Limit Tests

By / Method of Analysis, Quality control / LIMIT TESTS, QC /
What is limit tests ? | limit tests in Pharmaceuticals |
  • Article covers below limit tests procedure:
    • Limit Test for Aluminium
    • Limit Test for Ammonium
    • Limit Test for Arsenic
    • Limit Test for Calcium
    • Limit Test for Chlorides
    • Limit Test for Fluorides
    • Limit Test for Heavy Metals
    • Limit Test for Iron
    • Limit Test for Lead in Sugars
    • Limit Test for Magnesium
    • Limit Test for Magnesium and Alkaline-earth Metals
    • Limit Test for Nickel in Polyols
    • Limit Test for Phosphates
    • Limit Test for Potassium
    • Limit Test for Sulfates
What are limit tests?

Limit tests check if something is pure by finding impurities that can only be accepted in small amounts. They compare the substance being tested to another one that has a known amount of the impurity. This helps give an idea of how much impurity is in the test substance.

Limit tests in pharmacopoeia are analytical procedures to determine harmful or undesirable substances in pharmaceutical products. They establish acceptable limits for specified impurities to ensure product quality and safety.

Limit Test for Aluminium
  • Solution Preparation:
    • 5 g/L solution of hydroxyquinoline in chloroform: Dissolve 0.5 gm of hydroxyquinoline in 100 ml chloroform

Procedure : Place the prescribed solution in a separating funnel and shake with 2 quantities, each of 20 mL, and then with one 10 mL quantity of a 5 g/L solution of hydroxyquinoline in chloroform. Dilute the combined chloroform solutions to 50.0 mL with chloroform (test solution).

Prepare a standard in the same manner using the prescribed reference solution.

Prepare a blank in the same manner using the prescribed blank solution.

Measure the intensity of the fluorescence of the test solution (I1), of the standard (I2) and of the blank (I3) using an excitant beam at 392 nm and a secondary filter with a transmission band centred on 518 nm or a monochromator set to transmit at this wavelength.

Result: The fluorescence (I1–I3) of the test solution is not greater than that of the standard (I2–I3).

Limit Test for Ammonium

METHOD A

  • Solution Preparation:
    • 25% w/v solution of sodium hydroxide: Dissolve 25.0 gm of sodium hydroxide in 100 ml water.
    • Dilute sodium hydroxide solution: Dissolve 8.5 g of sodium hydroxide in water and dilute to 100 mL with the same solvent.
    • Ammonium standard solution (1 ppm NH4) : See Standard Solutions For Limit Tests
    • Potassium Tetraiodomercurate Solution, Alkaline: Dissolve 11 g of potassium iodide and 15 g of mercury(II) iodide in water and dilute to 100 mL with water. Immediately before use, mix the solution with an equal volume of a 25% w/v solution of sodium hydroxide.

Procedure: Introduce the prescribed solution into a test-tube or dissolve the prescribed quantity of the substance to be examined in 14 mL of water in a test-tube. Make the solution alkaline if necessary by the addition of dilute sodium hydroxide solution , dilute to 15 mL with water and add 0.3 mL of alkaline potassium tetraiodomercurate solution. Prepare a standard by mixing 10 mL of ammonium standard solution (1 ppm NH4) , 5 mL of water and 0.3 mL of alkaline potassium tetraiodomercurate solution. Stopper the test-tubes.

Result: After 5 min, any yellow colour in the test solution is not more intense than that in the standard.

METHOD B

Procedure : In a 25 ml jar fitted with a cap, place the prescribed quantity of the finely powdered substance to be examined and dissolve or suspend in 1 ml of water. Add 0.30 g of heavy magnesium oxide. Close immediately after placing a piece of silver manganese paper 5 mm square, wetted with a few drops of water , under the polyethylene cap. Swirl, avoiding projections of liquid, and allow to stand at 40 °C for 30 min. If the silver manganese paper shows a grey colour, it is not more intense than that of a standard prepared at the same time and in the same manner using the prescribed volume of ammonium standard solution (1 ppm NH4) , 1 mL of water and 0.30 g of heavy magnesium oxide.

