Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase
Nd. Weng et al., Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase, J CHROMAT B, 735(2), 1999, pp. 255-269
Morphine (MOR) is an opioid analgesic used for the treatment of moderate to
severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G
) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was a
ble to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was r
equired to define their pharmacokinetic profiles. An LC-MS-MS method was de
veloped in our laboratory to quantify all three analytes with the required
sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was
used to isolate MOR, M3G, M6G, and their corresponding deuterated internal
standards from heparinized plasma. The extract was injected on a LC tandem
mass spectrometer with a turbo ion-spray interface. Baseline chromatograph
ic separation among MOR, M3G, and M6G peaks was achieved on a silica column
with an aqueous organic mobile phase consisting of formic acid, water, and
acetonitrile. The total chromatographic run time was 3 min per injection,
with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respect
ively. Chromatographic separation of M3G and M6G from MOR was paramount in
establishing the LC-MS-MS method selectivity because of fragmentation of M3
G and M6G to MOR at the LC-MS interface. The standard curve range in plasma
was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G
. The inter-day precision and accuracy of the quality control (QC) samples
were <7% relative standard deviation (RSD) and <6% relative error (R.E.) fo
r MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Anal
yte stability during sample processing and storage were established. Method
ruggedness was demonstrated by the reproducible performance from multiple
analysts using several LC-MS-MS systems to analyze over one thousand sample
s from clinical trials. (C) 1999 Published by Elsevier Science B.V. All rig
hts reserved.