目的: 建立测定毛发样本中地西泮、去甲西泮和奥沙西泮的HPLC分析方法。方法: 采用超声法结合液液萃取法作为前处理方法。采用Athena C18-WP(250 mm×4.6 mm,5 μm)色谱柱;以甲醇-0.02 mol·L-1磷酸二氢钠溶液(磷酸调pH 3.4)-乙腈(30∶43∶27)为流动相;柱温50 ℃;流速0.7 mL·min-1;检测波长254 nm。内标为氯硝西泮。结果: 本方法在0.1~25 ng·mg-1范围内具良好的线性关系,定量限为0.1 ng·mg-1,检测限为0.05 ng·mg-1,批内准确度(n=6)在88.2%~103.9%,批间准确度(n=24)在88.5%~106.2%,精密度小于或等于9.19%,提取回收率稳定,符合方法学要求。将该方法应用于实际病人的毛发样本,测得地西泮及其代谢物去甲西泮、奥沙西泮浓度分别是(0.307±0.016)ng·mg-1、(0.244±0.012)ng·mg-1和(0.478±0.053) ng·mg-1,其中C去甲西泮/C地西泮为0.795,与文献中报道相符。结论: 优化后的方法操作快捷简便,重现性好,具适用性和可靠性。
Objective: To establish an HPLC analysis method for the determination of diazepam, nordiazepam and oxazepam in hair samples. Methods: The determination of drug content and related substances was performed by high performance liquid chromatography (HPLC). Ultrasonic method combined with liquid-liquid extraction method was used as the pretreatment method. The chromatographic column was Athena C18-WP column (250 mm×4.6 mm, 5 μm); the mobile phase was the mixture of methanol: 0.02 mol·L-1 sodium dihydrogen phosphate (adjusted to pH 3.4 with phosphoric acid): acetonitrile (30∶43∶27, v/v/v) in an isometric elution; the column temperature was 50 ℃; the flow rate was 0.7 mL·min-1; the running time was 30 min; the injection volume was 20 μL. The detection wavelength was 254 nm. The internal standard was clonazepam. Results: The linearity of all the analytes were good in the concentration range of 0.1 to 25 ng·mg-1(r>0.995). The limit of quantitative was 0.1 ng·mg-1 and the limit of detection was 0.05 ng·mg-1. The intra-batch accuracies were 88.2%-103.9% (n=6) and the inter-batch accuracies were 88.5%-106.2% with imprecisions ≤9.19%. The recoveries were stable for three anlytes, which met the requirement of methodology. The present method was applied to the authetic hair samples, in which the concentrations of diazepam, norazepam and oxazepam were (0.307±0.016) ng·mg-1, (0.244±0.012) ng·mg-1 and (0.478±0.053) ng·mg-1 respectively. Besides, Cnorazepam/Cdiazepam was 0.795, which was in consistent with the literature reported. Conclusion: The established HPLC method is fast and easy to operate, with good reproducibility, applicability, and reliability.
