Determination of methotrexate in human urine at trace levels by solid phase extraction and high‐performance liquid chromatography/tandem mass spectrometry
ABSTRACT For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highly sensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a precise and accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solid phase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol, the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validated by using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than 0.999, and the limit of detection was assessed at 0.2 microg L(-1). High specificity of the HPLC-MS/MS technique assures that no interfering substances are detected rather than the analyte of interest.
[Show abstract] [Hide abstract]
ABSTRACT: A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP18e column using an isocratic mobile phase of 5 mm ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 308.3, m/z 313.2 149.2 and m/z 180.3 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49–91.0 and 0.40–74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats. Copyright © 2014 John Wiley & Sons, Ltd.Biomedical Chromatography 10/2014; 29(5). DOI:10.1002/bmc.3348 · 1.66 Impact Factor
01/2015; 2(2):59-67. DOI:10.14205/2309-4435.2014.02.02.3
[Show abstract] [Hide abstract]
ABSTRACT: Different types of assays are used in clinical laboratories for determination of concentrations of various drugs in biological fluids for therapeutic drug monitoring. Historically, concentrations of various anticonvulsants such as phenytoin, carbamazepine, phenobarbital, and primidone in serum or plasma were measured using gas chromatography (GC) or high-performance liquid chromatography. Later, these assays were replaced by immunoassays because of automation as well as need for faster turnaround time. Minimal or no specimen pretreatment is needed for analysis of various drugs in sera using immunoassays. However, immunoassays are not available for all drugs monitored in clinical laboratories, for example lamotrigine, protease inhibitors, and new generation of anticonvulsants. For analysis of these drugs, GC, HPLC, or HPLC combined with tandem mass spectrometric techniques are used.10/2007: pages 67-86;