Development of HPLC methods for determination of citrulline malate impurities

Tóm tắt

Citrulline malate (CM) is a widely sought-after ingredient in the dietary supplement industry due to its ability to enhance exercise performance and promote muscle recovery. With the rising demand for CM, stringent quality control measures are essential, particularly in monitoring residual impurities in raw materials. An HPLC method was developed for the determination of fumaric acid (FA) and maleic acid (MA) impurities in CM raw material using a Shim-pack GIST C18 column (250 × 4.6 mm, 5 µm), detection at 210 nm, injection volume of 10 µL, and flow rate of 0.8 mL/min with a mobile phase consisting of acetonitrile and 3.3 mM potassium dihydrogen phosphate buffer (pH 2.0) (gradient). Another HPLC method was developed for the determination of N-acetylornithine (AO) impurity using the same C18 column, but with a detection wavelength of 200 nm, injection volume of 20 µL, flow rate of 1.0 mL/min, and a mobile phase consisting of 10 mM octane sulfonic acid sodium salt (pH 2.5) and acetonitrile under gradient condition. The methods were validated following AOAC International, confirming their accuracy and reliability (recovery not over than 100.0 ± 5.0% for all; RSD ≤ 1.1, 2.6, 3.0 % for FA, MA, AO respectively)  in the range of concentration from 0.1 to 70 µg/mL (FA); 0.1 to 3.5 µg/mL (MA); 0.7 to 5.0 µg/mL (AO) with the coefficients very close of 1 (0.9999 for FA and MA, 0.9994 for AO). LOD, LOQ were established at 0.03, 0.1 for both FA, MA, and 0.5, 0.7 for AO.