Quantification of midazolam, morphine and metabolites in plasma using 96-well solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

• Published in: Biomedical chromatography Vol. 24; no. 9; pp. 969 - 976
• Main Authors: Ahsman, Maurice J., van der Nagel, Bart C., Mathot, Ron A.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.09.2010
• Subjects: 96-well plates; Chromatography, High Pressure Liquid - methods; Humans; mass spectrometry; midazolam; Midazolam - blood; Molecular Structure; morphine; Morphine - blood; Solid Phase Extraction - methods; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry - methods; ultra performance liquid chromatography
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1394

Currently, pharmacokinetic–pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra‐performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1‐hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide in 100 μL of plasma. Cleanup consisted of 96 wells micro‐solid phase extraction, before reversed‐phase chromatographic separation (ultra‐performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid‐phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra‐ and interrun accuracy and precision were within 85–115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96‐well micro‐SPE and UPLC‐MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 μL of plasma. Copyright © 2010 John Wiley & Sons, Ltd.