Elsevier

Journal of Chromatography B

Volume 967, 15 September 2014, Pages 41-49
Journal of Chromatography B

A new HILIC-MS/MS method for the simultaneous analysis of carbidopa, levodopa, and its metabolites in human plasma

https://doi.org/10.1016/j.jchromb.2014.06.030Get rights and content

Highlights

  • This HILIC-MS/MS method is the first for simultaneous determination of levodopa, carbidopa, 3-o-methyldopa, and dopamine in human plasma.

  • A fast and simple sample preparation procedure using protein precipitation was also developed.

  • The method was fully validated according to US FDA and ANVISA bioanalytical method validation guidelines.

  • The method has been used successfully for therapeutic drug monitoring.

Abstract

Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC–MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 μm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75–800 ng/mL for LEV, 65–800 ng/mL for CAR and 3-OMD, and 20–400 ng/mL for DOPA (r > 0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision  13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.

Introduction

New drugs have become available in recent years for the treatment of Parkinson's disease. However, since the introduction of dopamine (DOPA) supplementation, levodopa (LEV) has been considered the gold standard treatment for motor symptoms [1], [2], [3].

Human plasma contains several catechols, including LEV and DOPA (plasmatic levels at 1.75 and 0.01 ng/mL, respectively) [4]. LEV is a natural precursor of DOPA, and it is used as a prodrug because, in contrast to DOPA, LEV can cross the brain-blood barrier (Fig. 1). Due to its extensive metabolization in extracerebral tissues, LEV is usually associated with enzymatic inhibitor drugs such as carbidopa (CAR) or benserazide (aromatic l-amino acid decarboxylase inhibitors) and tolcapone or entacapone (catechol-o-methyltransferase inhibitors) [5].

Prolonged use of LEV leads to fluctuations and motor complications such as the “wearing-off” phenomenon characterized by moments without the benefits provided by therapy and moments with its benefits but with added dyskinesias. Studies have shown that high doses of LEV are also related to dyskinesias presented by patients [6]. Therefore, it is recommended that LEV doses should be adjusted according to the individual needs of patients based on clinical response and the profile of adverse events [1].

Many analytical methods have been described in the literature for the determination of LEV and its metabolites in biological matrices using high-performance liquid chromatography (HPLC) and various detection techniques such as electrochemical detection [7], [8], [9], [10], [11], [12], [13], [14], [15], tandem mass spectrometry (MS/MS) [16], [17], [18], [19], [20], [21], and fluorescence [22], [23]. However, these methods use reversed-phase liquid chromatography (RPLC). Given the hydrophilic nature of these compounds, RPLC methods can result in low retention and usually necessitate the use of ion pair agents or derivative reagents that are incompatible with MS detection. For the most part, RPLC methods need high concentrations of aqueous phase (>90%) to obtain an acceptable retention [16], [17], [19]. Other problems include peak tailing following the presence of ionized silanol groups on the stationary phase and phase dewetting caused by the low concentrations of organic modifier that RPLC methods require for adequate retention of very polar analytes [24], [25].

Hydrophilic interaction liquid chromatography (HILIC) has been reported as an alternative to RPLC for the analysis of polar compounds [26], [27]. In HILIC, a hydrophilic stationary phase (bare silica or polar bonded silica) and a mobile phase consisting of >60% organic content, commonly acetonitrile, with a minimum of 2% of water are used [28]. The term HILIC was first used by Alpert, who considered the main retention mechanism to be the partitioning between the mobile phase and a mobile phase layer enriched with water partially immobilized on the stationary phase [29]. On the contrary, the retention mechanism in HILIC mode appears to be complex, involving also secondary electrostatic, hydrophobic, and hydrogen-bonding interactions dependent on the conditions used—for example, mobile phase additives [26], [30].

