipitate) + NO3-

• Nitric acid is added to prevent the precipitation of other impurities like sulphate, phosphate, etc.

• Standard Nessler cylinder contains 0.05845 chloride standard solution (25 ppm Cl).

• Opalescence in the test Nessler cylinder should not be more than in the standard Nessler cylinder.

Procedure: Amount of test samples of pharmacopoeial substance is prescribed by Pharmacopoeia. It depends upon Pharmacopoeial compounds.

Procedure to perform a limit test on magnesium sulphate I.P:

• 1. Preparation of Test sample:

  • · Dissolve 2 gm magnesium sulphate in 10 ml distilled water in a Nessler cylinder labelled as ‘Test’.

  • · Add 10 ml dilute nitric acid

  • · Dilute it with distilled water up to 50 ml.

  • · Add 1 ml 0.1M silver nitrate solution.

• 2. Preparation of Standard sample:

  • · Add 10 ml of chloride standard solution (25 ppm Cl) in Nessler cy;inder labelled as ‘standard’.

  • · Dilute it with distilled water up to 50 ml.

  • · Add 1 ml 0.1M silver nitrate solution.

  • Stir the reagents in each Nessler cylinder with a glass rod. Set aside for 5 minutes. Compare opalescence in both Nessler cylinders against a dark background.

• Interpretation: If the opalescence of the sample solution is greater than or equal to that of the standard solution, it indicates that the sample contains an excessive amount or amount of maximum limit of chloride ions. It does not pass the test. If the opalescence is less than that of the standard, the sample is considered to meet the opalescence limit and the test sample passes the test.

Limit Test for Sulphate

The limit test for sulfate is a semiqualitative analysis to determine the maximum permissible amount of sulfate ions (SO42-) in a sample. It involves the formation of a precipitate of barium sulfate (BaSO4), which is insoluble in dilute hydrochloric acid.

Principle: The interaction of sulfate with barium chloride in the presence of hydrochloric acid produces barium sulfate (precipitate).

• BaCl2 (soluble) + Na2SO4 (soluble) → BaSO4↓ (precipitate) + 2NaCl

Explanation of each step:

• 1. Hydrochloric acid is added to prevent the precipitation of other ions such as phosphate, oxalate, etc from the solution. This prevention is by the common ion effects that allow only sulfate precipitation.

• 2. A very small amount of sulfate impurity is present in the pharmacopoeial substances. Thus precipitation of the barium sulphate develops turbidity.

• 3. Turbidity in the test sample is compared with the standard sample in Nessler cylinders. The test sample passes the test if the turbidity in the test sample is less than the turbidity in the standard sample.

• 4. The Standard sample in the Nessler cylinder contains 0.1089% w/v potassium sulfate in 1 ml solution.

• Note: “Barium sulfate reagent” is used in the procedure in place of barium chloride. Barium sulfate reagent is a barium chloride solution in alcohol with a very small amount of potassium sulfate (0.905 gm in 100 ml). The presence of potassium sulfate in Barium sulfate reagent has the following advantages:

• 1. Potassium sulfate increases the sensitivity of the test. It increases the sulfate ion concentration above the solubility of barium sulfate. This develops turbidity.

• 2. Alcohol stops the formation of a supersaturated solution. This allows the precipitation of barium sulfate that develops turbidity.

Since 1995, Pharmacopoeia has allowed the use of barium chloride solution and alcohol in place of barium sulfate reagent to produce turbidity.

Procedure: (Limit test for sulfate in Sodium chloride IP)

The general steps involved are as follows:

• 1. Primary solution (Test solution) Preparation:

  • · Dissolve 2 gm of Sodium chloride IP in 10 ml Distilled water in a Nessler cylinder labeled as “Test”.

  • · Add 2 ml of dilute sulphuric acid

  • · Add distilled water to make a volume up to 45 ml.

  • · Add 5 ml Barium sulfate reagent.

  • · Stir the mixture with a glass rod.

  • · Set aside for 5 minutes to develop turbidity

• 2. Secondary solution (Auxiliary or Standard solution) preparation:

  • · Add 1 ml 0.1089% w/v potassium sulfate solution in Distilled water in a Nessler cylinder labeled as “Standard”.

  • · Add 9 ml Distilled water

  • · Add 2 ml of dilute sulphuric acid

  • · Add distilled water to make a volume up to 45 ml.

  • · Add 5 ml Barium sulfate reagent.

  • · Stirr the mixture with a glass rod.

  • Set aside for 5 minutes to develop turbidity

• 3. Comparison: Compare the turbidity of the test sample and standard sample. The standard sample contains a known concentration of sulfate ions.

• 4. Interpretation: If the turbidity of the sample solution is greater than or equal to that of the standard sulfate solution, it indicates that the sample contains an excessive amount of sulfate ions. It does not pass the test. If the turbidity is less than that of the standard, the sample is considered to meet the sulfate limit and the test sample passes the test.

