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

Assay of TNP-470 and its two major metabolites in human plasma by high-performance liquid chromatography-mass spectrometry.

In this study, a sensitive, specific assay for the determination of TNP-470 and its two major metabolites M-IV (also know as AGM-1883) and M-II in human plasma is reported. The assay involves liquid-liquid extraction of acidified plasma followed by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. A liquid-liquid extraction using an organic solvent mixture (methyl-tert-butyl-ether-hexane, 1:1, v/v) is used in place of solid-phase extraction because it provides consistent recoveries for all analytes, including the internal standard. Retention times for the analytes and internal standard are less than 7 min. Within- and between-day precision is < or = 6.5% and < or = 13.3% relative standard deviation, respectively, for the three analytes. The lower limits of quantitation are 0.25, 0.5, and 1.0 ng/mL for TNP-470, M-IV, and M-II, respectively.

Simultaneous determination of hydrocodone and hydromorphone in human plasma by liquid chromatography with tandem mass spectrometric detection.

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous analysis of hydrocodone (HYC) and its metabolite hydromorphone (HYM) in human plasma. A robotic liquid handler and a 96-channel liquid handling workstation were used to aliquot samples, to add internal standard (I.S.), and to extract analytes of interest. A 96-well mixed-mode solid-phase cartridge plate was used to extract the analytes and I.S. The chromatographic separation was on a silica column (50 x 3 mm, 5-microm) with a mobile phase consisting of acetonitrile, water and trifluoroacetic acid (TFA) (92:8:0.01, v/v). The run time for each injection was 2.5 min with the retention times of approximately 2.1 and 2.2 min for HYC and HYM, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transition 300-->199 (m/z) for HYC, and 28-->185 (m/z) for HYM. The validated calibration curve range was 0.100-100 ng/ml, based on a plasma volume of 0.3 ml. The correlation coefficients were greater than or equal to 0.9996 for both HYC and HYM. The low limit of quantitation (LLOQ) was 0.100 ng/ml for both HYC and HYM with signal-to-noise ratio (S/N) of 50 and 10. respectively. The deuterated analytes, used as internal standards, were monitored at mass transitions 303-->199 (m/z) for HYC-d3 and 289-->185 (m/z) for HYM-d3. The inter-day (n= 17) precision of the quality control (QC) samples were < or = 3.5% RSD (relative standard deviation) for HYC and < or = 4.7% RSD for HYM, respectively. The inter-day accuracy of the QC samples were < or = 2.1% RE (relative error) for HYC and < or = 1.8% RE for HYM. The intra-day (n=6) precision and accuracy of the QC samples were < or = 2.6% RSD and < or = 3.0% RE for HYC, and < or = 4.7% RSD and < or = 2.4% RE for HYM. There was no significant deviation from the nominal values after a 5-fold dilution of high concentration QC samples by blank matrix. The QC samples were stable when kept at room temperature for 24-h or experienced three freeze-thaw cycles. The extraction recoveries were 86% for HYC and 78% for HYM. No detectable carryover was observed when a blank sample was injected immediately after a 2500 ng/ml sample that was 25-fold more concentrated than the upper limit of quantitation (ULOQ).