Despite its complex mechanism, HILIC has the following advantages over RPLC: good peak shapes for bases, good retention of polar compounds, higher flow rates owing to the high organic content, and enhanced mass spectrometer sensitivity due to the high organic content in the mobile phase and the high efficiency of desolvation in electrospray (ESI) techniques [31]. In recent years, HILIC has been used to separate catecholamines [24] and neurotransmitters such as biogenic amines and amino acid precursors [28], but no technique has been developed for the simultaneous monitoring of LEV treatment with MS/MS detection. Considering the characteristics of these compounds and the advantages of HILIC, the aim of this study was to develop and validate a sensitive and selective HILIC-MS/MS method to quantify LEV, CAR, 3-o-methyldopa (3-OMD), and DOPA simultaneously in human plasma.

Section snippets

Chemicals, reagents, and samples

LEV (98.0%), CAR (98.0%), dopamine hydrochloride (98.0%), and internal standard (IS) deuterated LEV (98.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA); 3-OMD (92.0%) was obtained from United States Pharmacopoeia (Rockville, MD, USA). Acetonitrile and methanol (HPLC grade) were obtained from Tedia (Fairfield, CA, USA). Formic acid (88%) was obtained from J.T. Baker (Phillipsburg, NJ, USA). Ammonium formate (≥97%) was obtained from Acros Organics (Fair Lawn, NJ, USA). Hydrochloric

Method development

To develop a new sensitive and selective method for the simultaneous quantification of CAR, LEV, and its metabolites, we optimized various factors and parameters of MS/MS and LC. To the best of our knowledge, no HILIC-MS/MS method has been reported for the simultaneous determination of these compounds in plasma.

The signal intensity of each analyte was evaluated via the direct infusion of working standard solutions (200 ng/mL of LEV, CAR, DOPA, and 3-OMD) prepared in acetonitrile/water (50:50, v/v

Conclusion

The developed HILIC-MS/MS method was very efficient in the simultaneous determination of LEV, CAR, 3-OMD, and DOPA. To our best knowledge, the presented method for the first time allows the simultaneous determination of these compounds in human plasma in one analytical run. Parameters affecting HILIC separation and MS/MS detection were systematically investigated and optimized. Adequate retention was achieved using HILIC mode, demonstrating that it is an alternative to RPLC for the analysis of

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors thank the Department of Pharmacy of Universidade Federal do Paraná and Pelé Pequeno Príncipe Institute of Research for financial support and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for the scholarship.

References (45)

  • C.E. Clarke et al.

    Lancet

    (2002)
  • M. Rezak

    Dis. a Month

    (2007)
  • D. Gonçalves et al.

    Anal. Chim. Acta

    (2012)
  • J.M. Cedarbaum et al.

    J. Chromatogr. B: Biomed. Sci. Appl.

    (1987)
  • Y. Michotte et al.

    J. Pharm. Biomed. Anal.

    (1987)
  • D.C. Titus et al.

    J. Chromatogr. B: Biomed. Sci. Appl.

    (1990)
  • K.A. Sagar et al.

    J. Pharm. Biomed. Anal.

    (2000)
  • V. Rizzo et al.

    J. Pharm. Biomed. Anal.

    (1996)
  • T. Wikberg

    J. Pharm. Biomed. Anal.

    (1991)
  • M. Karimi et al.

    J. Chromatogr. B

    (2006)
  • F. Bugamelli et al.

    J. Pharm. Biomed. Anal.

    (2011)
  • K. Igarashi et al.

    J. Chromatogr. B

    (2003)
  • R.R. Gonzalez et al.

    J. Neurosci. Methods

    (2011)
  • C. Muzzi et al.

    Biomed. Pharmacother.

    (2008)
  • H.-X. Zhao et al.

    J. Pharm. Anal.

    (2011)
  • A. Kumar et al.

    J. Chromatogr. A

    (2011)
  • D.V. McCalley

    J. Chromatogr. A

    (2010)
  • D.V. McCalley

    J. Chromatogr. A

    (2007)
  • J. Heaton et al.

    J. Chromatogr. A

    (2012)
  • R.I. Chirita et al.

    J. Chromatogr. A

    (2010)
  • A.J. Alpert

    J. Chromatogr

    (1990)
  • H.L. Cai et al.

    Anal. Biochem.

    (2010)
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