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The need for a quantitative determination of zinc in the Pharmacopeial insulin preparations reflects the fact that the element is an essential component of zinc-insulin crystals. In common with lead, zinc may be determined either by the dithizone method or by atomic absorption.

Dithizone Method
Select all reagents for this test to have as low a content of heavy metals as practicable. If necessary, distill water and other solvents into hard or borosilicate glass apparatus. Rinse thoroughly all glassware with warm dilute nitric acid (1 in 2) followed by water. Avoid using on the separator any lubricants that dissolve in chloroform.
Special Solutions and Solvents—
ALKALINE AMMONIUM CITRATE SOLUTION— Dissolve 50 g of dibasic ammonium citrate in water to make 100 mL. Add 100 mL of ammonium hydroxide. Remove any heavy metals that may be present by extracting the solution with 20-mL portions of Dithizone Extraction Solution (see Lead 251) until the dithizone solution retains a clear green color, then extract any dithizone remaining in the citrate solution by shaking with chloroform.
CHLOROFORM— Distill chloroform in hard or borosilicate glass apparatus, receiving the distillate in sufficient dehydrated alcohol to make the final concentration 1 mL of alcohol for each 100 mL of distillate.
DITHIZONE SOLUTION— Use Standard Dithizone Solution (see Lead 251), prepared with the distilled Chloroform.
STANDARD ZINC SOLUTION— Dissolve 625 mg of zinc oxide, accurately weighed, and previously gently ignited to constant weight, in 10 mL of nitric acid, and add water to make 500.0 mL. This solution contains 1.0 mg of zinc per mL.
DILUTED STANDARD ZINC SOLUTION— Dilute 1 mL of Standard Zinc Solution, accurately measured, with 2 drops of nitric acid and sufficient water to make 100.0 mL. This solution contains 10 µg of zinc per mL. Use this solution within 2 weeks.
TRICHLOROACETIC ACID SOLUTION— Dissolve 100 g of trichloroacetic acid in water to make 1000 mL.
Procedure— Transfer 1 to 5 mL of the preparation to be tested, accurately measured, to a centrifuge tube graduated at 40 mL. If necessary, add 0.25 N hydrochloric acid, dropwise, to obtain a clear solution. Add 5 mL of Trichloroacetic Acid Solution and sufficient water to make 40.0 mL. Mix, and centrifuge.
Transfer to a hard-glass separator an accurately measured volume of the supernatant believed to contain from 5 to 20 µg of zinc, and add water to make about 20 mL. Add 1.5 mL of Alkaline Ammonium Citrate Solution and 35 mL of Dithizone Solution. Shake vigorously 100 times. Allow the chloroform phase to separate. Insert a cotton plug in the stem of the separator to remove any water emulsified with the chloroform. Collect the chloroform extract (discarding the first portion that comes through) in a test tube, and determine the absorbance at 530 nm, with a suitable spectrophotometer.
Calculate the amount of zinc present by reference to a standard absorbance-concentration curve obtained by using 0.5 mL, 1.0 mL, 1.5 mL, and, if the zinc content of the sample extracted exceeds 15 µg, 2.0 mL of the Diluted Standard Zinc Solution, corrected as indicated by a blank determination run concomitantly, using all of the reagents but no added zinc.

Auxiliary Information—
Staff Liaison : Kahkashan Zaidi, Ph.D., Senior Scientific Associate
Expert Committee : (GC05) General Chapters 05
USP29–NF24 Page 2616
Phone Number : 1-301-816-8269