Development of Analytical Method for Separation and Quantification of Cysteine Hydrochloride Monohydrate, Followed by Validation with TotalError Concept by Using Ultra Performance Liquid Chromatography with Pre-Column Derivatization

摘要

The aim of the work was to develop a UPLC method to separate the Cysteine (Cys) from other amino acids (AA) for quantification and validation of this method using total error concept. Separation was performed using Waters Cortecs C18+ UPLC (2.1 × 100 mm)1.6 μm column using Waters Acquity UPLC with Tunable UV detector with gradient mobile phase contains 0.1% TFA as mobile phase A and Acetonitrile: Water (90:10) as mobile phase B with a flow rate of 0.3 mL /min at 265 nm. Sample was pre-derivatized using Fluorenylmethyloxycarbonyl chloride (FMOC-Cl). Validation using total error concept was successfully achieved for the determination of Cys in Amino acid formulations. The validated dosing range covered from 0.1 mg/mL to 0.5 mg/mL. Accuracy profile and risk profile was found to be good throughout the range. Pre-column derivatization method proposed in this study could be presented as a suitable method for separation and quantification of Cys in AA formulations. The method specificity, accuracy, precision, linearity and robustness were proved in validation, this method can be used for the separation and quantification of Cys in AA formulations.