Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry

Abstract

A highly sensitive ultra-high-performance liquid chromatographic-tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantification of the synthetic peptide drug of exenatide in monkey plasma. Sample preparation was carried out by solid-phase extraction (SPE), and bivalirudin was used as the internal standard (IS). An excellent chromatographic separation was obtained on a reversed-phase C₁₈ column with a gradient elution. Detection utilized a Qtrap 5500 system operated in the positive ion mode with multiple reaction monitoring (MRM). The proposed method was validated by assessing the specificity, linearity, intra- and inter-day precision and accuracy, recovery, and stability. The method resulted in a linear calibration range of 0.10-30 ng/mL, extracting with only 50 μL monkey plasma aliquots. The intra- and inter-day precisions (as relative standard deviation) were less than 7.5% and 9.6%, respectively. The method could be successfully utilized for the pharmacokinetic study of exenatide in monkeys following a single subcutaneous injection of Byetta.

Keywords: Exenatide; Monkey; Pharmacokinetics; Plasma; Quantitation; UPLC/MS/MS.

Fig. 1

The product ion scans of [M+4H]⁴⁺ for exenatide (A) and [M+2H]²⁺ for bivalirudin (B).

Fig. 2

Representative MRM chromatograms of exenatide (m/z 1047.4→396.3) in four different animals blank plasma (human, monkey, dog and rat).

Fig. 3

Representative MRM chromatograms of exenatide and bivalirudin in monkey plasma. (A) Blank plasma; (B) blank plasma spiked with 0.1 ng/mL of exenatide (I) and 8.5 ng/mL bivalirudin (II); (C) plasma sample from a monkey volunteer 15 min of exenatide after the subcutaneous injection of exenatide (5 μg for each monkey).

Fig. 4

Plasma concentration–time profile of subcutaneous injection of exenatide (5 μg for each monkey) to healthy monkey. Data are mean±SD for three monkeys (three males).