Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An accurate and reliable liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.). The analyte and the I.S. were extracted from acidified plasma by methyl-t-butyl ether. Separation was achieved on a YMCbasic column using acetonitrile–water–formic acid mobile phase. The MS–MS detection was by monitoring fragmentation 372.1→326.2 (m/z) for COL-3 and 255.1→153.1 (m/z) for the I.S. on a Sciex API 365 using a Turbo Ionspray in positive ion mode. The retention times were approximately 1.7 min for COL-3 and 1.8 min for the I.S. The validated dynamic range was 0.03–10.0 μg/ml using 0.25-ml plasma with correlation coefficients of ≥0.9985. The precision and accuracy for the calibration standards (n=3) were RSD≤5.3% and RE≤4.0%. The precision and accuracy for low-, mid- and high-concentration QC samples were RSD≤2.8% and RE≤5.1% for intra-batch (n=6) and RSD≤2.3% and RE≤3.4% for inter-batch (n=18), respectively. The extraction recoveries were 99% for COL-3 and 93% for I.S. The results showed that the quality control plasma samples were stable for at least 1 year if stored at approximately −70 °C. The presented method is simple, fast, specific and rugged. This method has been successfully used for supporting human pharmacokinetic studies.