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A validated UPLC-MS/MS method for the quantitation of an unstable peptide, monocyte locomotion inhibitory factor

Xuemei Liu1, Pei Hu1, Yongsheng Wang2

  • 1Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, PR China; Beijing Key Laboratory of Clinical PK and PD Investigation for Innovative Drugs, PR China.

Journal of Pharmaceutical and Biomedical Analysis
|May 19, 2018
PubMed

Insights

A new ultra-performance liquid chromatography-tandem mass spectrometry method accurately measures Monocyte Locomotion Inhibitory Factor (MLIF) in human plasma. This validated assay is crucial for pharmacokinetic studies of MLIF, a peptide used for neural protection in stroke.

Area of Science:

  • Analytical Chemistry
  • Pharmacokinetics
  • Neuroscience

Background:

  • Monocyte Locomotion Inhibitory Factor (MLIF) is a pentapeptide with anti-inflammatory properties, investigated for neural protection in acute ischemic stroke.
  • Accurate quantification of MLIF in human plasma is essential for pharmacokinetic evaluation in clinical trials.
  • Peptide instability in biological matrices presents a significant challenge for bioanalytical method development.

Purpose of the Study:

  • To develop and validate a sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for measuring MLIF in human plasma.
  • To address and overcome the challenge of MLIF instability in plasma samples.
  • To apply the validated method in a first-in-human study.

Main Methods:

  • Human plasma samples were prepared using protein precipitation followed by acidification with sulfuric acid to ensure MLIF stability.
  • Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column using a gradient elution with acetonitrile and formic acid in water.
  • MLIF was detected using a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring (MRM) mode.

Main Results:

  • The UPLC-MS/MS method was fully validated for MLIF quantification over a concentration range of 0.5-40 ng/mL, with a lower limit of quantification (LLOQ) of 0.5 ng/mL.
  • The method demonstrated excellent precision (inter- and intra-batch ≤ 8.8%) and accuracy (88.7-104.2%), with a mean extraction recovery of 43.3% and no significant matrix effect.
  • The developed method confirmed MLIF stability under various handling and storage conditions post-acidification.

Conclusions:

  • A robust and sensitive UPLC-MS/MS method was successfully established for the accurate determination of MLIF in human plasma.
  • The method's validation and stability assessments confirm its suitability for pharmacokinetic studies.
  • The method was successfully applied to a first-in-human study of MLIF in healthy Chinese subjects, enabling crucial pharmacokinetic evaluations.
Keywords:
First-in-humanIschemic strokeMLIFPharmacokineticsUPLC–MS/MS

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