Quantification of midazolam, morphine and metabolites in plasma using 96-well solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry.
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 microL 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 microL of plasma.
Ahsman
Maurice J
MJ
Department of Hospital Pharmacy, Erasmus University Medical Center, Rotterdam, The Netherlands.
van der Nagel
Bart C
BC
Mathot
Ron A
RA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008874:Midazolam / Q000097:blood*; D015394:Molecular Structure; D009020:Morphine / Q000097:blood*; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009020:Morphine; D008874:Midazolam