Modified gas chromatographic-mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma.

ABSTRACT

A modified method for the determination of gacyclidine enantiomers in human plasma by GC-MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d3-gacyclidine) as internal standard was developed. Following a single-step liquid-liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d3-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (-)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (-)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) < 14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r2 > 0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were < 9% at 5, 10 and 20 ng/ml, and < 16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d3-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.