A Liquid Chromatographic-Electrospray Ionization-Tandem Mass Spectrometric Method for Determination of Buprenorphine, Its Metabolite, norBuprenorphine, and a Coformulant, Naloxone, That Is Suitable for in Vivo and in Vitro Metabolism Studies

Moody, David E.; Slawson, Matthew H.; Strain, Eric C.; Laycock, John D.; Spanbauer, Alan C.; Foltz, Rodger L.

doi:10.1006/abio.2002.5673

Cited by 123 publications

(107 citation statements)

References 23 publications

(29 reference statements)

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“…Consistent with our previous study using 21 nM buprenorphine (Moody et al, 2002), incubation of 10 M buprenorphine with 12 human baculovirus insect cell-expressed P450 isoenzymes (25 pmol) showed that the 3A family and 2C8 were the major enzymes involved in buprenorphine elimination and norbuprenorphine formation (data not shown). The most efficient enzyme for M1 formation was P450 3A5, followed by 2C8, 3A4, and 3A7.…”

Section: Novel Metabolites Of Buprenorphinesupporting

confidence: 90%

“…In our previous studies on in vitro metabolism of buprenorphine, we focused on use of substrate concentrations that approached therapeutic plasma concentrations (Moody et al, 2002;Chang and Moody, 2005).…”

Section: Resultsmentioning

confidence: 99%

“…The quantification of buprenorphine and norbuprenorphine (or semiquantification of hydroxylated metabolites) in incubation samples and urine samples was performed using a modification of our previously described LC-ESI-MS/MS method (Moody et al, 2002). The incubation samples were made basic (pH Ͼ10) by the addition of 50 l of 1 N NaOH and extracted with a 4-ml mixture of n-butyl chloride and acetonitrile (4:1, v/v); the organic layer was dried under N 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Under the CID-MS/MS conditions, the characterized product ions generated from protonated molecular ions of buprenorphine (m/z 468) were at m/z 414, m/z 396, and m/z 101 (Moody et al, 2002). A [M Ϫ 54] ϩ peak at m/z 414 (referred to as the a-moiety) showed the removal of a cyclopropylmethyl group.…”

Section: Novel Metabolites Of Buprenorphinementioning

confidence: 99%

“…However, a recent study by Picard et al (2005) using liquid chromatography-tandem mass spectrometry identified the presence of two hydroxylated metabolites, one of buprenorphine and one of norbuprenorphine, in human liver microsomes (HLMs) and urine samples from patients treated with buprenorphine. Buprenorphine N-dealkylation is mainly catalyzed by cytochrome P450 (P450) 3A4 (Iribarne et al, 1997;Kobayashi et al, 1998), with involvement of P450 3A5 and 2C8 (Moody et al, 2002;Picard et al, 2005). The involvement of specific P450s in production of the hydroxylated metabolites was limited to a finding that trace amounts of hydroxy-buprenorphine were produced by P450 3A4-, 3A5-, and 3A7-transfected cell lines (Picard et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Novel Metabolites of Buprenorphine Detected in Human Liver Microsomes and Human Urine

Chang¹,

Moody²,

McCance‐Katz³

2005

Drug Metab Dispos

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ABSTRACT:The in vitro metabolism of buprenorphine was investigated to explore new metabolic pathways and identify the cytochromes P450 (P450s) responsible for the formation of these metabolites. The resulting metabolites were identified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In addition to norbuprenorphine, two hydroxylated buprenorphine (M1 and M2) and three hydroxylated norbuprenorphine (M3, M4, and M5) metabolites were produced by human liver microsomes (HLMs), with hydroxylation occurring at the tert-butyl group (M1 and M3) and at unspecified site(s) on the ring moieties (M2, M4, and M5). Time course and other data suggest that buprenorphine is N-dealkylated to form norbuprenorphine, followed by hydroxylation to form M3; buprenorphine is hydroxylated to form M1 and M2, followed by N-dealkylation to form M3 and M4 or M5. The involvement of selected P450s was investigated using cDNA-expressed P450s coupled with scaling models, chemical inhibition, monoclonal antibody (MAb) analysis, and correlation studies. The major enzymes involved in buprenorphine elimination and norbuprenorphine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8, whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb analysis and chemical inhibition, the contribution of 2C8 was higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a more important role in HLMs with higher 3A4/5 activity. Examination of human urine from subjects taking buprenorphine showed the presence of M1 and M3; most of M1 was conjugated, whereas 60 to 70% of M3 was unconjugated.Buprenorphine, a semisynthetic derivative of the alkaloid thebaine (Lewis, 1973), is a partial -opioid agonist and -opioid antagonist (Cowan et al., 1977). It was first developed as an analgesic for moderate to severe pain in the early 1970s, but is currently more widely used as a replacement therapy for opiate dependence. Buprenorphine has comparable effects to methadone in regard to treatment of opiate-dependent patients (Strain et al., 1996;Johnson et al., 2000), but has reduced risk because of the "ceiling effect" associated with its partial -opioid agonist properties (Walsh et al., 1994(Walsh et al., , 1995.Absorption, distribution, metabolism, and excretion studies of buprenorphine have been carried out in humans using gas chromatography-mass spectrometry (Cone et al., 1984), and in animals using thin-layer chromatography of tritiated buprenorphine (Brewster et al., 1981;Pontani et al., 1985). These studies suggested that buprenorphine was mainly metabolized by N-dealkylation and glucuronidation of both buprenorphine and norbuprenorphine. A tentative 6-O-demethyl norbuprenorphine in free and conjugated form was observed in rat urine (Pontani et al., 1985), and some unknown polar metabolites were found in rat bile samples (Brewster et al., 1981). No evidence was given for additional metabolites in humans (Cone et al., 1984). However, a recent study by Picard et al. (2005) using liquid chromatography-tandem mass spectrometry identified the pres...

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Section: Novel Metabolites Of Buprenorphinesupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Novel Metabolites Of Buprenorphinementioning

confidence: 99%