Result: Test solution is not more intense than that in the standard.

Limit Test for Arsenic

METHOD A

  • Solution Preparation:
    • Stannous chloride solution: Heat 20 g of tin with 85 mL of hydrochloric acid until no more hydrogen is released. Allow to cool.
    • Potassium iodide solution: A 16.6% w/v solution of potassium iodide.
    • Arsenic standard solution (1 ppm As): See Standard Solutions For Limit Tests

Procedure : In the conical flask dissolve the prescribed quantity of the substance to be examined in 25 mL of water, or in the case of a solution adjust the prescribed volume to 25 mL with water. Add 15 mL of hydrochloric acid, 0.1 mL of stannous chloride solution and 5 mL of potassium iodide solution, allow to stand for 15 min and introduce 5 g of activated zinc. Assemble the two parts of the apparatus  immediately and immerse the flask in a bath of water at a temperature such that a  uniform evolution of gas is maintained. Prepare a standard in the same manner, using 1 mL of arsenic standard solution (1 ppm As), diluted to 25 mL with water.

Result: After not less than 2 hours the stain produced on the mercuric bromide paper in the test is not more intense than that in the standard.

METHOD B

  • Solution Preparation:
    • Hypophosphorous reagent: Dissolve, with the aid of gentle heat, 10 g of sodium hypophosphite in 20 mL of water and dilute to 100 mL with hydrochloric acid. Allow to settle and decant or filter through glass wool.

Procedure : Introduce the prescribed quantity of the substance to be examined into a test-tube containing 4 mL of hydrochloric acid and about 5 mg of potassium iodide and add 3 mL of hypophosphorous reagent. Heat the mixture on a water-bath for 15 min, shaking occasionally. Prepare a standard in the same manner, using 0.5 mL of arsenic standard solution (10 ppm As).

Result: After heating on the water-bath, any colour in the test solution is not more intense than that in the standard.

Limit Test for Calcium
  • Solution Preparation:
    • Alcoholic calcium standard solution (100 ppm Ca): See Standard Solutions For Limit Tests
    • Ammonium oxalate solution:  Dissolve 4.0 gm of ammoniumoxalate in 100 ml water.
    • Dilute acetic acid: Dilute 11.4 ml of glacial acetic acid to 100 mL with water.

Note: All solutions used for this test should be prepared with distilled water.

Procedure: To 0.2 mL of alcoholic calcium standard solution (100 ppm Ca), add 1 mL of ammonium oxalate solution. After 1 min, add a mixture of 1 mL of dilute acetic acid and 15 mL of a solution containing the prescribed quantity of the substance to be examined and shake. Prepare a standard in the same manner using a mixture of 10 mL of aqueous calcium standard solution (10 ppm Ca), 1 mL of dilute acetic acid and 5 mL of distilled water.

 Results: After 15 min, any opalescence in the test solution is not more intense than that in the standard.

Limit Test for Chlorides
  • Solution Preparation:
    • Dilute nitric acid : Dilute 14.3 ml of nitric acid  to 100 mL with water.
    • Silver nitrate solution : Dissolve 1.7 gm of Silver nitrate in 100 ml water.
    • Chloride standard solution (5 ppm Cl): See Standard Solutions For Limit Tests

Procedure: To 15 mL of the prescribed solution add 1 mL of dilute nitric acid and pour the mixture as a single addition into a test-tube containing 1 mL of silver nitrate solution. Prepare a standard in the same manner using 10 mL of chloride standard solution (5 ppm Cl) and 5 mL of water. Examine the tubes laterally against a  black background.

Results: After standing for 5 min protected from light, any opalescence in the test solution is not more intense than that in the standard.

