Journal of Chromatography B, 848 (2007) 264–270
Low-level quantitation of oxycodone and its oxidative metabolites,
noroxycodone, and oxymorphone, in rat plasma by high-performance
liquid chromatography–electrospray ionization–tandem
mass spectrometry
Stephen R. Edwards, Maree T. Smith
∗
School of Pharmacy, The University of Queensland, St. Lucia, Brisbane, Qld. 4072, Australia
Received 25 July 2006; accepted 20 October 2006
Available online 13 November 2006
Abstract
A method was developed for quantification of oxycodone, noroxycodone, and oxymorphone in small volumes (50 l) of rat plasma by high-
performance liquid chromatography–electrospray ionization–tandem mass spectrometry using turbo ion-spray. Deuterated (d
3
) opioid analogues
acted as internal standards. Sample preparation involved protein precipitation with acetonitrile, centrifugal evaporation, and reconstitution in mobile
phase; analyte separation was performed on a C18 (5 m, 2.1 mm × 50 mm) column using a linear gradient program. Lower limits of quantitation
(ng/ml) and their between-day accuracy and precision were—oxycodone, 0.9 (-0.2 and 7.8%); noroxycodone, 1.0 (0.6 and 6.2%); oxymorphone
1.0 (-1.8 and 9.5%).
© 2006 Elsevier B.V. All rights reserved.
Keywords: Oxycodone; Noroxycodone; Oxymorphone; Rat; High-performance liquid chromatography (HPLC); Electrospray ionization (ESI); Tandem mass
spectrometry (MS-MS)
1. Introduction
The semisynthetic opioid, oxycodone (OXY), has been in
clinical use since 1917, and for the past decade it has been used in
controlled-release preparations for the treatment of chronic pain
[1]. Oxycodone undergoes oxidative metabolism to form norox-
ycodone and oxymorphone (Fig. 1). In human liver microsomes,
the predominant oxidative metabolic pathway for OXY is via N-
demethylation to noroxycodone (NOR), with O-demethylation
to oxymorphone (OXM) accounting for only 13% [2]. How-
ever, following administration of a 15mg oral dose of OXY to
human volunteers, maximal plasma concentrations (C
max
) of
OXY, and NOR were 38 and 26 ng/ml, respectively, while C
max
for OXM was only 1.1 ng/ml [3]. This finding is consistent with
other studies, where oxycodone’s pharmacodynamic effects in
humans were shown to be produced by the parent drug, rather
than its O-demethylated metabolite, oxymorphone [4,5]. Addi-
∗
Corresponding author. Tel.: +61 7 3365 2554; fax: +61 7 3365 1688.
E-mail address: m.smith@pharmacy.uq.edu.au (M.T. Smith).
tionally, recent in vivo studies in the rat have found evidence for
the active influx of OXY across the blood–brain barrier [6].
OXY has been reported to have a high oral bioavailability
(60–87%) in humans [7,8]. However, there are no studies that
have investigated the oral bioavailability of OXY in the rat,
despite this species being widely used to study the in vivo phar-
macology of OXY. Hence, the purpose of the present study was
to develop and validate a highly sensitive and specific method
for the quantitation of OXY and its oxidative metabolites, NOR
and OXM, in small volumes of rat plasma.
We have previously reported a method employing high-
performance liquid chromatography (HPLC) combined with
electrospray ionization–tandem mass spectrometry for low-level
quantitation of both OXY and MOR, and their respective pri-
mary metabolites NOR and morphine-3-glucuronide (M3G) in
rat plasma [9]. This method which involved the use of a standard
nebulizer ion-spray source was subsequently utilized to simulta-
neously quantify plasma OXY, NOR, and OXM concentrations
in diabetic and non-diabetic rats [10]. In the improved method
described herein, turbo ion-spray was used in concert with a
linear gradient program for solvent delivery to achieve greater
1570-0232/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2006.10.039