Development of a simultaneous liquid–liquid extraction and chiral derivatization method for stereospecific GC‐MS analysis of amphetamine‐type stimulants in human urine using fractional factorial design

Abstract

A stereospecific gas chromatography-mass spectrometry analysis method for amphetamine-type stimulants in human urine was recently developed. For maximum efficiency, liquid-liquid extraction and chiral derivatization of the analytes using (R)-(-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride were performed simultaneously. The effects of (1) use of saturated sodium chloride in 2.0 M sodium hydroxide, (2) extraction solvent volume, (3) percentage of triethylamine, (4) derivatization reagent volume, (5) sample mixing time, (6) incubation temperature and (7) incubation time on method sensitivity and variability were assessed using a two-level, eight-run Plackett-Burman design followed by a fold-over design. The use of saturated sodium chloride solution and the derivatization reagent volume were significant factors (ANOVA, p<0.01). The saturated sodium chloride solution decreased sensitivity whereas an increased volume of derivatization reagent increased sensitivity. Calibration curves for all analytes were linear between 5 and 500 microg/L, with correlation coefficients of >0.99. Detection limits were <or=2.3 microg/L and quantitation limits <or=7.7 microg/L. Reproducibility was good, with relative standard deviation values at <20%. Recovery exceeded 100% for most analytes. The experimental design enabled easy and rapid identification of significant factors using a minimal number of samples. This method has good potential for studies requiring rapid and sensitive stereospecific quantification of amphetamine-type stimulants.