The development and validation of quantitative methods for the determination of stavudine and alfuzosin in plasma and monic acid in urine

[摘要] English: The development and validation of bio-analytica) assay methods suitable for the quantification ofstavudine in plasma, alfuzosin in plasma and monic acid in urine is discussed. A short summary ofthese methods are given:•A sensitive method for the determination of stavudine in plasma was developed, using highperformanceliquid chromatographic separation with tandem mass spectrometric detection. Thesamples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18®solid phaseextraction (SPE) columns. Chromatography was performed on a Supelco Discovery C18, 5urn, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M):acetonitrile: methanol (800:100:100, v/v/v) at a flow rate of 0.3 ml/min. Detection wasachieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS/MS) set at unitresolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemicalionization (APCl) was used to obtain deprotonated ions (molecular ion m/z 223.1 to the production m/z 42.01). The mean recovery for stavudine was 94 % with a lower limit of quantificationset at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of massspectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography)and selective method for the determination of stavudine in human plasma than has previouslybeen published. The assay metod was used to quantitatively determine stavudine concentrationsin plasma samples to follow the concentration vs. time profile for at least five half lives of thedrug after a single 40 mg oral dose of stavudine was given to healthy adult male humansubjects.•A selective, sensitive and rapid liquid chromatography-tandem mass speetrometry method forthe determination of alfuzosin in plasma was developed. An Applied Biosystems API 2000triple quadrupole mass speetrometer in multiple reaction monitoring (MRM) mode, usingTurboIonSpray (TIS) with positive ionisation was used (molecular ion of alfuzosin m/z 390.2 tothe product ion m/z 71.2; molecular ion of prazosin m/z 384.2 to the product ion m/z 95.0).Using prazosin as an internal standard, liquid-liquid extraction was followed by CI8 reversedphase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosinwas 82.9 % with a lower limit of quantification set at 0.298 ng/ml, the calibration range beingbetween 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity andselectivity of tandem mass spectrometric (MS/MS) detection to allow for a more rapid(extraction and chromatography) and selective method for the determination of alfuzosin inhuman plasma than has previously been published. The assay method was used to quantifyalfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steadystate.•Two selective methods for the determination of monic acid (a metabolite of mupirocin) in urinewere developed, using high-performance liquid chromatographic separation with tandem massspectrometric detection. An Applied Biosystems API 2000 triple quadrupole mass spetrometerin multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positiveionisation, was used (molecular ion of monic acid m/z 345.2 to the product ion m/z 327.0) Theminimal sample preparation and short chromatography time (retention time ~ 3.8 min.) makesthese methods suitable for the assay of large numbers of samples per day. Linearity (weighted1/concentratiorr²) was established from 50.1 to 1001 ng/ml for the direct injection method, andlinearity (weighted 1/concentration) was established from 15.8 to 1013 ng/ml for the SPEmethod. The assay method was used for the determination of monic acid concentrations inhuman urine in order to detect and quantify the absorption of mupirocin after multiple topicalapplications ofO.5 g of a 2 % ointment.Analytical data that were generated during these three research projects are discussed in thisdissertation, improvements and novelties to existing methods are elucidated. The methods for thedetermination of stavudine and alfuzosin have been published. Both full-length publications areincluded in this dissertation, together with correspondence between myself and the journal editorsand referees. The assay methods for monic acid will soon be submitted for publication in theJournal of Chromatography B.

[发布机构] University of the Free State