Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical...

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol-water-formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H](+) m/z 483.3 for heteroclitin D and [M + H](+) m/z 629.3 for the IS. The standard curve was linear (R(2) ≥0.995) over the concentration range 9.98-2080 ng/mL and had good back-calculated accuracy and precision. The intra- and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and -8.9-3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague-Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin.