For the quantification of the sedative and anesthetic drug midazolam and its main (active) metabolites 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, human EDTA plasma, human heparin plasma and human urine a single accurate method by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed. Protein precipitation as sample preparation, without the need of a time-consuming deglucuronidation step for the quantification of 1-hydroxymidazolam glucuronide, resulted in a simple and rapid assay suitable for clinical practice with a total runtime of only 1.1 min. The four components and the isotope-labeled internal standards were separated on a C₁₈ column and detection was performed with a triple-stage quadrupole mass spectrometer operating in positive ionization mode.

The method was validated based on the “Guidance for Industry Bioanalytical Method Validation” (Food and Drug Administration, FDA) and the “Guideline on bioanalytical method validation” of the European Medicines Agency (EMA). Linearity was proven over the ranges of 5–1500 μg/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 25–5000 μg/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 μL. Matrix comparison indicated that the assay is also applicable to other human matrices like EDTA and heparin plasma and urine. Stability experiments showed good results for the stability of midazolam, 1-hydroxymidazolam and 1-hydroxymidazolam glucuronide in serum, EDTA and heparin plasma and urine stored for 7 days under different conditions. At room temperature, 4-hydroxymidazo-lam is stable for 7 days in EDTA plasma, but stable for only 3 days in serum and heparin plasma and less than 24 h in urine. All four compounds were found to be stable in serum, EDTA plasma, heparin plasma and urine for 7 days after sample preparation and for 3 freeze–thaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.

为了定量测定人血清，人EDTA血浆，人肝素血浆和人尿液中镇静和麻醉药咪达唑仑及其主要（活性）代谢物1-羟基咪达唑仑，4-羟基咪达唑仑和1-羟基咪达唑仑葡糖醛酸的单一准确方法高效液相色谱-串联质谱（UHPLC-MS / MS）已开发出来。无需进行费时的去葡糖醛酸化步骤即可定量1-羟基咪达唑仑葡糖醛酸化物，即可将蛋白质沉淀作为样品前处理，从而实现了简单，快速的测定方法，适用于临床实践，总运行时间仅为1.1分钟。四个成分和同位素标记的内标在C ₁₈上分离用正离子化模式运行的三级四极杆质谱仪进行检测。

该方法已根据“工业生物分析方法验证指南”（食品和药物管理局，FDA）和欧洲药品管理局（EMA）的“生物分析方法验证指南”进行了验证。使用100μL的样品量，对咪达唑仑，1-羟基咪达唑仑和4-羟基咪达唑仑的线性证明在5–1500μg/ L范围内，而1-羟基咪达唑仑葡糖醛酸在25–5000μg/ L的范围内。基质比较表明，该测定法还适用于其他人类基质，如EDTA，肝素血浆和尿液。稳定性实验表明，咪达唑仑，1-羟基咪达唑仑和1-羟基咪达唑仑葡糖苷酸在不同条件下的血清，EDTA和肝素血浆及尿液中的稳定性良好，结果为7天。在室温下，4-羟基咪唑-lam在EDTA血浆中稳定7天，但在血清和肝素血浆中仅稳定3天，而在尿中少于24小时。样品制备后7天和3个冻融循环中，所有四种化合物在血清，EDTA血浆，肝素血浆和尿液中均稳定。该测定法已用于咪达唑仑对（儿科）重症监护患者的治疗药物监测。