[1] LÓPEZ-MUÑOZ F, ALAMO C, GARCÍA-GARCÍA P. The discovery of chlordiazepoxide and the clinical introduction of benzodiazepines: half a century of anxiolytic drugs[J]. J Anxiety Disord, 2011, 25(4): 554
[2] 叶晓楠, 向甜甜, 王艳艳, 等. 炒酸枣仁对地西泮诱导的睡眠及宿醉效应的影响[J]. 中南药学, 2021, 19(11): 2280
YE XN, XIANG TT, WANG YY, et al. Effect of fried Semen Ziziphi spinosae on sleep and hangover induced by diazepam in mice[J]. Cent South Pharm, 2021, 19(11): 2280
[3] 柯浩堃, 吕赛男, 郝红霞, 等. 苯二氮卓类化合物的检测方法研究进展[J]. 分析试验室, 2020, 39(9): 1110
KE HK, LÜ SN, HAO HX, et al. Advances in the detection of benzodiazepines[J]. Chin J Anal Lab, 2020, 39(9): 1110
[4] MADRY MM, KRAEMER T, BAUMGARTNER MR. Large scale consumption monitoring of benzodiazepines and z-drugs by hair analysis[J]. J Pharm Biomed Anal, 2020, 183: 113
[5] 蒋文慧, 王朝虹, 赵蒙, 等. 新型微流体液相色谱(micro-LC)串联四极杆质谱检测头发中5种苯二氮卓类药物[J]. 中国法医学杂志, 2020, 35(1): 29
JIANG WH, WANG CH, ZHAO M, et al. Ultrasensitive analysis of 5 benzodiazepines in hair samples using microfluidic tile technology and LC-MS/MS[J]. Chin J Forensic Med, 2020, 35(1): 29
[6] MORINI L, POZZI F, GROPPI A. Stability of benzodiazepines in hair after prolonged exposure to chlorinated water[J]. Forensic Sci Int, 2017, 278: 217
[7] KIM J, IN S, CHOI H, et al. Illegal use of benzodiazepines and/or zolpidem proved by hair analysis[J]. J Forensic Sci, 2013, 58(2): 548
[8] COBO-GOLPE M, DE-CASTRO-RÍOS A, CRUZ A, et al. Determination of antidepressants and benzodiazepines in paired hair and nail samples[J]. Forensic Sci Int, 2021, 326: 110935
[9] SHIN Y, KONG TY, CHEONG JC, et al. Simultaneous determination of 75 abuse drugs including amphetamines, benzodiazepines, cocaine, opioids, piperazines, zolpidem and metabolites in human hair samples using liquid chromatography-tandem mass spectrometry[J]. Biomed Chromatogr, 2019, 33(9): e4600
[10] WANG X, JOHANSEN SS, NIELSEN MKK, et al. Targeted analysis of 116 drugs in hair by UHPLC-MS/MS and its application to forensic cases[J]. Drug Test Anal, 2017, 9(8): 1137
[11] KIM J, IN S, CHOI H, et al. Illegal use of benzodiazepines and/or zolpidem proved by hair analysis[J]. J Forensic Sci, 2013, 58(2): 548
[12] ESMAEILI-SHAHRI E, ES'HAGHI Z. Superparamagnetic Fe3O4@SiO2 core-shell composite nanoparticles for the mixed hemimicelle solid-phase extraction of benzodiazepines from hair and wastewater samples before high-performance liquid chromatography analysis[J]. J Sep Sci, 2015, 38(23): 4095
[13] ES'HAGHI Z, NEZHADALI A, BAHAR S, et al. [PMIM]Br@TiO2 nanocomposite reinforced hollow fiber solid/liquid phase microextraction:An effective extraction technique for measurement of benzodiazepines in hair, urine and wastewater samples combined with high-performance liquid chromatography[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2015, 980: 55
[14] PEREZ ER, KNAPP JA, HORN CK, et al. Comparison of LC-MS-MS and GC-MS analysis of benzodiazepine compounds included in the drug demand reduction urinalysis program[J]. J Anal Toxicol, 2016, 40(3): 201
[15] 乐浩鸣. 人体毛发中硝甲西泮的HPLC-MS/MS检测[J]. 刑事技术, 2021, 46(2): 178
LE HM. HPLC-MS/MS Identifying nimetazepam in human hair[J]. Forensic Sci Tech, 2021, 46(2): 178
[16] 沈敏, 向平. 毛发分析基础及应用[M]. 北京: 科学出版社, 2020: 112, 363
SHEN M, XIANG P. Fundamentals and Applications of Hair Analysis[M]. Beijing: Science Press, 2020: 112, 363
[17] MAHJOUB AE, STAUB C. Determination of benzodiazepines in human hair by on-line high-performance liquid chromatography using a restricted access extraction column[J]. Forensic Sci Int, 2001, 123: 17
[18] VILLAIN M, CONCHEIRO M, CIRIMELE V, et al. Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2005, 825(1): 72