Limit Test for Fluorides

  • Solution Preparation:

Procedure: Introduce into the inner tube of the apparatus (see Figure 2.4.5.-1) the prescribed quantity of the substance to be examined, 0.1 g of acid-washed sand and 20 mL of a mixture of equal volumes of sulfuric acid and water. Heat the jacket containing tetrachloroethane maintained at its boiling point (146 °C). Heat the steam generator and distil, collecting the distillate in a 100 mL volumetric flask containing 0.3 mL of 0.1 M sodium hydroxide and 0.1 mL of phenolphthalein solution. Maintain a constant volume (20 mL) in the tube during distillation and ensure that the distillate remains alkaline, adding 0.1 M sodium hydroxide if necessary. Dilute the distillate to 100 mL with water (test solution). Prepare a standard in the same manner by distillation, using 5 mL of fluoride standard solution (10 ppm F) instead of the substance to be examined. Into two glass-stoppered cylinders introduce 20 mL of the test solution and 20 mL of the standard and 5 mL of aminomethylalizarindiacetic acid reagent.

Results : After 20 min, any blue colour in the test solution (originally red) is not more intense than that in the standard.

Limit Test for Heavy Metals

The methods described below require the use of thioacetamide reagent. As an alternative, sodium sulfide solution (0.1 mL) is usually suitable. Since tests prescribed in monographs have been developed using thioacetamide reagent, if sodium sulfide solution is used instead, it is necessary to include also for methods A, B and H a monitor solution, prepared from the quantity of the substance to be examined prescribed for the test, to which has been added the volume of lead standard solution prescribed for preparation of the reference solution. The test is invalid if the monitor solution is not at least as intense as the reference solution.

Solution Preparation:

  • Thioacetamide Reagent: Add 1 mL of a mixture of 15 mL of 1M sodium hydroxide, 5 mL of water and 20 mL of glycerol (85%) to 0.2 mL of thioacetamide solution, heat in a water bath for 20 seconds, cool and use immediately.
  • Thioacetamide solution: Dissolve 4.0 gm of thioacetamide in 100 ml water.
  • 1M sodium hydroxide: Dissolve 4.0 gm of sodium hydroxide in 100 ml water.
  • Sodium Sulfide Solution:  Dissolve 12 g of sodium sulfide with heating in 45 mL of a mixture of 10 volumes of water and 29 volumes of glycerol (85%), allow to cool and dilute to 100 mL with the same mixture of solvents.  The solution should be colourless.
  • xM hydrochloric acid: Solutions of molarity xM should be prepared by diluting 85x mL of hydrochloric acid to 1000 mL with water
  • xM ammonia : Solutions of molarity xM should be prepared by diluting 75x mL of 13.5M ammonia or 56x mL of 18M ammonia to 1000 mL with water.
  • Buffer solution pH 3.5: Dissolve 25 g of ammonium acetate in 25 mL of water and add 38 mL of 7M hydrochloric acid. Adjust the pH to 3.5 with either 2M hydrochloric acid or 6M ammonia and dilute to 100 mL with water.

METHOD A

Test solution: 12 mL of the prescribed aqueous solution of the substance to be examined.

Reference solution (standard): A mixture of 10 mL of lead standard solution (1  ppm Pb) or lead standard solution (2 ppm Pb), as prescribed, and 2 mL of the  prescribed aqueous solution of the substance to be examined.

Blank solution: A mixture of 10 mL of water and 2 mL of the prescribed aqueous solution of the substance to be examined.

Procedure: To each solution, add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately. Examine the solutions after 2 min.  

System suitability: The reference solution shows a slight brown colour compared to the blank solution.  

Result: Any brown colour in the test solution is not more intense than that in the reference solution.

If the result is difficult to judge, filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying moderate and constant pressure to the piston. Compare the spots on the filters obtained with the different solutions.

METHOD B

Test solution: 12 mL of the prescribed solution of the substance to be examined prepared using an organic solvent containing a minimum percentage of water (for example, dioxan containing 15 per cent of water or acetone containing 15 per cent of water).

Reference solution (standard): A mixture of 10 mL of lead standard solution (1 or 2 ppm Pb), as prescribed, and 2 mL of the prescribed solution of the substance to be examined in an organic solvent. Prepare the lead standard solution (1 or 2 ppm Pb) by dilution of lead standard solution (100 ppm Pb) with the solvent used for the  substance to be examined.

Blank solution: A mixture of 10 mL of the solvent used for the substance to be  examined and 2 mL of the prescribed solution of the substance to be examined in an  organic solvent.

