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Extraction and purification of 1-hydroxymidazolam glucuronide from ultrafiltrate
© BioMed Central Ltd. 2010
Published: 1 March 2010
We developed a technique for detection of MDZ and 1-hydroxymidazolam glucuronide (1-OHMG). No commercial source of 1-OHMG prevented assay development. 1-OHMG had pharmacological activity and is renally excreted. We postulated that ultrafiltrate (UFR) would be rich in 1-OHMG. We describe a method to purify 1-OHMG from UFR.
Ethics approval was granted. To purify, UFR was extracted on a C-18 column, it was washed and eluted. HPLC-MS separation was performed. The 1-OHMG (mol. wt 517/518 in MS) was desiccated and re-dissolved four times to maximise purity. Electrospray (ESI) mass spectrometry (MS) characterised 1-OHMG. The purity was calculated using NMR. A calibration plot of 1-OHMG, MDZ and DZP (internal standard) for HPLC-MS was performed.
We were able to extract and purify an active drug metabolite from UFR. Five litres of UFR resulted in 2 mg 1-OHMG. This is a potentially rich source of drugs or drug metabolites, allowing pharmacokinetic studies greatly required in critical illness.