Limit test for Iron

Principle: Iron interacts with thioglycolic acid in the presence of citric acid in the ammonical alkaline media. This forms ferrous salt of thioglycollic acid (A purple-colored salt).

• 2HSCH2COOH (Thioglycolic acid) + Fe++ (Ferrous ion) = Fe(HSCH2COO)2 (Ferrous sulphate) + 2H+. All reagents should be free from iron impurities.

• Thioglycolic acid is used for the following purposes

  • 1. Iron impurity in pharmacopeial substances is in ferrous salt (Fe++) and ferric salt (Fe++). Thioglycolic acid reduces Ferric salt to ferrous salt.

    • 2HSCH2COOH (Thioglycolic acid) + Fe+++ (Ferric ion) = Fe++ + 2 2HSCH2COO- + 2H+

  • 2. Thioglycolic acid interacts with ferrous salt to develop a purple color.

  • Citric acid is used for the following purpose

    • Iron reacts with ammonia to form iron hydroxide precipitate. Citric acid prevents iron interaction with ammonia and keeps iron in solution form by forming a complex.

      • 2Fe + 10NH3 = 2Fe(NH2)5 + 5H2

      • 2C6H8O4 + Fe = Fe(C6H6O4)2 + 2H2

Procedure (Limit test for iron in Sodium chloride IP)

The general steps involved are as follows:

• 1. Primary solution (Test solution) Preparation:

  • Dissolve 1 gm sodium chloride in 40 ml distilled water inside Nessler Cylinder labeled as “Test”.

  • Add 2 ml of 20% iron-free citric acid solution

  • Add 0.1 ml of thioglycolic acid

  • Add iron-free ammonia solution to make the solution in the Nessler cylinder alkaline

  • Add sufficient distilled water to make the volume up to 50 ml.

  • Stir the solution with a glass rod

  • Set aside for 5 minutes

• 2. Secondary solution (Auxiliary or Standard solution) Preparation:

  • Add 2 ml of standard iron solution (20 ppm Fe) in 40 ml distilled water inside Nessler Cylinder labeled as “Standard”.

  • Add 2 ml of 20% iron-free citric acid solution

  • Add 0.1 ml of thioglycolic acid

  • Add iron-free ammonia solution to make the solution in the Nessler cylinder alkaline

  • Add sufficient distilled water to make the volume up to 50 ml.

• Stir the solution with a glass rod

• Set aside for 5 minutes

• 3. Comparison: Compare the color of the test sample and standard sample by viewing vertically downward. The standard sample contains a known concentration of iron ions.

• Interpretation: If the color intensity of the sample solution is greater than or equal to that of the standard iron solution, it indicates that the sample contains an excessive amount of iron ions. It does not pass the test. If the color intensity is less than that of the standard, the sample is considered to meet the iron limit and test sample pass the test.

Limit Test for Lead

Two methods are used to perform limit tests for lead.

• 1. B.P. Method

• 2. I.P./U.S.P. Method

B.P. Method for Lead Limit Test

• Lead salt reacts with sodium sulfide to form a black precipitate of lead sulfide. A dilute solution of lead salt (10 PPM) develops a black precipitate of lead sulfide in the presence of ammonia and potassium cyanide.

• Lead salt (Soluble) + Na2S (Soluble) = Sodium salt (Soluble) + PbS (precipitate)

• Pb(No3)2 + Na2S = 2NaNO3 + PbS.

Explanation of each step

• 1. Metals like copper, iron, etc also react with sodium sulfide to form black precipitate. Ammonia and potassium cyanide block copper, iron, and other metals from reacting with sodium sulfide, and only lead will interact with sodium sulfide to form a sulfide precipitate.

• 2. The Nessler cylinder is made of glass. This glass must be free from lead impurities.

• 3. All reagents used must be free from lead impurities and labeled as ‘PbT’.

Procedure

• 1. Primary solution (Test solution)

  • · Add a specified amount (Specified in pharmacopeia) of the test sample in the Nessler cylinder. Label as Test or Primary.

  • · Dissolve the sample in distilled water or as specified in the pharmacopeia.

  • · Make the solution alkaline using ammonia solution.

  • · Add 1 ml potassium cyanide solution.

• 2. Secondary solution (Auxiliary solution): The preparation method is the same as the primary solution. But it also contains a specified volume of standard lead solution (Lead nitrate solution).

  • · Add a specified amount (Specified in pharmacopeia) of the test sample in the Nessler cylinder. Label as Test or Primary.

  • · Dissolve the sample in distilled water or as specified in the pharmacopeia.

  • · Add 5 ml of dilute solution of lead,

  • · Make the solution alkaline using ammonia solution.

  • · Add 1 ml potassium cyanide solution.

Alok Bains