Procedure: To each solution, add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately. Examine the solutions after 2 min.  

System suitability: The reference solution shows a slight brown colour compared to the blank solution.  

Result: Any brown colour in the test solution is not more intense than that in the reference solution.  

If the result is difficult to judge, filter the solutions through a suitable membrane filter  (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying  moderate and constant pressure to the piston. Compare the spots on the filters obtained with the different solutions.

METHOD C

  • Solution Preparation:
    • Dilute sulfuric acid: Add 5.5 mL of sulfuric acid to 60 mL of water, allow to cool and dilute to 100 mL with the same solvent.
    • Dilute hydrochloric acid: Dilute 16.9 ml of hydrochloric acid in 100 ml water.
    • Lead standard solution (10 ppm Pb) : See Standard Solutions For Limit Tests
    • 250 g/L solution of magnesium sulfate in dilute sulfuric acid: Dissolve 25 gm of magnesium sulfate in 100 ml dilute sulfuric acid

Test solution: Place the prescribed quantity (not more than 2 g) of the substance  to be examined in a silica crucible with 4 mL of a 250 g/L solution of magnesium sulfate in dilute sulfuric acid. Mix using a fine glass rod. Heat cautiously. If the  mixture is liquid, evaporate gently to dryness on a water-bath. Progressively heat to ignition and continue heating until an almost white or at most greyish residue is  obtained. Carry out the ignition at a temperature not exceeding 800 °C. Allow to cool. Moisten the residue with a few drops of dilute sulfuric acid. Evaporate, ignite again  and allow to cool. The total period of ignition must not exceed 2 h. Take up the residue in 2 quantities, each of 5 mL, of dilute hydrochloric acid. Add 0.1 mL of  phenolphthalein solution, then concentrated ammonia until a pink colour is  obtained. Cool, add glacial acetic acid until the solution is decolorised and add 0.5 mL in excess. Filter if necessary and wash the filter. Dilute to 20 mL with water.  

Reference solution (standard): Prepare as described for the test solution, using  the prescribed volume of lead standard solution (10 ppm Pb)  instead of the  substance to be examined. To 10 mL of the solution obtained add 2 mL of the test solution.

Monitor solution: Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) prescribed for preparation of the reference solution. To 10 mL of the solution obtained add 2 mL of the test solution.

Blank solution: A mixture of 10 mL of water and 2 mL of the test solution.

Procedure: To 12 mL of each solution, add 2 mL of buffer solution pH 3.5. Mix and add to 1.2  mL of thioacetamide reagent. Mix immediately. Examine the solutions after 2 min.  

  •  System suitability: 
    •  The reference solution shows a slight brown colour compared to the blank  solution,
    •  The monitor solution is at least as intense as the reference solution.

Result: Any brown colour in the test solution is not more intense than that in the reference solution.

If the result is difficult to judge, filter the solutions through a suitable membrane filter  (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying  moderate and constant pressure to the piston. Compare the spots on the filters  obtained with the different solutions.

METHOD D

Test solution: In a silica crucible, mix thoroughly the prescribed quantity of the substance to be examined with 0.5 g of magnesium oxide. Ignite to dull redness until a homogeneous white or greyish-white mass is obtained. If after 30 min of ignition the mixture remains coloured, allow to cool, mix using a fine glass rod and repeat the ignition. If necessary repeat the operation. Heat at 800 °C for about 1 h. Take up the residue in 2 quantities, each of 5 mL, of a mixture of equal volumes of  hydrochloric acid  and water. Add 0.1 mL of phenolphthalein solution and then concentrated ammonia until a pink colour is obtained. Cool, add glacial acetic acid until the solution is decolorised and add 0.5 mL in excess. Filter if necessary and wash the filter. Dilute to 20 mL with water.  

Reference solution (standard): Prepare as described for the test solution using the prescribed volume of lead standard solution (10 ppm Pb) instead of the  substance to be examined and drying in an oven at 100-105 °C. To 10 mL of the solution obtained add 2 mL of the test solution.

