Midazolam (MDZ) is routinely employed as a marker compound of cytochrome P450 3A (CYP3A) activity. Despite the many HPLC–UV methods described to quantify MDZ in plasma, all of them use acetonitrile (ACN) or a mixture of methanol–isopropanol as organic solvent of the mobile phase. Since the ACN shortage in 2008, efforts have been made to replace this solvent during HPLC analysis. A simple, sensitive, accurate and repeatable HPLC–UV method (220 nm) was developed and validated to quantify MDZ in rat plasma using methanol instead. The method was applied during a herb–drug interaction study involving Maytenus ilicifolia, a Brazilian folk medicine used to treat gastric disorders. Plasma samples were alkalinized and MDZ plus alprazolam (internal standard) were extracted with diethyl ether. After solvent removal, the residue was reconstituted with methanol–water (1:1). The analyte was eluted throughout a C₁₈ column using sodium acetate buffer (10 mm, pH 7.4)–methanol (40:60, v/v). The precision at the lower limit of quantification never exceeded 19.40%, and 13.86% at the higher levels of quality control standards, whereas the accuracy ranged from −19.81 to 14.33%. The analytical curve was linear from 50 to 2,000 ng/ml. The activity of the hepatic CYP3A enzymes was not affected by the extract.

中文翻译：

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高效液相色谱法在大鼠血浆中咪达唑仑定量的验证：在Maytenus ilicifolia-药物相互作用研究中的应用

咪达唑仑（MDZ）通常用作细胞色素P450 3A（CYP3A）活性的标记化合物。尽管描述了许多定量血浆中MDZ的HPLC-UV方法，但所有方法都使用乙腈（ACN）或甲醇-异丙醇的混合物作为流动相的有机溶剂。自从2008年ACN短缺以来，已努力在HPLC分析中更换这种溶剂。开发了一种简单，灵敏，准确和可重复的HPLC-UV方法（220 nm），并验证了使用甲醇代替大鼠血浆中MDZ的定量。该方法在涉及Maytenus ilicifolia的草药-药物相互作用研究中应用，一种用于治疗胃部疾病的巴西民间药物。血浆样品被碱化，MDZ和阿普唑仑（内标）用乙醚萃取。除去溶剂后，残留物用甲醇-水（1：1）复溶。使用乙酸钠缓冲液（10 m m，pH 7.4）-甲醇（40：60，v / v）在整个C ₁₈色谱柱上洗脱分析物。定量下限的精度从未超过19.40％，而在更高水平的质量控制标准中则为13.86％，而精度在-19.81至14.33％之间。分析曲线在50至2,000 ng / ml之间呈线性关系。肝脏CYP3A酶的活性不受提取物的影响。