Simultaneous bioanalysis of rasagiline and its major metabolites in human plasma by LC–MS/MS: Application to a clinical pharmacokinetic study

doi:10.1016/j.jpba.2016.04.003

Journal of Pharmaceutical and Biomedical Analysis

Volume 125, 5 June 2016, Pages 280-285

https://doi.org/10.1016/j.jpba.2016.04.003 Get rights and content

Highlights

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An HPLC–MS/MS assay for simultaneous determination of concentrations of rasagiline and it three metabolites in plasma was validated.
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This method has been successfully applied to a pharmacokinetic study of oral administration of rasagiline tablets.
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It is the first time to report the pharmacokinetic profiles of rasagiline metabolites.

Abstract

Rasagiline is a selective, irreversible inhibitor of monoamine oxidase type-B (MAO-B) and has been used both as a monotherapy and in addition to levodopa in the treatment of Parkinson’s disease (PD). Rasagiline is metabolized by the cytochrome P450 (CYP) system, and the following three major metabolites with potential neuroprotective activity have been identified: 1-aminoindan (AI), 3-hydroxy-N-propargyl-1-aminoindan (3-OH-PAI) and 3-hydroxy-1-aminoindan (3-OH-AI). In this study, a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of rasagiline and its major metabolites in human plasma. This method was validated in terms of specificity, linearity, precision, accuracy, recovery, matrix effect and stability. The validated method was then applied to a clinical pharmacokinetic study after the oral administration of 1 mg rasagiline mesylate tablets to six healthy Chinese volunteers.

Graphical abstract

Introduction

Rasagiline (N-propargyl-1(R)-aminoindan) is a monoamine oxidase type-B (MAO-B) inhibitor that is usually used as a monotherapy in early Parkinson’s disease (PD) and as an adjunct medication in advanced PD to decrease off-time and improve the symptoms of PD in levodopa-treated patients with motor fluctuations [1], [2], [3], [4]. Rasagiline also provides neuroprotective effects in laboratory models of neurodegeneration [5], [6], [7], [8].

Rasagiline undergoes almost complete biotransformation in the liver, and two main pathways are involved in the metabolism of rasagiline, which include N-dealkylation and/or hydroxylation to yield 1-aminoindan (AI), 3-hydroxy-N-propargyl-1-aminoindan (3-OH-PAI) and 3-hydroxy-1-aminoindan (3-OH-AI) [9], [10], [11]. In vitro experiments indicate that both pathways are dependent on the cytochrome P450 (CYP) system, with CYP1A2 as the major isoenzyme involved in the rasagiline metabolism [9], [10], [11]. In addition, several studies have shown the potential neuroprotective effects of AI, suggesting that it might contribute to the overall neuroprotective and antiapoptotic effects of its parent compound rasagiline [12], [13], [14], [15], moreover, as derivatives of rasagiline and AI, 3-OH-PAI and 3-OH-AI might also have potential neuroprotective effects [16], [17]. Because metabolites may contribute to the pharmacology or toxicity of the parent drug [18], it is essential to establish a reliable method for the determination of rasagiline and its major metabolites in human plasma.

Several analytical methods have been developed to detect rasagiline in biological fluids or pharmaceutical preparations using HPLC [19], [20], [21], [22], LC–MS/MS [23], [24], [25], GC–MS [26] or crystallographic analysis [3]; however, few studies have sought to determine its metabolite AI [26], [27], [28]. Thébault et al. described a method for the simultaneous determination of rasagiline and AI by GC–MS, but the lower limits of quantitations (LLOQs) of 0.25 and 0.5 ng/ml for rasagiline and AI (>1/20 of respective C_max), respectively, could not guarantee the pharmacokinetic valuation, and the sample preparation procedure involving derivatization is too time-consuming [26]. Furthermore, no studies to date have clarified the exposure of 3-OH-PAI and 3-OH-AI in humans. Considering the potential effects of these metabolites, a sensitive and simple assay to simultaneous determine the concentrations of rasagiline and its metabolites (AI, 3-OH-PAI and 3-OH-AI) would be highly useful and could provide support for the comprehensive evaluation of their pharmacokinetic profiles.

In this study, we developed and validated a simple and rapid LC–MS/MS method for the simultaneous determination of rasagiline and its major metabolites in human plasma. This method was successfully applied to a pharmacokinetic study following the oral administration of 1 mg rasagiline mesylate tablets to six healthy Chinese volunteers.