Monitor solution: Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) prescribed for preparation of the reference solution and drying in an oven at 100-105 °C. To 10 mL of the solution obtained add 2 mL of the test solution.

Blank solution: A mixture of 10 mL of water and 2 mL of the test solution.

Procedure: To 12 mL of each solution, add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately. Examine the solutions after 2 min.  

  • System suitability
    •  The reference solution shows a slight brown colour compared to the blank solution,
    •  The monitor solution is at least as intense as the reference solution.

Result: Any brown colour in the test solution is not more intense than that in the reference solution.

If the result is difficult to judge, filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying moderate and constant pressure to the piston. Compare the spots on the filters obtained with the different solutions.

METHOD E

Test solution: Dissolve the prescribed quantity of the substance to be examined in 30 mL of water or the prescribed volume.   

Reference solution (standard): Unless otherwise prescribed, dilute the prescribed volume of lead standard solution (1 ppm Pb) to the same volume as the test solution.

Procedure : Prepare the filtration apparatus by adapting the barrel of a 50 mL syringe without its piston to a support containing, on the plate, a membrane filter (nominal pore size 3 µm) and above it a prefilter (Figure 2.4.8.-1).  

Transfer the test solution into the syringe barrel, put the piston in place and then apply an even pressure on it until the whole of the liquid has been filtered. In opening the support and removing the prefilter, check that the membrane filter remains uncontaminated with impurities. If this is not the case replace it with another membrane filter and repeat the operation under the same conditions.

To the prefiltrate or to the prescribed volume of the prefiltrate add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately and allow to stand for 10 min and again filter as described above, but inverting the order of the filters, the liquid passing first through the membrane filter before passing through the prefilter (Figure 2.4.8.-1). The filtration must be carried out slowly and uniformly by applying moderate and constant pressure to the piston of the  syringe. After complete filtration, open the support, remove the membrane filter, and dry using filter paper.  

 In parallel, treat the reference solution in the same manner as the test solution.

Result: The colour of the spot obtained with the test solution is not more intense than that obtained with the reference solution.

METHOD F

Test solution: Place the prescribed quantity or volume of the substance to be examined in a clean, dry, 100 mL long-necked combustion flask (a 300 mL flask  may be used if the reaction foams excessively). Clamp the flask at an angle of 45°. If  the substance to be examined is a solid, add a sufficient volume of a mixture of 8 mL of sulfuric acid and 10 mL of nitric acid  to moisten the substance thoroughly; if  the substance to be examined is a liquid, add a few millilitres of a mixture of 8 mL of  sulfuric acid and 10 mL of nitric acid. Warm gently until the reaction commences, allow the reaction to subside and add additional portions of the same acid mixture, heating after each addition, until a total of 18 mL of the acid mixture has been added. Increase the amount of heat and boil gently until the solution darkens. Cool, add 2 mL of nitric acid and heat again until the solution darkens. Continue the heating, followed by the addition of nitric acid until no further  darkening occurs, then heat strongly until dense, white fumes are produced. Cool,  cautiously add 5 mL of water, boil gently until dense, white fumes are produced  and continue heating to reduce to 2-3 mL. Cool, cautiously add 5 mL of water and  examine the colour of the solution. If the colour is yellow, cautiously add 1 mL of  strong hydrogen peroxide solution and again evaporate until dense, white fumes  are produced and reduce to a volume of 2-3 mL. If the solution is still yellow in colour, repeat the addition of 5 mL of water and 1 mL of strong hydrogen peroxide solution until the solution is colourless. Cool, dilute cautiously with water and  rinse into a 50 mL colour comparison tube, ensuring that the total volume does not  exceed 25 mL. Adjust the solution to pH 3.0-4.0, using short range pH indicator paper as external indicator, with concentrated ammonia (dilute ammonia may be used, if desired, as the specified range is approached), dilute with water to 40  mL and mix. Add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately. Dilute to 50 mL with water and mix.  

Reference solution (standard): Prepare at the same time and in the same manner as the test solution, using the prescribed volume of lead standard solution (10 ppm Pb).  

Monitor solution: Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) prescribed for the preparation of the reference solution.

