10. LIMIT TESTS

Introduction to Limit Tests

Limit Tests are semi-quantitative or quantitative tests designed to identify and control very small amounts of impurities present in pharmaceutical substances. These impurities, though minute, can affect the quality and safety of the product; hence, their limits are prescribed in pharmacopoeias.

Each limit test is developed to be specific, sensitive, and reproducible. The tests ensure that the impurity level does not exceed the permissible limit.

Important Factors in Limit Test Design

  • Specificity: The test should produce a definite reaction with the impurity to be detected.
  • Sensitivity: The test should detect the smallest permissible quantity of impurity as per pharmacopoeial standards.
  • Personal Errors: Proper handling and standardized methods are essential to avoid operator-related variations.

Limit Test for Chloride

Aim:

To perform the limit test for chloride impurity.

Principle:

The test is based on the precipitation of chloride ions as silver chloride (AgCl) by reaction with silver nitrate in the presence of dilute nitric acid. The turbidity produced is compared with a standard solution of known chloride concentration.

Reagents:

  • 10% w/w HNO₃: Add 8.6 ml of concentrated nitric acid (70%) to about 50 ml of distilled water, then make up to 100 ml.
  • 5% w/w AgNO₃: Dissolve 5 g silver nitrate in distilled water and make up to 100 ml.
  • 25 ppm Chloride Solution: Dilute 5 volumes of 0.0824% sodium chloride to 100 volumes with distilled water.

Procedure:

  1. Dissolve the sample in distilled water or take liquid directly into a Nessler cylinder.
  2. Prepare a standard with 10 ml of 25 ppm chloride solution.
  3. Add 10 ml dilute nitric acid and dilute both to 50 ml with distilled water.
  4. Add 1 ml of 0.1 M silver nitrate to each cylinder.
  5. Observe the opalescence against a black background. If the sample turbidity is less than the standard, it passes the test.

Limit Test for Arsenic

Aim:

To perform the limit test for arsenic impurity.

Principle:

Based on the Gutzeit test, where arsenic is converted into arsine gas (AsH₃) by reaction with zinc and hydrochloric acid. Arsine reacts with mercuric chloride paper to produce a yellow stain proportional to the arsenic content.

Reagents:

  • 1 M Potassium Iodide: Dissolve 16.6 g KI in 100 ml distilled water.
  • Arsenic Standard Solution (10 ppm): Dissolve 0.330 g As₂O₃ in 5 ml 2 M NaOH, dilute to 250 ml, then further dilute 1:100 with water.

Procedure:

  1. Add 5 ml 1 M KI and 10 g Zn to the sample in a conical flask.
  2. Assemble the apparatus and maintain uniform heating in a water bath.
  3. Repeat the same steps for the standard solution.
  4. Compare the yellow stains on mercuric chloride papers. If the test stain is lighter, the sample passes.

Limit Test for Sulphate

Aim:

To perform the limit test for sulphate impurity.

Principle:

Based on precipitation of sulphate ions as barium sulphate (BaSO₄) using barium chloride in acidic conditions. The turbidity formed is compared with a standard.

Reagents:

  • 25% w/v BaCl₂: Dissolve 25 g in 100 ml distilled water.
  • 5 M Acetic Acid: Add 28.73 ml glacial acetic acid to water and make up to 100 ml.
  • 10 ppm SO₄²⁻ Solution: Dilute 0.181% K₂SO₄ in water to 100 volumes.

Procedure:

  1. In a Nessler cylinder, add 1 ml BaCl₂ solution and 1.5 ml standard sulphate solution.
  2. Add 15 ml of the sample, 0.15 ml acetic acid, and dilute to 50 ml.
  3. Prepare standard similarly using 15 ml of standard sulphate solution.
  4. Compare opalescence against a black background. The test passes if less turbid than the standard.

Limit Test for Iron

Principle:

Iron reacts with thioglycollic acid in ammonium citrate buffer to form a purple complex of ferrous thioglycollate. The intensity is compared with a standard containing 0.04 mg Fe.

Reagents:

  • 20% w/v Citric Acid: Dissolve 20 g citric acid in 100 ml water.
  • 20 ppm Iron Standard: Dilute 0.1726% ferric ammonium sulphate solution 1:10 with 0.05 M H₂SO₄.

Procedure:

  1. Dissolve the test sample in water, add 2 ml citric acid and 0.1 ml thioglycollic acid.
  2. Make alkaline with ammonia and dilute to 50 ml.
  3. Prepare the standard similarly.
  4. Compare the purple color intensity; if lighter than the standard, it passes.

Limit Test for Heavy Metals

Heavy metals, including lead, copper, arsenic, and mercury, may be present as trace impurities. The test is based on reaction with hydrogen sulphide to form brown or black sulphides, compared with a lead standard (usually 20 ppm).

Methods (As per IP)

Method A:

  • Used for clear, colorless solutions.
  • Add 10 ml hydrogen sulphide solution to test and standard (pH 3–4), dilute to 50 ml, and compare color after 5 min.

Method B:

  • Used for substances not giving clear solutions.
  • Sample is charred with H₂SO₄, treated with HCl, filtered, and adjusted to pH 3–4 before testing.

Method C:

  • For substances soluble in sodium hydroxide medium.
  • Both test and standard receive 5 drops of Na₂S solution; compare colors after 5 minutes.

Limit Test for Lead

Principle:

The test is based on formation of a red-violet lead-dithizone complex when lead reacts with dithizone in chloroform. The color intensity corresponds to lead concentration.

Procedure (IP):

  1. Add ammonium citrate, hydroxylamine HCl, and phenol red to the sample; make alkaline with ammonia.
  2. Add potassium cyanide and extract with dithizone-chloroform solution until green color persists.
  3. Combine extracts, shake with 1% HNO₃ and fresh dithizone; compare violet color with standard lead solution.

Procedure (BP):

  1. Prepare primary and auxiliary solutions of the test substance.
  2. Add a known lead quantity to auxiliary solution, adjust pH, and add sodium sulphide.
  3. Compare color intensity—if auxiliary is darker, the lead content is within limits.

Detailed Notes:

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