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Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain

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Abstract

PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 μL plasma or rat brain homogenate by protein precipitation using 0.1 % formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1 mm × 50 mm) column with 1.7 μm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5 % (v/v) containing 0.1 % formic acid (solvent A) and 100 % acetonitrile containing 0.1 % formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35 → 209.30 for PF-5190457 and m/z 518.47 → 214.43 for the internal standard. The recovery ranged from 102 to 118 % with coefficient of variation (CV) less than 6 % for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R 2 ≥ 0.998, n = 3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115 % and percent imprecision was ≤15 %. The assays were successfully utilized to measure the concentration of PF-5190457 in pre-clinical and clinical pharmacology studies of the compound.

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Abbreviations

ACN:

Acetonitrile

CV:

Coefficient of variation

IS:

Internal standard

LLOQ:

Lower limit of quantification

ME:

Matrix effect

MeOH:

Methanol

MRM:

Multiple reaction monitoring

MS:

Mass spectrometry

Mw:

Molecular weight

PLs:

Phospholipids

QCs:

Quality controls

UPLC-MS/MS:

Ultra-performance liquid chromatography-tandem mass spectrometry

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Acknowledgments

The authors would like to thank Professor Deyu Li for his help in elucidating the fragmentation pattern of PF-5190457. This work was supported by grant number 1UH2TR000963 (PIs: Akhlaghi and Leggio) from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), and by NIH intramural funding ZIA-AA000218 (PI: Leggio) jointly supported by the Division of Intramural Clinical and Biological Research of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and the Intramural Research Program of the National Institute on Drug Abuse (NIDA).

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Authors and Affiliations

  1. Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 495A College of Pharmacy, 7 Greenhouse Road, Kingston, RI, 02881, USA

    Mwlod Ghareeb & Fatemeh Akhlaghi

  2. Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology, Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, USA

    Lorenzo Leggio

  3. Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

    Lorenzo Leggio

  4. Center for Alcohol and Addiction Studies, Department of Behavioral and Social Sciences, Brown University, Providence, RI, 02903, USA

    Lorenzo Leggio

  5. Pfizer Inc., 610 Main St, Cambridge, MA, 02139, USA

    Ayman El-Kattan

Authors
  1. Mwlod Ghareeb
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  2. Lorenzo Leggio
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  3. Ayman El-Kattan
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  4. Fatemeh Akhlaghi
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Correspondence to Fatemeh Akhlaghi.

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Ghareeb, M., Leggio, L., El-Kattan, A. et al. Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain. Anal Bioanal Chem 407, 5603–5613 (2015). https://doi.org/10.1007/s00216-015-8730-2

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  • DOI: https://doi.org/10.1007/s00216-015-8730-2

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