Blank solution: Prepare as described for the test solution, omitting the substance  to be examined.

Examine the solutions vertically against a white background after 2 min.  

  •  System suitability:
    • the reference solution shows a brown colour compared to the blank solution,
    • the monitor solution is at least as intense as the reference solution.

Result: Any brown colour in the test solution is not more intense than that in the  reference solution.

If the result is difficult to judge, filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying  moderate and constant pressure to the piston. Compare the spots on the filters  obtained with the different solutions.

METHOD G

CAUTION: when using high-pressure digestion vessels the safety precautions and operating instructions given by the manufacturer must be followed. The digestion cycles have to be elaborated depending on the type of microwave oven to be used  (for example, energy-controlled microwave ovens, temperature-controlled microwave  ovens or high-pressure ovens). The cycle must conform to the manufacturer’s instructions. The digestion cycle is suitable if a clear solution is obtained.

Test solution: Place the prescribed amount of the substance to be examined (not more than 0.5 g) in a suitable, clean beaker. Add successively 2.7 mL of sulfuric  acid, 3.3 mL of nitric acid and 2.0 mL of strong hydrogen peroxide solution using a magnetic stirrer. Allow the substance to react with a reagent before adding the next one. Transfer the mixture to a dry high-pressure-resistant digestion vessel  (fluoropolymer or quartz glass).

Reference solution (standard): Prepare as described for the test solution, using  the prescribed volume of lead standard solution (10 ppm Pb) instead of the  substance to be examined.

Monitor solution: Prepare as prescribed for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) prescribed for the preparation of the reference solution.

Blank solution: Prepare as described for the test solution, omitting the substance  to be examined.

Close the vessels and place in a laboratory microwave oven. Digest using a sequence of 2 separate suitable programmes. Design the programmes in several steps in order to control the reaction, monitoring pressure, temperature or energy  depending on the type of microwave oven available. After the first programme allow  the digestion vessels to cool before opening. Add to each vessel 2.0 mL of strong hydrogen peroxide solution and digest using the second programme. After the  second programme allow the digestion vessels to cool before opening. If necessary to obtain a clear solution, repeat the addition of strong hydrogen peroxide solution  and the second digestion programme.

Cool, dilute cautiously with water and rinse into a flask, ensuring that the total  volume does not exceed 25 mL.

Using short-range pH indicator paper as external indicator, adjust the solutions to pH 3.0-4.0 with concentrated ammonia (dilute ammonia may be used as the  specified range is approached). To avoid heating of the solutions use an ice-bath and a magnetic stirrer. Dilute to 40 mL with water and mix. Add 2 mL of buffer solution pH 3.5. Mix and add to 1.2 mL of thioacetamide reagent. Mix immediately. Dilute to 50 mL with water, mix and allow to stand for 2 min.

Filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying moderate and constant  pressure to the piston. Compare the spots on the filters obtained with the different solutions.  

  • System suitability 
    • the spot obtained with the reference solution shows a brown colour compared to the spot obtained with the blank solution,
    • the spot obtained with the monitor solution is at least as intense as the spot obtained with the reference solution.

Result: The brown colour of the spot obtained with the test solution is not more intense than that of the spot obtained with the reference solution.

METHOD H

Test solution: Dissolve the prescribed quantity of the substance to be examined in 20 mL of the solvent or solvent mixture prescribed.

Reference solution: Dilute the prescribed volume of lead standard solution (10  ppm Pb) to 20 mL with the solvent or solvent mixture prescribed.

Blank solution: 20 mL of the solvent or solvent mixture prescribed.

Procedure: To each solution, add 2 mL of buffer solution pH 3.5. Mix. (In some cases precipitation occurs, in which case the specific monograph would describe re- dissolution in a defined volume of a given solvent.) Add to 1.2 mL of thioacetamide reagent. Mix immediately and allow to stand for 2 min. Filter the solutions through  a suitable membrane filter (nominal pore size 0.45 µm). Compare the spots on the  filters obtained with the different solutions.

System suitability: The spot obtained with the reference solution shows a brownish-black colour compared to the spot obtained with the blank solution.  

