Liquid chromatography tandem mass spectrometry determination of total budesonide levels in dog plasma after inhalation exposure
Finnish Food Safety Authority Evira, Chemistry and Toxicology Research Unit, Mustialankatu 3, 00790 Helsinki, Finland.Analytical and Bioanalytical Chemistry (Impact Factor: 3.44). 11/2011; 402(3):1209-15. DOI: 10.1007/s00216-011-5549-3
A sensitive and selective method to quantify budesonide in dog plasma samples was developed and fully validated. Liquid-liquid extraction was followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry with electrospray ionization. After reconstitution of the analytes in the mobile phase, samples were analysed by reversed-phase liquid chromatography with isocratic elution. d8-Budesonide was used as an internal standard, and characteristic transitions of d8-budesonide and budesonide were used for quantification. The method was validated with respect to selectivity, specificity, linearity, recovery, repeatability, reproducibility and limits of detection and quantification. The validated method was successfully applied to monitor the plasma levels of budesonide in dogs exposed to clinical doses of inhaled and intravenous drug.
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ABSTRACT: Budesonide (BUD) is used as a mixture of 22R and 22S epimers for the topical treatment of asthma, rhinitis, and inflammatory bowel disease. To study stereoselectivity in the pharmacokinetics of each epimer, we developed a stereoselective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of 22R and 22S epimers of BUD in human plasma. The epimers of BUD were extracted from plasma using n-hexane/dichloromethane/isopropanol (2:1:0.1, v/v/v) under alkaline conditions. Baseline separation was obtained within 7min on an Acquity UPLC BEH C (50mm×2.1mm, 1.7μm) column using an isocratic mobile phase consisting of acetonitrile/5mM ammonium acetate/acetic acid (29:71:0.142, v/v/v) at a flow rate of 0.7mL/min. Mass spectrometric detection was performed in a multiple reaction monitoring mode using the m/z 489→357 transition for BUD epimers and the m/z 497→357 transition for the internal standard d-BUD epimers. Calibration curves were linear over the concentration ranges of 5.0-500 and 5.0-3000pg/mL for 22R-BUD and 22S-BUD, respectively. The lower limit of quantification was 5.0pg/mL for both epimers. The method was successfully applied in a pharmacokinetic study of BUD controlled-release capsules in humans. Consistent differences in the pharmacokinetics of the 22R and 22S epimers were observed, the AUC of 22S-BUD was approximately six times higher than that of 22R-BUD, and the 22S-/22R-BUD ratio of total body clearance was 0.17.
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ABSTRACT: A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5μL injected) were analyzed with selected reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50-5000pg/mL with and LOD 24pg/mL. Calibration response showed good linearity (correlation coefficients<0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n=95).
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