전혈과 소변에서 액체-액체추출법과 UPLC-MS/MS에 의한Benzodiazepines 및 그 대사체들의 확인과 정량
Identification and Quantification of Benzodiazepines and Their Metabolites by Liquid-Liquid Extraction and UPLC-MS/MS in Whole Blood and Urine
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전혈과 소변에서 benzodiazepines와 그 대사체들, zolpidem 및 zopiclone의 확인과 정량을 액체-액체추출법(LLE)과 초고성능 액체크로마토그래피/탄뎀질량분석법(UPLC-MS/MS)으로 수행하였다. 이약물들은 2M Trizma-HCl (pH 9.2)로 완충된 전혈 또는 소변으로부터 1 mL 1-chlorobutane을 사용하여추출하고 UPLC로 분리하였다. 확인시험은 MRM 모드에서 머무름 시간, 정량이온 대 타겟이온의 이온세기비로 결정하였다. 이 시험방법은 미국 FDA와 한국 식품의약품안전처 및 법독성학 분야에서 권고하는 생체시료에 대한 시험방법 유효화에 대한 가이드라인을 적용하여 검증하였다. 시험방법 유효성은선택성, LLOQ, 작업범위, LOD, 직선성, 정확도, 정밀도, 매질효과, 회수율, 단기 안정성, 냉·해동 안정성 및 장기간 저장 안정성을 확인하였다. 결론적으로 법독성학에서 benzodiazepines와 그 대사체들, zolpidem 및 zipiclone 등을 정확하게 확인하고 정량하는 시험방법과 분석과정이 유용함을 증명하였다
Abstract
The confirmation and quantification of benzodiazepines and their metabolites, zolpidem and zopiclone in whole blood and urine were carried out by liquid-liquid extraction (LLE) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). These drugs were extracted from 0.2 mL whole blood or urine buffered with 2M Trizma-HCl (pH 9.2) using 1 mL 1-chlorobutane and were separated by UPLC. The confirmation was determined by retention time and ion ratio of quant. ion to target ion in MRM mode. This method was verified by the guideline of bioanalytical method validation recommended by US FDA, Korean FDA and the field of forensic toxicology. The method validation are confirmed by the selectivity, LLOQ, working range, LOD, linearity, accuracy, precision, matrix effect, recovery, processed sample stability, freeze/thaw stability, and long term stability. In conclusion, this analytical method and procedure have been proven to be useful for the routine and accurate confirmation and quantification of benzodiazepines and metabolites, zolpidem and zopiclone for the forensic toxicology.
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