Result: The brownish-black colour of the spot obtained with the test solution is not more intense than that of the spot obtained with the reference solution.

Limit Test for Iron
  • Solution Preparation:

Procedure: Dissolve the prescribed quantity of the substance to be examined in water and dilute to 10 mL with the same solvent or use 10 mL of the prescribed solution. Add 2 mL of a 200 g/L solution of citric acid and 0.1 mL of thioglycollic acid. Mix, make alkaline with ammonia and dilute to 20 mL with water. Prepare a standard in the same manner, using 10 mL of iron standard solution (1 ppm Fe).

Results: After 5 min, any pink colour in the test solution is not more intense than that in the standard.

Limit Test for Lead in Sugars

 Determine the lead by atomic absorption spectrometry.

  • Solution Preparation:
    • 10 g/L solution of ammonium pyrrolidinedithiocarbamate: Dissolve 1 g of ammonium pyrrolidinedithiocarbamate in 100 ml water.

Test solution: Dissolve 20.0 g of the substance to be examined in a mixture of  equal volumes of dilute acetic acid and water and dilute to 100.0 mL with the  same mixture of solvents. Add 2.0 mL of a clear 10 g/L solution of ammonium pyrrolidinedithiocarbamate and 10.0 mL of methyl isobutyl ketone and then shake for 30 s protected from bright light. Allow the layers to separate and use the  methyl isobutyl ketone layer.

Reference solutions: Prepare 3 reference solutions in the same manner as the test solution but adding 0.5 mL, 1.0 mL and 1.5 mL respectively of lead standard solution (10 ppm Pb) in addition to the 20.0 g of the substance to be examined.

Procedure: Set the zero of the instrument using methyl isobutyl ketone treated as described  for the test solution without the substance to be examined. Measure the absorbance  at 283.3 nm using a lead hollow-cathode lamp as source of radiation and an air- acetylene flame.

Results: The substance to be examined contains not more than 0.5 ppm of lead, unless  otherwise prescribed.

Limit Test for Magnesium
  • Solution preparation:
    • Dilute hydrochloric acid: Dilute 16.9 ml of hydrochloric acid in 100 ml water.
    • Dilute sodium hydroxide solution: Dissolve 8.5 g of sodium hydroxide in water and dilute to 100 mL with the same solvent.  
    • Magnesium standard solution (10 ppm Mg): See Standard Solutions For Limit Tests
    • 1 g/L solution of hydroxyquinoline in chloroform: Dissolve 0.1 g of hydroxyquinoline in 100 ml of chloroform

Procedure: To 10 mL of the prescribed solution add 0.1 g of disodium tetraborate. Adjust the  solution, if necessary, to pH 8.8 to pH 9.2 using dilute hydrochloric acid or dilute sodium hydroxide solution. Shake with 2 quantities, each of 5 mL, of a 1 g/L solution of hydroxyquinoline in chloroform, for 1 min each time. Allow to stand. Separate and discard the organic layer. To the aqueous solution add 0.4 mL of butylamine and 0.1 mL of triethanolamine. Adjust the solution, if necessary, to pH 10.5 to pH 11.5. Add 4 mL of the solution of hydroxyquinoline in chloroform, shake for 1 min, allow to stand and separate. Use the lower layer for comparison. Prepare a standard in the same manner using a mixture of 1 mL of magnesium standard solution (10 ppm Mg) and 9 mL of water.

Results: Any colour in the solution obtained from the substance to be examined is not more  intense than that in the standard.

Limit Test for Magnesium and Alkaline-earth Metals
  • Solution preparation:
    • Ammonium chloride buffer solution pH 10.0 : Dissolve 5.4 g of ammonium chloride in 20 mL of water,  add 35.0 mL of ammonia  and dilute to 100.0 mL with water.
    • Mordant Black 11 Triturate: Mix 1 g of mordant black 11 with 99 g of sodium chloride.
    • 0.01 M sodium edetate solution: Dissolve 3.722 g of disodium edetate in sufficient water to produce 1000 mL.
    • 0.1 M zinc sulfate: Dissolve 29 g of zinc sulfate in sufficient water to produce 1000 mL.

