High-performance liquid chromatography with mass spectrometry detection for quantitating COL-3, a chemically modified tetracycline, in human plasma

Abstract

COL-3, 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline, is a matrix metalloproteinase inhibitor. A specific and sensitive analytical method was necessary to quantitate the analyte in human plasma. High-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection was utilized to quantitate COL-3 from 30 to 10 000 ng/ml in two calibration curves: 30–1500 and 400–10000 ng/ml. The sample preparation consisted of acetonitrile precipitation for all plasma samples. COL-3 is separated on a Waters Symmetry® C-18 (2.1×150 mm) column with oxalic acid (0.01 M, pH 2.2)–acetonitrile mobile phase. The total run time was 23 min. Identification of COL-3 and the internal standard was through positive chemical ionization and selective ion monitoring. A quantifying and qualifying ion for COL-3 is used to verify the presence of COL-3 in patient samples. Inter- and intra-run mean percent errors for all of the quality controls were less than 18.3% and relative standard deviations were all less than 14.9%. Recovery of COL-3 and the internal standard was approximately 55 and 72%, respectively. Freeze–thaw stability of COL-3 was variable. This method is suitable for quantifying COL-3 in patient samples and to further characterize the clinical pharmacology of this compound.