Establishment And Application Of Chiral And Achiral High-performance Liquid Chromatography For Doxazosin

Doxazosin, a quinazoline-derivative, is a long acting and selectivepostsynaptic a1-adrenoceptor antagonist. Doxazosin is a chiral drug with twoenantiomers for one asymmetric carbon atom in its molecule. Now it has beenused as a racemic for clinical treatment of benign prostatic hyperplasia/lowerurinary tract symptoms (BPH/LUTS) as a first line drug and essentialhypertension as a common add-on drug.As is well known, the efficacy of drugs is closely related to their plasmaconcentration. Therefore, it is essential to study the changes of the plasmaconcentration of (±)doxazosin for assessing its clinical curative effect andadverse reactions during the treatment. An achiral HPLC method wasdeveloped for the determination of (±)doxazosin in rat plasma. Then themethod was successfully applied to study the pharmacokinetics of(±)doxazosin after an oral and intravenous administration to rats.Since a lot of chiral biological macromolecules in biosystem, differentdrugs will interact with different macromolecules according to theirstereochemical structure, which will generate different biological responsesand display different pharmacokinetics and pharmacodynamics. As a result,chiral drugs, such as doxazosin, often exist stereoselective disposition in vivo.So it is important to study the relationship between the plasma concentrationand their clinical curative effect of the two enantiomers of doxazosin. In ourstudy, we established a chiral HPLC method using Ultron ES-OVM chiralchromatographic column for the determination of doxazosin enantiomers in ratplasma. We also investigated their chiral inversion in vitro and vivo.Part one Establishment of achiral HPLC method for doxazosinObjective: To establish an achiral HPLC method using fluorescencedetector for the determination of (±)doxazosin. Methods: Optimize the wavelength for fluorescence detection of(±)doxazosin. Prazosin was used as the internal standard. Investigate the effectof different mobile phase, flow rate, buffer concentration and pH on theresolution.Results: The chromatographic conditions of HPLC-FLD method wasestablished. The chromatographic column was Agilent C18and the columntemperature was at30°C. Isocratic elution was carried out using a mobilephase of buffer-methanol (44:56, v/v; buffer:30mmol·L-1phosphate buffer,pH3.1) at a flow rate of1.0mL·min-1. The fluorescence detection was set atλEx=255nm and λEm=385nm. Prazosin was used as the internal standard.Conclusion: Under the chromatographic condition, a baseline separationof prazosin and doxazosin was obtained with good shapes.Part two The pharmacokinetics study after an oral and intravenousadministration of (±)doxazosin to ratsObjective: To establish an HPLC method using fluorescence detector forthe determination of (±)doxazosin in rat plasma and study itspharmacokinetics after an oral and intravenous administration of (±)doxazosinto rats.Methods:14Sprague-Dawley male rats were randomly divided into oraland intravenous group, which administered (±)doxazosin at a dosage of6mg·kg-1, respectively. The animal plasma was pretreated by liquid-liquidextraction, which use mixtures of n-hexane and ethyl acetate (1:1, v/v) asextraction solvent. The plasma concentration of (±)doxazosin was assayed byHPLC with fluorescence detection. Chromatographic separation wasperformed on an Agilent C18column, and the column temperature was at30°C. Isocratic elution was carried out using a mobile phase of buffer-methanol(44:56, v/v; buffer:30mmol·L-1phosphate buffer, pH3.1) at a flow rate of1.0mL·min-1. The fluorescence detection was set at λEx=255nm and λEm=385nm.Prazosin was used as the internal standard. WinNonlin software was used tocalculate the pharmacokinetic parameters after oral and intravenousadministration. We also evaluate the oral bioavailability of (±)doxazosin in rats.Results: A good linear relationship was achieved when the concentrationof (±)doxazosin was within the range of4-4000ng·mL-1. The averagerecovery for (±)doxazosin was101.8%with RSD5.7%. After an oraladministration, the t1/2and AUC were (2.19±0.35) h and (1939.14±0.19)h·ng·mL-1, respectively; after an intravenous administration, the t1/2and AUCwere (1.41±0.15) h and (4271.80±0.09) h·ng·mL-1, respectively.Conclusion: The method is selective, accurate and reproducible, which issuitable for the detection of (±)doxazosin in rat plasma. Based on thepharmacokinetic parameters after oral and intravenous administration, the oralbioavailability of (±)doxazosin in rats is45.4%.Part three Establishment of chiral HPLC method for doxazosinObjective: To establish a chiral HPLC method using fluorescencedetector for the determination of doxazosin enantiomers.Methods: The effect of mobile phase pH, flow rate and different organicmodifier on the resolution of doxazosin enantiomers was investigated.Results: The chiral chromatographic conditions of HPLC-FLD methodwere established. Ultron ES-OVM was taken as the chiral chromatographiccolumn, and the column temperature was at30°C. Isocratic elution wascarried out using a mobile phase of buffer-acetonitrile (85:15, v/v; buffer:20mmol·L-1phosphate buffer, pH5.3) at a flow rate of0.8mL·min-1. Thefluorescence detection was set at λEx=255nm and λEm=385nm. Prazosin wasused as the internal standard.Conclusion: A chiral HPLC-FLD method of (±)doxazosin wasestablished on the Ultron ES-OVM chiral chromatographic column. A baselineseparation of the two enantiomers was obtained within11min.Part four Determination of doxazosin enantiomers in rat plasma andinvestigation of their chiral inversionObjective: To establish an HPLC method using fluorescence detector forthe determination of doxazosin enantiomers and investigate their chiralinversion in vitro and vivo. Methods: The animal plasma was pretreated by liquid-liquid extraction,which use mixture of n-hexane and ethyl acetate (1:1, v/v) as extractionsolvent. Ultron ES-OVM was taken as the chiral chromatographic column,and the column temperature was at30°C. Isocratic elution was carried outusing a mobile phase of buffer-acetonitrile (85:15, v/v; buffer:20mmol·L-1phosphate buffer, pH5.3) at a flow rate of0.8mL·min-1. The fluorescencedetection was set at λEx=255nm and λEm=385nm. Prazosin was used as theinternal standard.(-)Doxazosin or (+)doxazosin added into rat plasma in vitrowas determined after incubating in37°C water bath for2,5and10days.(-)Doxazosin or (+)doxazosin was administered orally to the rats for onemonth. Plasma samples were taken at8h after the last administration.Results: A good linear relationship was achieved when the concentrationof doxazosin enantiomers was within the range of4-2000ng·mL-1. Theaverage recovery for (-)doxazosin was99.5%with RSD3.6%, and for(+)doxazosin was99.3%with RSD4.3%. Chiral inversion was observedneither in vitro nor in vivo studies.Conclusion: The method is selective, accurate and reproducible, which issuitable for the detection of doxazosin enantiomers in rat plasma. The in vitroand in vivo studies indicate that chiral inversion occurs uneasily between(-)doxazosin and (+)doxazosin in rats.