ZINC DETERMINATION

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 INTRODUCTION 

A quantitative determination of zinc in drug substance and drug product monographs containing zinc is achieved by the Dithizone Method, Ion Chromatographic Method, or Atomic Absorption Method. Unless a specific method is indicated in the individual monograph, any of these methods can be used. 

2 PROCEDURE 

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 a hard or borosilicate glass apparatus. Rinse thoroughly all glassware with warm diluted 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 the Dithizone extraction solution (see Lead 〈251〉) until the Dithizone extraction solution retains a clear green color, then extract any dithizone remaining in the citrate solution by shaking with chloroform. 

Chloroform: Distill chloroform in a hard or borosilicate glass apparatus, receiving the distillate in sufficient dehydrated alcohol to make the nal concentration 1 mL of alcohol for each 100 mL of distillate. 

Dithizone solution: Use the Standard dithizone solution (see 〈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/mL of zinc. 

Diluted standard zinc solution: Dilute 1 mL of the Standard zinc solution, accurately measured, with 2 drops of nitric acid and sufficient water to make 100.0 mL. This solution contains 10 µg/mL of zinc. Use this solution within 2 weeks. 

Trichloroacetic acid solution: Dissolve 100 g of trichloroacetic acid in water to make 1000 mL. 

Procedure: Transfer 1–5 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 the 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 5–20 µg of zinc, and add water to make about 20 mL. Add 1.5 mL of the Alkaline ammonium citrate solution and 35 mL of the 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, 1.0, 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. 

Ion Chromatographic Method 

The following ion chromatographic general procedure is provided for the determination of zinc in compendial articles. See Ion Chromatography 〈1065〉 for discussion of the theory and principles of measurements using ion chromatography. 

Use water with a resistivity of not less than 18 megohm-cm to prepare the solutions. 

Diluent: 0.2% (w/v) hydrochloric acid 

Mobile phase: 7.0 mM dipicolinic acid, 66.0 mM potassium hydroxide, 5.6 mM potassium sulfate, and 74.0 mM formic acid in water; adjust with 2 N potassium hydroxide TS to a pH of 4.2. Pass through a suitable filter of 0.2-µm pore size. 

Post-column derivatization reagent: 0.5 mM 4-(2-pyridylazo)resorcinol monosodium salt, 1.0 M 2-dimethylaminoethanol, 0.50 M ammonium hydroxide, and 0.30 M sodium bicarbonate in water. Stir and sonicate until the solid is completely dissolved. Pass through a suitable filter of 0.2-µm pore size. 

Standard stock solution: 1500 µg/mL of zinc from USP Zinc oxide RS prepared as follows. Transfer an appropriate portion of USP Zinc Oxide RS to a suitable volumetric flask. Add 6 N hydrochloric acid to about 10% of the nal flask volume to dissolve. Dilute with water to volume. 

Standard solution: 15.0 µg/mL of zinc in Diluent from the Standard stock solution 

Sample stock solution: Prepare as directed in the monograph. 

Sample solution: Equivalent to 15.0 µg/mL of zinc in Diluent from the Sample stock solution, unless otherwise stated in the monograph Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: Vis 530 nm 

Columns 

Guard: 4.0-mm × 5-cm; 9-µm packing L100 

Analytical: 4.0-mm × 25-cm; 9-µm packing L100 

Column temperature: 30° 

Flow rate: 1.2 mL/min 

Flow rate of post-column reagent: 0.6 mL/min. Introduce using a pulseless flow of reagent through a 375-µL polymeric mixing coil or other suitable volume coil.¹ 

Injection volume: 10 µL 

Run time: Not less than 2 times the retention time of zinc 

System suitability 

Sample: Standard solution 

Suitability requirements 

Tailing factor: Not more than 2.0 

Relative standard deviation: Not more than 0.73%, unless otherwise stated in the monograph 

Analysis 

Samples: Standard solution and Sample solution 

Unless otherwise stated in the monograph, calculate the concentration of zinc in the portion of Sample solution taken: 

Result = (r_U/r_S) × C_S 

r_U = peak response from the Sample solution 

r_S = peak response from the Standard solution 

C_S = concentration of zinc in the Standard solution (µg/mL) 

Change to read: 

Atomic Absorption Method 

The following atomic absorption general procedure is provided for the determination of zinc in compendial articles. Diluent: 0.01 N hydrochloric acid 

Zinc stock standard solution: Use a certified 1000-µg/mL standard. 

Zinc calibration standard solutions: Prepare not less than 3 calibration standard solutions within the range of 0.2–1.6 μg/mL, or suitable concentrations to span anticipated zinc concentrations in the sample(s). Prepare these solutions by two-step dilutions of the Zinc stock standard solution with the Diluent. 

Drug substance sample solution: Accurately weigh an amount of the sample and dissolve in Diluent. Dilute with Diluent to volume in a volumetric flask; dilute the solution such that the zinc concentration is within the concentration range of the prepared Zinc calibration standard solutions. 

Drug product sample solution: If the sample is a suspension, resuspend and add 4–7 µL of 6 N hydrochloric acid,to each milliliter of the suspension  as needed to dissolve prior to dilution. Dilute the sample with Diluent to a zinc concentration within the concentration range of the prepared Zinc calibration standard solutions. 

Instrumental conditions 

(See Atomic Absorption Spectroscopy 〈852〉.) 

Mode: Atomic absorption spectrophotometry 

Analytical wavelength: 213.9 nm 

Lamp: Zinc hollow-cathode, 5–15 mA 

Flame: Air–acetylene of suitable composition 

Blank: 0.01 N hydrochloric acid 

Analysis: Zero the instrument using the Blank. Determine the absorbance in triplicate, of the Blank,   the Zinc calibration standard solutions, and the Sample solutions. 

Calibration: Prepare a calibration curve from the mean of the readings of the absorbance of the Blank and the Zinc calibration standard solutions (linear or quadratic t). 

System suitability 

Samples: Zinc calibration standard solutions 

Suitability requirements 

Correlation coefficient: NLT 0.997, determined from the standard curve 

Relative standard deviation: NMT 2.5%, for the triplicate measurements for each aspiration of the Zinc calibration standard solutions with the highest concentration  

Calculations: Read the concentration of zinc in the sample solution using the calibration curve and calculate the concentration of zinc in the sample. 

3 ADDITIONAL REQUIREMENTS 

USP Reference Standards 〈11〉 

USP Zinc Oxide RS 

1 A knitted reaction coil, part number 043700, available from ThermoFisher Scientific (www.thermosher.com), may be suitable.