An Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantification of Vancomycin Requiring Only 2 µL of Rabbit Serum

Abstract

A highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantifi-cation of vancomycin (VAN) in low volumes of rabbit serum. For each analysis , 2 µL of rabbit serum was precipitated with methanol that contained the internal standard teicoplanin (TEI). The supernatant was transferred into a 384 well-plate, diluted with water, covered with a pierceable silicone mat and 5 µL was analyzed in positive ionization mode. The UHPLC-MS/MS consisted of an Agilent 1290 Infinity UHPLC system connected to an AB Sciex QTrap ® 5500 hybrid linear ion-trap triple quadrupole mass spectrometer equipped with a Turbo Spray source. Chromatographic separation was achieved using a Waters Acquity UPLC BEH C 18 (1.7 µm, 2.1 mm × 100 mm) column, a VanGuard (1.7 µm, 2.1 × 5 mm) guard column and a mobile phase of water and methanol both containing 5 mM ammonium acetate with 0.1% formic acid. VAN was quantified with multiple reaction monitoring using the transitions of m/z 725.5/144.2, and TEI was monitored at m/z 940.6/316.4. The accuracy, precision, linearity, range and lower limit of quantification (LLOQ) were determined. The accuracy was ≤9.93% and the precision was ≤10.6%. The range was established as 0.1 to 40 µg•mL −1. The LLOQ was 0.1 µg•mL −1 VAN requiring 2 µL of sample with an accuracy of −20.2% and precision of 8.39%. The method was applied successfully to determine the VAN concentrations in rabbit serum after the i.v. administration of VAN.