Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

Katagi, Munehiro; Tatsuno, Michiaki; Miki, Akihiro; Nishikawa, Mayumi; Nakajima, Kunio; Tsuchihashi, Hitoshi

doi:10.1016/s0378-4347(01)00213-4

Cited by 45 publications

(22 citation statements)

References 32 publications

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“…SGO was synthesized according to the procedure described in our previous paper. 6) Standard solutions (each concentration 10 mM) of these compounds were prepared in distilled water and diluted to appropriate concentrations with potassium phosphate buffer 50 mM or a control rat liver microsomal solution immediately before use. An internal standard (IS), ethylamphetamine, was synthesized according to the method of Crossley and Moore 18) and the solution was prepared in distilled water In order to investigate the conversion of selegiline (SG), a drug used in the treatment of Parkinson's disease, to selegiline N-oxide (SGO) as a major metabolic pathway for SG, rat liver microsomal incubations were carried out in vitro in the presence of NADPH.…”

Section: Methodsmentioning

confidence: 99%

“…The chromatographic separation was accomplished on a semi-micro SCX column (2.0 mm i.d.ϫ150 mm; Shiseido, Tokyo, Japan) with acetonitrile/ammonium formate 10 mM (pH 3.0) (70 : 30, v/v) as the mobile phase at a flow rate of 0.1 ml/min. 6) Incubation Procedure The incubation medium usually contained potassium phosphate buffer 50 mM adjusted to a specific pH (pH 7-9), 1 mM NADPH, and the rat liver microsomal protein (final concentration 1 mg protein/ml) in a final volume of 500 ml. The mixture was preincubated at 37°C for 3 min and the reaction was initiated by the addition of substrate SG solution in buffer to give a final substrate concentration of 100 mM.…”

Section: Methodsmentioning

confidence: 99%

“…6,7) With regard to the metabolism of SG to MA, DM-SG, and AP by N-dealkylation, several studies, mainly in Europe, have suggested that hepatic cytochrome P450s (CYPs) are involved, and some reports have proposed that CYP2D6, 8) CYP1A2, and CYP3A4 play major roles in the metabolism of SG. 9) A recent report has also revealed that CYP2B6 and CYP2C19 are of major importance.…”

Section: Selegiline (Sg) [(R)-(ϫ)-na-dimethyl-n-2-propynylphenethylamentioning

confidence: 99%

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In Vitro Confirmation of Selegiline N-Oxidation by Flavin-Containing Monooxygenase in Rat Microsome Using LC-ESI MS

Tsutsumi

Katagi

Nishikawa

et al. 2004

Biological & Pharmaceutical Bulletin

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Selegiline (SG) [(R)-(Ϫ)-N,a-dimethyl-N-2-propynylphenethylamine] developed in Hungary in 1964, is a potent, irreversible, and selective inhibitor of monoamine oxidase type-B (MAO-B) predominantly found in the human brain and it inhibits the breakdown of dopamine in the brain.1) It has been widely used alone or in combination with levodopa, a dopamine precursor, in the treatment of Parkinson's disease in Europe and America. It was also approved in Japan as a prescription medicine (under the trade name FP) in 1998 and it has been used clinically for Parkinson's disease.As shown in Fig. 1, it is well established that SG is predominantly metabolized into methamphetamine (MA) and amphetamine (AP).2-5) Therefore the clear discrimination of the legitimate therapeutic use of SG from the illicit use of MA is indispensable in the forensic science field. Thus Japanese authorities designated it as a precursor of the illicit stimulant MA in 1996, and began to regulate its possession and use.More recently, our study of the urinary excretion profiles of SG and its metabolites in human has demonstrated that SGO can serve as a new indicator of SG administration in place of a characteristic SG metabolite desmethyl selegiline (DM-SG) for the discrimination of SG use from MA abuse and that the main metabolic pathways for SG to SGO (by Noxidation) as well as to MA, DM-SG, and AP (by dealkylation) exist in humans.6,7) With regard to the metabolism of SG to MA, DM-SG, and AP by N-dealkylation, several studies, mainly in Europe, have suggested that hepatic cytochrome P450s (CYPs) are involved, and some reports have proposed that CYP2D6, 8) CYP1A2, and CYP3A4 play major roles in the metabolism of SG.9) A recent report has also revealed that CYP2B6 and CYP2C19 are of major importance.10) However, the N-oxidation of SG was not mentioned in those studies.Studying the metabolism of an irreversible MAO inhibitor, pargyline, that exhibits the same a-acetylenic amine moiety as SG in vitro in rat liver microsomes Weli et al. suggested that the N-oxidation of pargyline occurred mainly by a P450-independent pathway. 11,12) However, the enzymes responsible for its N-oxidation were not investigated in detail. Thus no research has described the enzymes responsible for the N-oxidation of SG. It is generally known that amine compounds are readily N-oxidized in humans by flavin-containing monooxygenase (FMO) and/or CYPs. Particularly for the Noxidation of various kinds of tertiary amines including nicotine, benzydamine, etc., some studies have demonstrated that FMO rather than the CYPs was responsible. [13][14][15][16][17] In the current study, to clarify the enzyme system responsible for the N-oxidation of SG to SGO, the in vitro metabolism of SG was studied using rat liver microsomes, and the involvement of FMO in the N-oxidation of SG was demonstrated. MATERIALS AND METHODSMaterials SG (l-deprenyl) hydrochloride and DM-SG hydrochloride were kindly provided by Fujimoto Pharmaceuticals (Osaka, Japan), and MA hydrochloride was obtained from Dainippon ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Selegiline (Sg) [(R)-(ϫ)-na-dimethyl-n-2-propynylphenethylamentioning

confidence: 99%

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In Vitro Confirmation of Selegiline N-Oxidation by Flavin-Containing Monooxygenase in Rat Microsome Using LC-ESI MS

