Development of MEPS–UHPLC/PDA methodology for the quantification of clozapine, risperidone and their major active metabolites in human urine

Abstract

A new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the simultaneous determination of clozapine and risperidone, and their active metabolites, in urine samples. Important parameters affecting MEPS performance, including type of sorbent material (C2, C8, C18, SIL, M1, R-AX, R-CX, PGC, PEP, HLB-DVB and H-DVB), elution solvent system, number of extraction cycles, sample volume, elution volume, and sample pH, were evaluated. The optimal experimental conditions involved the loading of 500μl of sample through a C18 sorbent in a five extraction cycles and in a short time period (5min for the entire sample preparation step). The obtained extract was analyzed by UHPLC, using a binary mobile phase, composed of aqueous 0.1% formic acid and methanol in the gradient elution mode (10min total analysis). Good results were obtained under the optimized conditions, in terms of linearity with correlation coefficients (r2) values higher than 0.9986. Low detection limits, ranging between 0.052 and 0.055μg ml−1 were achieved, whereas the lowest quantification limit was 0.1μg ml−1, corresponding to the lowest concentration of the standard curve. Satisfactory results were found with accuracy values ranging from 83% to 116%, and with precision results from 2.65% to 18%. Recoveries ranged between 76% and 96%, which was in agreement with results reported by other studies.