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dc.contributor.advisorSwart, K. J.
dc.contributor.advisorHundt, H. K. L.
dc.contributor.authorWiesner, Joachim Lubbe
dc.date.accessioned2017-05-16T07:24:38Z
dc.date.available2017-05-16T07:24:38Z
dc.date.issued2003-05
dc.identifier.urihttp://hdl.handle.net/11660/6219
dc.description.abstractEnglish: The development and validation of bio-analytica) assay methods suitable for the quantification of stavudine in plasma, alfuzosin in plasma and monic acid in urine is discussed. A short summary of these methods are given: • A sensitive method for the determination of stavudine in plasma was developed, using highperformance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18®solid phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery" C18, 5 urn, 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 was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCl) was used to obtain deprotonated ions (molecular ion m/z 223.1 to the product ion m/z 42.01). The mean recovery for stavudine was 94 % with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (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 previously been published. The assay metod was used to quantitatively determine stavudine concentrations in plasma samples to follow the concentration vs. time profile for at least five half lives of the drug after a single 40 mg oral dose of stavudine was given to healthy adult male human subjects. • A selective, sensitive and rapid liquid chromatography-tandem mass speetrometry method for the determination of alfuzosin in plasma was developed. An Applied Biosystems API 2000 triple quadrupole mass speetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation was used (molecular ion of alfuzosin m/z 390.2 to the 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 reversed phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9 % with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been published. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state. • Two selective methods for the determination of monic acid (a metabolite of mupirocin) in urine were developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. An Applied Biosystems API 2000 triple quadrupole mass spetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation, was used (molecular ion of monic acid m/z 345.2 to the product ion m/z 327.0) The minimal sample preparation and short chromatography time (retention time ~ 3.8 min.) makes these methods suitable for the assay of large numbers of samples per day. Linearity (weighted 1/concentratiorr²) was established from 50.1 to 1001 ng/ml for the direct injection method, and linearity (weighted 1/concentration) was established from 15.8 to 1013 ng/ml for the SPE method. The assay method was used for the determination of monic acid concentrations in human urine in order to detect and quantify the absorption of mupirocin after multiple topical applications ofO.5 g of a 2 % ointment. Analytical data that were generated during these three research projects are discussed in this dissertation, improvements and novelties to existing methods are elucidated. The methods for the determination of stavudine and alfuzosin have been published. Both full-length publications are included in this dissertation, together with correspondence between myself and the journal editors and referees. The assay methods for monic acid will soon be submitted for publication in the Journal of Chromatography B.en_ZA
dc.description.abstractAfrikaans: Die ontwikkeling en validering van bioanalitiese metodes vir die kwantifisering van geneesmiddels (stavudien in plasma, alfuzosin in plasma en "monic acid" in urien) word bespreek. 