Analysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresis

Abstract

A capillary electrophoresis (CE) method for the separation of the diastereoisomers of 6-oxycodol (6OCOL) and nor-6-oxycodol (N6OCOL), the 6-keto-reduced metabolites of oxycodone (OCOD) and noroxycodone (NOCOD), respectively, is reported and employed to assess the stereoselectivity of these metabolic steps in vivo, in vitro, and in chemical synthesis. CE in an untreated fused-silica capillary with acidic buffers containing 2-hydroxypropyl-beta-cyclodextrin, randomly sulfated beta-cyclodextrin, or single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) is shown to permit the simultaneous separation of the stereoisomers of 6OCOL and N6OCOL. A 100 mM phosphate buffer of pH 2.0 containing 2.05% w/v HDAS-beta-CD provides a medium for rapid analysis and unambiguous identification of these stereoisomers in solid-phase extracts of (i) urines stemming from patients under pharmacotherapy with OCOD, (ii) incubations of OCOD and NOCOD with human liver cytosol and the human liver S9 fraction, and (iii) after chemical synthesis from OCOD and NOCOD using NaBH(4). In all cases, alpha-N6OCOL is shown to be the predominant stereoisomer of N6OCOL. For 6OCOL, the same is true for in vitro formation and for chemical synthesis. In urine, however, beta-6OCOL is observed to be excreted in a higher amount than alpha-6OCOL. For the urinary alpha-/beta-isomer ratio of 6OCOL and N6OCOL, there are no differences between the data obtained for nonhydrolyzed and enzymatically hydrolyzed urines. The data document the stereoselectivity of the 6-keto-reduction of OCOD and NOCOD in man.

Capillary electrophoresis contributions to the hydromorphone metabolism in man

“…1). Previous work dealing with the CE separation of a-and b-oxycodol [32], which are structural analogs of a-and b-HMOROL, revealed the need for a chiral selector. The same selector, HDAS-b-CD, was tested for the analysis of the 6-keto-reduced metabolites of HMOR using a 50 mm id capillary of 45.4 cm length (50 cm total length) together with 100 mM H 3 PO 4 at pH 2 containing 1.6 or 2.05% w/v HDAS-b-CD.…”

“…The synthesis of HMOROL was performed according to a protocol employed by Hahn and Fishman [33] for the reduction of naloxone that was recently used in our laboratory for the reduction of oxycodone [31,32]. Briefly, 250 mL of HMOR methanolic solution (1.0 mg/mL) were diluted with 1.5 mL of methanol in a disposable 5 mL glass tube.…”

“…Furthermore, drug and metabolite monitoring is a topic of high interest in forensic science. Our laboratory has been involved in the elucidation of the metabolism of various opioids in humans by means of different CE techniques [25][26][27][28][29][30][31][32]. CE assays with UV absorption and MS detection were shown to represent attractive approaches for the detection of these compounds.…”

“…Furthermore, computer simulated fragmentation was shown to be a useful complementary technique for structure clarification [31]. As the metabolism of HMOR in man has not yet been completely elucidated, the aim of this work was to apply our CE technology that was previously developed for the analysis of oxycodone and its metabolites [30][31][32] to the investigation of the HMOR metabolism. This paper presents CE-MS n and CE-UV data obtained for urine samples of a patient under pharmacotherapy with HMOR.…”

“…For that purpose, HMOROL and hydromorphone-N-oxide (HMORNO) were synthesized and NHMOR was produced in vitro using recombinant CYP3A4 from baculovirusinfected insect cells, whereas all other attempts for the assignments had to be done without standards. Furthermore, in analogy to the recent work with oxycodone [32], CE data obtained in the presence of a chiral selector are employed to identify the diastereoisomers of 6-ketoreduced HMOR.…”