Section snippets

Chemicals and reagents

Rasagiline (99.68% purity), AI (98.2% purity), 3-OH-PAI (98.5% purity) and 3-OH-AI (98.1% purity) standards were provided by Sichuan Fangxiang Pharmaceutical Co., Ltd (Sichuan, China). Clopidogrel (internal standard, IS, 99.5% purity) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade methanol was obtained from Merck (Darmstadt, Germany), ethyl acetate from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China),

MS spectrometry

The structures of rasagiline, AI, 3-OH-PAI and 3-OH-AI could be ionized under the appropriate conditions to give the corresponding indane and hydroxyindan. These analytes were found to respond best to positive ionization with the protonated ions [M+H]⁺, which are presented as major peaks for all these compounds. Their product ion mass spectra are shown in Fig. 1. The transitions of m/z 172.4 → 116.8, 134.3 → 116.8, 188.4 → 132.3, 150.1 → 132.3 and 321.9 → 184.7 were therefore used to analyze samples

Conclusions

We have developed and validated a simple, highly sensitive and specific LC–MS/MS method for the simultaneous determination of rasagiline and its metabolites AI, 3-OH-PAI and 3-OH-AI in human plasma. This method was developed in accordance with the regulatory guidelines provided by the FDA and EMA, and this work represents the first systematic evaluation of a rasagiline assay.

Acknowledgements

The authors are grateful to the subjects who were involved in this study, the investigators, and the research staff. The authors further appreciate the collaboration and help from Dr. Yi He, Chengdu Blood Center (Chengdu, China).

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Citation Excerpt :
R-AI is the major metabolite of rasagiline and has potential neuroprotective effects [24]. Several analytical methods such as GC–MS [25] and LC–MS/MS [26] for the analysis of AI in plasma have been described. However, up to now, no reports have described the analysis of AI enantiomers in biological samples.
(S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid (Pro-PPZ) was employed as a chiral derivatization reagent (CDR) for the efficient enantioseparation and ultrasensitive mass spectrometric detection of chiral amines. Pro-PPZ was prepared from the one-step reaction of 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) and l-proline. Two amines and two amino acid methyl esters were selected as model chiral amines, which were easily labeled with Pro-PPZ under mild reaction conditions (35 °C for 10 min) generating Pro-PPZ-amine derivatives. The resulting diastereomers were completely separated by reversed-phase liquid chromatography (RP-LC) using an ODS column (R_s = 3.4–17.0 for amines). Ultrasensitive detection limits on femtomolar level were obtained for the tested amines using multiple reaction monitoring (MRM) chromatograms at a single monitoring ion, m/z 289 (0.1–5.0 fmol for amines). The practical metabolite analysis of (R)-1-aminoindan (R-AI) in saliva samples was performed by LC–MS/MS using the Pro-PPZ derivatization method. The method was validated in terms of precision, accuracy, and linearity. Using this method, R-AI concentrations in saliva were determined after a single oral administration of the drug rasagiline to healthy male and female subjects, but no (S)-1-aminoindan (S-AI) was detected, which suggesting that R-AI was not converted into S-enantiomer in the metabolic process. R-AI concentrations in four healthy volunteers ranged from 32.85 nM to 49.45 nM, with an average value of 43.76 nM. To date, there is no LC–MS (or MS/MS) method reported for the enantioselective determination of R-AI in human saliva samples.
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Short communicationSimultaneous bioanalysis of rasagiline and its major metabolites in human plasma by LC–MS/MS: Application to a clinical pharmacokinetic study

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and reagents

MS spectrometry

Conclusions

Acknowledgements

Lancet

Exp. Neurol.

Neurobiol. Dis.

Exp. Neurol.

J. Pharmaceut. Biomed.

J. Chromatogr. B

J. Chromatogr. B

J. Pharm. Anal.

A controlled, randomized, delayed-start study of rasagiline in early Parkinson disease

Arch. Neurol.

Binding of rasagiline-related inhibitors to human monoamine oxidases: a kinetic and crystallographic analysis

J. Med. Chem.

A double-blind, delayed-start trial of rasagiline in Parkinson's disease

New Engl. J. Med.

Rasagiline is neuroprotective in an experimental model of brain ischemia in the rat

J. Neural Transm.

ADMET considerations when prescribing novel therapeutics to treat restless legs syndrome

Expert Opin. Drug Metab.

Anti-Parkinson's disease drugs and pharmacogenetic considerations

Expert Opin. Drug Metab.

From depression to neurodegeneration and heart failure: re-examining the potential of MAO inhibitors

Expert Opin. Drug Metab.

The neuroprotective mechanism of 1-(R)-aminoindan, the major metabolite of the anti-parkinsonian drug rasagiline

J. Neurochem.

Short communication
Simultaneous bioanalysis of rasagiline and its major metabolites in human plasma by LC–MS/MS: Application to a clinical pharmacokinetic study