Tsutsumi

Katagi

Nishikawa

et al. 2004

Biological & Pharmaceutical Bulletin

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“…L-Deprenyl and its major metabolites (L-nordeprenyl, L-methamphetamine, and L-amphetamine) can be determined in serum and urine using various separation methods, such as gas chromatography (GC) [4,5], gas chromatography-mass spectrometry (GC-MS) [6,7], high-performance liquid chromatography (HPLC) [8,9], HPLC-MS [10][11][12], and capillary electrophoresis [13,14]. Specificity for the parent compound is given by the use of selected ion monitoring of either GC-MS or HPLC-MS.…”

Section: Introductionmentioning

confidence: 99%

HPLC analysis and detection ofl-deprenyl

Csomor¹,

Laufer²,

Pöstényi³

et al. 2014

Acta Chromatographica

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Summary. L-Deprenyl and its major metabolites were subjected to reversed-phase highperformance liquid chromatography (HPLC). The separation was monitored using both ultraviolet (UV) and electrochemical detectors. Ultraviolet absorbance detected L-deprenyl, L-nordeprenyl, L-methamphetamine, and L-amphetamine. Amperometric detection was specific and sensitive to the parent compound (L-deprenyl, a tertiary amine) only. Peaks of the major L-deprenyl metabolites did not give any comparable signal using amperometric monitoring.

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“…Deprenyl-N-oxide was identified in human [15,16] and rat urine [17,18] in in vivo metabolism studies. However, the contribution of the various FMO or CYP450 isoforms to this metabolic pathway has not yet been clarified.…”

Section: Introductionmentioning

confidence: 99%

Assessment of the N‐oxidation of deprenyl, methamphetamine, and amphetamine enantiomers by chiral capillary electrophoresis: An in vitro metabolism study

et al. 2004

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A chiral capillary electrophoresis method using hydroxypropyl-beta-cyclodextrin as chiral selector was developed and validated for the quantification of the N-oxygenated metabolites of deprenyl, methamphetamine, and amphetamine enantiomers, formed in vitro. The influence of various parameters (selector concentration, buffer pH, temperature, polymer additive, etc.) on the simultaneous separation of the optical isomers of the parent drugs and their metabolites has been evaluated. The buffer pH had the greatest impact on the separation selectivity of the N-oxygenated compounds. Linear calibration curves were obtained over the concentration range of 2.5-50 microM for the enantiomers of amphetamine-hydroxylamine, methamphetamine-hydroxylamine, and deprenyl-N-oxide. The inter- and intra-assay precision and accuracy varied by less than 15% for all analytes at concentrations of 5, 10, and 30 microM, and less than 20% at the lower limit of quantitation (2.5 microM). The sample extraction recovery ranged between 109 and 129% at the three concentration levels. The drug enantiomers were incubated with recombinant human flavin-containing monooxygenase enzymes (FMO3 and FMO1), and human liver microsomes, respectively. The enantioselectivity of the substrate preference, as well as the stereoselective formation of the new chiral center upon the oxidation of the prochiral tertiary nitrogen of deprenyl were assessed. FMO1, the extrahepatic form of the enzyme in man, was shown to be more active in the N-oxygenation of both deprenyl and methamphetamine isomers than FMO3. Deprenyl enantiomers and S-methamphetamine were substrates of human recombinant FMO3. Conversion of amphetamine to its hydroxylamine derivative could not be observed on incubation with either FMO1 or FMO3. Formation of the new chiral center on the nitrogen, during N-oxidation of the tertiary amine deprenyl, was found stereoselective. The two FMO isoforms have shown opposite preference in the formation of this chiral center. Methamphetamine-hydroxylamine formed from methamphetamine was further transformed by FMO, amphetamine-hydroxylamine was identified as the product of a demethylation reaction.

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Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography–electrospray ionization mass spectrometry

Cited by 45 publications

References 32 publications

In Vitro Confirmation of Selegiline N-Oxidation by Flavin-Containing Monooxygenase in Rat Microsome Using LC-ESI MS

In Vitro Confirmation of Selegiline N-Oxidation by Flavin-Containing Monooxygenase in Rat Microsome Using LC-ESI MS

HPLC analysis and detection ofl-deprenyl

Assessment of the N‐oxidation of deprenyl, methamphetamine, and amphetamine enantiomers by chiral capillary electrophoresis: An in vitro metabolism study

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