Rapid and simple method to determine morphine and its metabolites in rat plasma by liquid chromatography-mass spectrometry.
A rapid and simple method for the determination of morphine (M), normorphine (NM), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma by high-performance liquid chromatographic separation with mass spectrometric detection (HPLC-MS) has been developed. Samples (40 microl) were cleaned-up by protein precipitation with two volumes (80 microl) of acetonitrile and reconstituted in formic acid 0.1% in water. Naloxone was used as internal standard. Analytes were separated on a phenyl-hexyl column using a step-gradient (1 ml/min) of acetonitrile and formic acid in water. Acetonitrile was added post-column (0.3 ml/min). Quantification of morphine and its metabolites was achieved with an Agilent 1100 series HPLC-MS system equipped with electrospray interface set to selected ion-monitoring (SIM) mode. Calibration curves covered a wide range of concentrations (2.44-10,000 nM) and were best fitted with a weighed quadratic equation. The limits of quantification achieved with this method were 2.44 nM for M and 4.88 nM for NM, M3G and M6G. The method proved accurate (85-98%), precise (C.V.<10%) and was successfully applied to a wide range of in vitro and in vivo pharmacokinetic studies in rodents.
Projean
Denis
D
Faculté de Pharmacie, Université de Montréal, Montréal, Québec, Canada.
Minh Tu
The
T
Ducharme
Julie
J
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D009020:Morphine / Q000097:blood*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009020:Morphine