Procedure: To 200 mL of water add 0.1 g of hydroxylamine hydrochloride ,10 mL of ammonium chloride buffer solution pH 10.0 , 1 mL of 0.1 M zinc sulfate and about 15 mg of mordant black 11 triturate. Heat to about 40 °C. Titrate with 0.01 M sodium edetate until the violet colour changes to full blue. To the solution add the prescribed quantity of the substance to be examined dissolved in 100 mL of water  or use the prescribed solution. If the colour of the solution changes to violet, titrate with 0.01 M sodium edetate until the full blue colour is again obtained.

The volume of 0.01 M sodium edetate used in the second titration does not exceed the prescribed quantity.

Limit Test for Nickel in Polyols

Determine the nickel by atomic absorption spectrometry

  • Solution preparation:

Test solution: Dissolve 20.0 g of the substance to be examined in a mixture of equal volumes of dilute acetic acid and water and dilute to 100.0 mL with the same mixture of solvents. Add 2.0 mL of a saturated solution of ammonium pyrrolidinedithiocarbamate (about 10 g/L) and 10.0 mL of methyl isobutyl ketone and then shake for 30 s protected from bright light. Allow the layers to separate and use the methyl isobutyl ketone layer.

Reference solutions: Prepare 3 reference solutions in the same manner as the test solution but adding 0.5 mL, 1.0 mL and 1.5 mL respectively of nickel standard solution (10 ppm Ni) in addition to the 20.0 g of the substance to be examined.

Procedure: Set the zero of the instruments using methyl isobutyl ketone treated as described for preparation of the test solution omitting the substance to be examined. Measure the absorbance at 232.0 nm using a nickel hollow-cathode lamp as source of radiation and an air-acetylene flame.

Results: The substance to be examined contains not more than 1 ppm of nickel, unless otherwise prescribed.

Limit Test for Phosphates
  • Solution preparation:
    • Phosphate standard solution (5 ppm PO4) : See Standard Solutions For Limit Tests
    • Sulphomolybdic reagent: Dissolve with heating 2.5 g of ammonium molybdate in 20 mL of water. Dilute 28 mL of sulfuric acid  in 50 mL of water, then  cool. Mix the two solutions and dilute to 100 mL with water.
    • Stannous Chloride Solution: Heat 20 g of tin with 85 mL of hydrochloric acid until no more  hydrogen is released. Allow to cool

Procedure: To 100 mL of the solution prepared and, if necessary, neutralised as prescribed add 4 mL of sulfomolybdic reagent Shake and add 0.1 mL of stannous chloride solution Prepare a standard in the same manner using 2 mL of phosphate standard solution (5 ppm PO4) and 98 mL of water. After 10 min, compare the colours using 20 mL of each solution.

 Results: Any colour in the test solution is not more intense than that in the standard.

Limit Test for Potassium
  • Solution preparation:
    • Potassium standard solution (20 ppm K): See Standard Solutions For Limit Tests
    • 10 g/L solution of sodium tetraphenylborate: Dissolve 1.0 gm of sodium tetraphenylborate in 100 ml water.

Procedure: To 10 mL of the prescribed solution add 2 mL of a freshly prepared 10 g/L solution of sodium tetraphenylborate. Prepare a standard in the same manner using a mixture  of 5 mL of potassium standard solution (20 ppm K) and 5 mL of water.

Results: After 5 min, any opalescence in the test solution is not more intense than that in the  standard.

Limit Test for Sulfates
  • Solution preparation:

 Note: All solutions used for this test must be prepared with distilled water.

Procedure: Add 3 mL of a 250 g/L solution of barium chloride to 4.5 mL of sulfate standard solution (10 ppm SO4). Shake and allow to stand for 1 min. To 2.5 mL of this suspension, add 15 mL of the solution to be examined and 0.5 mL of acetic acid.  Prepare a standard in the same manner using 15 mL of sulfate standard solution (10 ppm SO4) instead of the solution to be examined.

Results: After 5 min, any opalescence in the test solution is not more intense than that in the standard.

Reference : British Pharmacopoeia