Sensitive and specific determination of midazolam and 1-hydroxymidazolam in human serum by liquid chromatography-electrospray mass spectrometry.
A liquid chromatographic-mass spectrometric technique was designed for the determination of the anaesthetic benzodiazepine midazolam (MID) and its active metabolite 1-hydroxymidazolam (1-OHM), with the aim of conducting pharmacokinetic/pharmacodynamic studies. MID and 1-OHM were extracted from alkalinised (pH 9.5) spiked and clinical serum samples using a single step, liquid-liquid extraction procedure with diethyl ether-2-propanol (98:2, v/v). The chromatographic separation was performed on a Nucleosil C18, 5 microm (150x1 mm I.D.) column, using a gradient of acetonitrile in 5 mM ammonium formate, pH 3.0 as the mobile phase, delivered at a flow-rate of 50 microl/min. The compounds were ionised in the ionspray source of an atmospheric pressure mass spectrometer, fragmented by in-source collisions and the pseudomolecular and fragment ions detected in the selected ion monitoring mode. The recovery was between 79 and 87% for MID, between 83 and 87% for 1-OHM and 81.5% for methylclonazepam. The limit of detection was 0.2 microg/l for MID and 0.5 microg/l for 1-OHM, the limit of quantitation (LOQ) was 0.5 microg/l for both. Linearity was verified from these LOQs up to 2000 microg/l and the method was found accurate and precise over this range. It was successfully applied to a preliminary study to establish the concentration versus time curve of MID and 1-OHM in a patient administered midazolam by continuous infusion.
Marquet
P
P
Department of Pharmacology and Toxicology, University Hospital, Limoges, France. marquet@unilim.fr
Baudin
O
O
Gaulier
J M
JM
Lacassie
E
E
Dupuy
J L
JL
François
B
B
Lachâtre
G
G
D018686:Anesthetics, Intravenous / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006993:Hypnotics and Sedatives / Q000097:blood*; D013058:Mass Spectrometry / Q000379:methods*; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D012121:Respiration, Artificial; D012680:Sensitivity and Specificity
D018686:Anesthetics, Intravenous; D006993:Hypnotics and Sedatives; C040081:1-hydroxymethylmidazolam; D008874:Midazolam