'n Kort opsomming van hierdie metodes word bespreek: • 'n Sensitiewe metode vir die bepaling van stavudien in plasma is ontwikkel deur gebruik te maak van hoë-verrigting vloeistofchromatografiese skeiding en massa-spektrometriese bepaling. Die analiet is uit plasma geëkstraheer deur gebruik te maak van Waters, Sep-Pak® Vae, 100 mg, tCls® soliede fase ekstraksiekolomme. Chromatografie is uitgevoer op 'n Supelco Discovery® Cls,5 urn, 150 x 2 mm kolom met 'n mobiele fase wat bestaan uit ammonium asetaat (0.01 M) : asetonitriel : metanol (800:100:100, v/v/v) teen 'n vloei-tempo van 0.3 ml/min. ·Die bepaling van stavudien is gedoen met 'n Applied Biosystems API 2000 massaspektrometer (LC-MS/MS) wat op eenheid resolusie in die meervoudige reaksiebepalings modus (MRM) gestel is (molekulêre ioon m/z 223.1 na die produkioon m/z 42.01). Atmosferiese druk-chemiese ionisering (APCl) is as ioniseringsproses gebruik om gedeprotoneerde ione te produseer. Die gemiddelde opbrengs vir stavudien was 94 % met 'n kwantifiseringslimiet van 4 ng/ml. In hierdie metode word 'n massa-spektrometriese bepaling met verhoogde sensitiwiteit en selektiwiteit beskryf. Daar word dus voorsiening gemaak vir 'n vinniger en meer selektiewe metode as wat voorheen gepubliseer is vir die bepaling van stavudien in menslike plasma. Die metode is aangewend vir die bepaling van stavudien konsentrasies in plasma monsters om die konsentrasie-tyd profiel van die middel vir ten minste vyf half1eeftye te bepaal, nadat 'n 40 mg stavudien dosering mondelings aan gesonde, volwasse, manlike proefpersone toegedien is. • 'n Selektiewe, sensitiewe en vinnige vloeistofchromatografiese-tandem-massa-spektrometriese metode is ontwikkel vir die bepaling van alfuzosin in plasma. 'n Applied Biosystems API 2000 massaspektrometer met 'n turbo-ioonsproeibron is gebruik. Die spektrometer is in die positiewe ioniseringsmodus gestel en meervoudige reaksiebepalings is gedoen (molekulêre ioon van alfuzosin m/z 390.2 na die produkioon m/z 71.2; molekulêre ioon van prazosin m/z 384.2 na die produkioon m/z 95.0). 'n Vloeistof-vloeistof ekstraksieprosedure is uitgevoer en prazosin is as interne standaard gebruik. Chromatografiese skeiding is op 'n CIS omgekeerde fase kolom uitgevoer. Die gemiddelde opbrengs vir alfuzosin was 82.9 % met 'n kwantifiseringslimiet van 0.298 ng/ml. Die kalibrasie reikwydte was tussen 0.298 en 38.1 ng/ml. In hierdie metode word 'n tandem massa-spektrometriese bepalingsmetode met verhoogde sensitiwiteit en selektiwiteit beskryf. Daar word dus voorsieninig gemaak vir 'n metode wat vinniger en meer selektief is as metodes wat voorheen gepubliseer is vir die bepaling van alfuzosin in menslike plasma. Hierdie metode is aangewend om alfuzosin in menslike plasma te kwantifiseer nadat veelvoudige doserings (5 mg) aan die proefpersone toegedien is. • Twee selektiewe metodes vir die bepaling van "monic acid" (metaboliet van mupirocin) in urien is ontwikkel deur gebruik te maak van hoë-verrigting vloeistofchromatografiese skeiding en massa-spektrometriese bepaling. 'n Applied Biosystems API 2000 massaspektrometer met 'n turbo-ioonsproei bron is gebruik. Die instrument is in die positiewe ioniseringsmodus gebruik en bepalings is in die meervoudige reaksiebepalingsmodus gedoen (molekulêre ioon van "monic acid" mJz 345.2 na die produkioon mJz 327.0). Die minimale monstervoorbereiding en kort chromatografietyd (retensie-tyd - 3.8 min.) maak hierdie metode uiters geskik vir die bepaling van 'n groot aantal monsters per dag. Die direkte-inspuitingsmetode het 'n lineêre kromme (l/c2 ) tussen 50.1 en 1001 ng/ml gelewer en die soliede-fase-metode het 'n lineêre kromme (lie) tussen 15.8 en 1013 ng/ml gelewer. Analitiese data wat in die drie navorsingsprojekte gegenereer is word bespreek en verbeteringe sowel as nuwighede word uitgelig. Die metodes vir die bepaling van stavudien en alfuzosin is reeds as vollengte artikels gepubliseer. Beide die twee gepubliseerde artikels tesame met die korrespondensie wat plaasgevind het tussen myself en die redakteurs en beoordelaars is ingesluit in die verhandeling. Die metodes vir die bepaling van "monic acid" sal binnekort vir publikasie aan die "Journal of Chromatography Bil voorgelê word.af
dc.description.sponsorshipFARMOVS-PAREXELen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectMethod developmenten_ZA
dc.subjectValidationen_ZA
dc.subjectHigh-performance liquid chromatographyen_ZA
dc.subjectMass spectrometryen_ZA
dc.subjectStavudineen_ZA
dc.subjectAlfuzosin and monic aciden_ZA
dc.subjectAntibioticsen_ZA
dc.subjectAntiretroviral agentsen_ZA
dc.subjectBiological assayen_ZA
dc.subjectDissertation (M.Med.Sc. (Pharmacology))--University of the Free State, 2003en_ZA
dc.titleThe development and validation of quantitative methods for the determination of stavudine and alfuzosin in plasma and monic acid in urineen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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