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 microg/ml using 0.25-ml plasma with correlation coefficients of >or=0.9985. The precision and accuracy for the calibration standards (n=3) were RSD<or=5.3% and RE<or=4.0%. The precision and accuracy for low-, mid- and high-concentration QC samples were RSD<or=2.8% and RE<or=5.1% for intra-batch (n=6) and RSD<or=2.3% and RE<o=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 degrees C. The presented method is simple, fast, specific and rugged. This method has been successfully used for supporting human pharmacokinetic studies.