Development and validation of a method for the determination of a therapeutic peptide with affinity for the human B1
1Amgen, Department of Pharmacokinetics and Drug Metabolism, Thousand Oaks, CA 93021, USA.
Abstract:
The peptide described in this report (MW 1180 Da; 10-amino acid synthetic peptide) is a potent and selective antagonist of the human B1 receptor (B1) that has been investigated for the treatment of chronic pain. A method to quantitate this peptide in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics, and efficacy of this compound. Plasma samples (0.2 mL) were extracted using a Waters Oasis MAX (10 mg) 96-well plate and the resulting samples were analyzed using an Applied Biosystems API-5000 HPLC-MS/MS with an electrospray ionization (ESI) source. The method was validated for the determination of the B1 peptide in human plasma over the concentration range of 1-50 ng/mL. Isotopically labeled B1 peptide ((13)C6(15)N(2)-B1 peptide) was used as an internal standard. Interday precision and accuracy, determined from analysis of quality control (QC) samples, yielded coefficients of variation (CV) of less than 5.3% and accuracy within a 2.4%. Within batch precision and accuracy determinations provided CV values of less than 7.3% and accuracy within a 6.0% bias. Precautions had to be taken to prevent B1 peptide loss to container surfaces and contamination of the HPLC-MS/MS. The validated assay was used in support of human clinical trials.
