The metabolic and pharmacokinetic studies for HM-3 in rats based on LC-Q-TOF/MS and LC–MS/MS combing a convenient biological sample processing method

• The metabolites in rat plasma were identified using LC-Q-TOF/MS.
• Built a LC–MS/MS method for the measurement of HM-3 and metabolites.
• Heating at 70 °C could avoid the degradation of HM-3 in fresh blood sample.
• Pharmacokinetics of HM-3 was studied in rats.

In this contribution, the metabolic and pharmacokinetic characteristics for the therapeutic peptide HM-3 were investigated using LC-Q-TOF/MS and LC–MS/MS systems combing a fast biological sample processing method. According to the accurate MS1 and MS2 data generated by LC-Q-TOF/MS, a total of 6 metabolites in rats were detected and tentatively identified as the degradation products which formed by successive loss of amino acid from HM-3. The structures of the 2 main metabolites (M1 and M2) were confirmed by comparing the chromatographic and mass spectrographic characteristics with the corresponding synthetic standards. Then, an absolute quantitative analysis method based on LC–MS/MS system was built and fully validated with respect to linearity, sensitivity, accuracy, precision, matrix effect, stability, etc. The results indicated that HM-3, M1 and M2 were linear in peak area ratios over the concentration range of 0.5–200.0 ng/mL with a correlation coefficient > 0.99. The intra-day and inter-day precisions (RSD%) were less than 15%, and the accuracy was below 10% in terms of RE%. The validated method was then successfully applied to the studies of preclinical pharmacokinetics for HM-3.