He W, Parissis N
Laboratory of Toxicology, University of Ghent, Belgium.
J Pharm Biomed Anal. 1997 Dec;16(4):707-15. doi: 10.1016/s0731-7085(97)00185-4.
A high performance liquid chromatography (HPLC) assay was developed for the determination of flunitrazepam (FNZ) and its metabolites in urine and plasma. The analytes and the internal standard (triazolam, TRZ) were extracted by Sep-Pak C18 SPE-cartridge and separated utilizing a 5 microm ChromSpher C8 glass column with a gradient mobile phase containing methanol and 0.125% (v/v) of isopropylamine in water. Diode array detection (DAD) was carried out at a monitoring wavelength of 240 nm and a reference wavelength of 550 nm. Standard curves were linear from their quantitation limits until 200 ng ml(-1) urine or 250 ng ml(-1) plasma for 7-amino-desmethyl-flunitrazepam (ADF), 7-amino-flunitrazepam (AF), 7-acetamino-flunitrazepam (ACF) and until 400 ng ml(-1) urine or 500 ng ml(-1) plasma for FNZ, 1-desmethyl-flunitrazepam (DF), and 3-hydroxyl-flunitrazepam (HF). The intraday and interday coefficients of variation ranged from 2.04 to 9.07% and from 2.64 to 14.10%, respectively in urine and from 5.13 to 8.60% and from 7.27 to 10.46%, respectively in plasma. The developed method is used in forensic toxicology and is also applicable to pharmacokinetic studies in man.
建立了一种高效液相色谱（HPLC）分析法，用于测定尿液和血浆中的氟硝西泮（FNZ）及其代谢产物。分析物和内标（三唑仑，TRZ）通过Sep-Pak C18固相萃取柱提取，并使用5微米的ChromSpher C8玻璃柱进行分离，流动相为含甲醇和0.125%（v/v）异丙胺的水的梯度洗脱。二极管阵列检测（DAD）在监测波长240 nm和参比波长550 nm下进行。对于7-氨基去甲基氟硝西泮（ADF）、7-氨基氟硝西泮（AF）、7-乙酰氨基氟硝西泮（ACF），标准曲线在定量限至200 ng/ml尿液或250 ng/ml血浆范围内呈线性；对于FNZ、1-去甲基氟硝西泮（DF）和3-羟基氟硝西泮（HF），标准曲线在定量限至400 ng/ml尿液或500 ng/ml血浆范围内呈线性。日内和日间变异系数在尿液中分别为2.04%至9.07%和2.64%至14.10%，在血浆中分别为5.13%至8.60%和7.27%至10.46%。所建立的方法用于法医毒理学，也适用于人体药代动力学研究。
