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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 31, Number 8, October,
pp.453-461
Quantitative Analysis of Naltrexone and
6b-Naltrexol in Human, Rat, and Rabbit Plasma by Liquid Chromatography–Electrospray
Ionization Tandem Mass Spectrometry with Application to the
Pharmacokinetics of Depotrex® in Rabbits
Matthew H. Slawson[1], Meng Chen[1],
David E. Moody[1], Sandra D. Comer[2], Elie S. Nuwayser[3], Wenfang
B. Fang[1], and Rodger L. Foltz[1]
[1]Center for Human Toxicology, Department of Pharmacology and
Toxicology, University of Utah, Salt Lake City, Utah 84112;
[2]New
York State Psychiatric Institute, College of Physicians and Surgeons
of Columbia University, Department of Psychiatry, New York, New
York 10032; and
[3]Biotek, Inc., Woburn, Massachusetts 01801
To improve the analysis of naltrexone and its
primary metabolite 6b-naltrexol, a sensitive and specific method
for the analysis of subnanogram-per-milliliter concentrations
of these analytes in human, rat, and rabbit plasma was developed
utilizing liquid chromatography (LC) coupled to electrospray
ionization (ESI) tandem mass spectrometry (MS–MS). Plasma
samples were extracted utilizing a liquid–liquid extraction
technique. Chromatographic separation was achieved using an isocratic
solvent system consisting of dilute formic acid and methanol
pumped through an ODS-AQ HPLC column. ESI-MS–MS was in
the positive ion mode followed by collision-induced dissociation
of the protonated molecular ions for naltrexone, 6b-naltrexol,
and their deuterated analogues. This method was validated using
Good Laboratory Practice approved methods and was compared to
an existing gas chromatography (GC)–MS method by analyzing
plasma samples collected from a clinical study. Specificity determined
from comparing blank plasma fortified with internal standard
to samples fortified with internal standard and analyte at the
lower limit of quantitation (LLOQ) from six different human,
rat, and rabbit sources demonstrated sufficient signal-to-noise
to set the LLOQ at 0.1 ng/mL. This assay has a quantitative range
of 0.1–100 ng/mL. The inter- (human only) and intra-assay
precision and accuracy in plasma varied by less than 13, 11,
and 16% at the LLOQ for both analytes and by less than 10, 10,
and 9% at higher concentrations for human, rat, and rabbit plasma,
respectively. No loss of analyte was observed after 24 h of room
temperature storage in human, rat, and rabbit plasma or three
cycles of freezing and thawing of human plasma prior to extraction.
Human samples that had been extracted were stable for at least
five days when stored frozen at –20°C or for at least
two days when stored at room temperature on an autosampler. The
GC–MS and LC–MS–MS methods correlated in the
measured plasma concentrations of both naltrexone and 6b-naltrexol.
This method has been validated and subsequently used in the determination
of the pharmacokinetics of Depotrex in rabbits. In rabbits, the
parent compound shows dose-dependent pharmacokinetics as seen
in humans, but rabbits have much lower unconjugated metabolite,
6b-naltrexol, than that seen in humans.
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