An LC–MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10–5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51–7.70% with accuracy of 95.1–104.2%. The average recovery of cTP6 for three concentration levels was 59.6–64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6–8 h after intra-muscular injection. The half-life was 2.24–2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 μg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 μg/kg for 7 consecutive days in rhesus monkeys.