Reversed-phase ion-pair HPLC method for the direct analysis of 1-OH midazolam glucuronide in human serum

In recent years, it has become clear that the presence of high concentratio ns of 1-OH midazolam glucuronide is probably the cause of unexplained prolo nged midazolam comas in patients with poor renal function. Until recently, only indirect methods for the analysis of this glucuronide were known, whic h had several disadvantages, such as a long analysis period (>6 hours). Thi s article describes the validation of a method for the direct analysis of t his compound in human serum, using reversed-phase ion-pair high-performance liquid chromatography (HPLC) in combination with solid phase extraction. T he intraday and interday coefficients of variation have values below 6% for different possible serum concentrations. The limit of quantification (0.1 mg/L) is much lower than concentrations found in patients with a coma cause d by the accumulation of 1-OH midazolam glucuronide. Recovery of 1-OH midaz olam glucuronide is almost 100% at three different serum concentrations. Li nearity is confirmed for normal serum levels (<1 mg/L) and for serum levels that might occur in patients with impaired renal function (<20 mg/L). Dete ction is performed at 254 nm with a diode array detector, which can also be used to check the peak purity in case of unexpected impurities.