All the reagents used were chemically pure, analytical reagent grade and were used without further purification. Triple distilled water was used throughout this study. Zinc standard stock solution (1000 μg Zn/mL), 6 M and 0.01 M hydrochloric acid solutions were prepared according to the standard procedure. Zinc working solution (0.20–1.20 μg/mL of Zn) was freshly prepared by diluting zinc standard solution (1 mg/mL Zn) with 0.01 M hydrochloric acid. Humulin N Vial (5 mL, 100 IU/mL) of Lilly Pharmaceuticals, Egypt was used. Well shaken insulin vial (1.0 mL containing 100 IU of insulin) was dilute to 100.0 mL with 0.01 M hydrochloric acid.