Establishment Of SPE-LC-MS/MS Method For Doxazosin And Metabolism Of Doxazosin In Rats

Doxazosin mesylate(DOX),a highly selective and long-acting α1 adrenoreceptor antagonist and piperazine derivative,is the frist-line drug in the treatment of benign prostatic hyperplasia(BPH)complicated with lower urinary tract symptoms(LUTS).In the meantime,it has demonstrated efficacy for the treatment of hypertension as a common add-on drug.Containing a chiral carbon atom,doxazosin is a chiral drug consisting of two optical isomers,(+)doxazosin and(-)doxazosin,and is used clinically as a racemate which is composed of an equal amount of(+)doxazosin and(-)doxazosin.Studies on(+)doxazosin and(-)doxazosin in animals have shown that the two optical isomers have significant differences in pharmacokinetics,pharmacological action and toxic effect.(-)Doxazosin may have certain advantages over(+)doxazosin in the treatment of BPH.The efficacy of drugs is known to be closely related to the blood concentration,including doxazosin.Previous studies have shown that both hypotensive effect and α1-adrencoceptor inhibitor activity are directly related to the concentration of doxazosin in blood.In addition,the monitoring of drug blood concentration can provide the basis for safe,rational and effective use of drugs in clinical practice.Over the past decade,advanves in instrumental analysis techniques contribute to monitoring of drug blood concentration,thereby promoting the development of pharmacokinetics research in vivo.Most importantly,mass spectrometry has repeatedly been proven to be a powerful technique for the rapid,quantitative determination of drugs and metabolites in physiologic fluids,which lays the foundation for the development of pharmacokinetics and metabonomics.HPLC-UVD,HPLC-FLD and HPLC-MS methods are generaly used for quantitative determination of doxazosin in biological samples.However,the value of lowest limit of quantification(LLOQ)for doxazosin in the current method is no more than 0.1 ng·mL-1.Neverthless,in terms of studies on the pharmacokinetics of doxazosin,it is of graet significance to explore the lower plasma concentration and the more sensitive method determining doxazosin concentration in plasma.In our study,we established a new solid phase extraction-liquid chromatography-tandem mass spectrometry(SPE-LC-MS/MS)method for the quantification of(-)doxazosin at very low concentration in rat plasma.And the lowest limit of quantification was 10.4 pg·mL-1.The new SPE-LC-MS/MS method was also suitable for determining concentration of(+)doxazosin and(±)doxazosin in biological samples.In additon,a LC-MS/MS method was established to study the pharmacokinetics and metabolites of(±)doxazosin in rats after intravenous administration of(±)doxazosin(0.686 mg·kg-1).Part one Establishment of SPE-LC-MS/MS method for(-)doxazosin at very low concentration in rat plasma samplesObjective: Previous publications have shown that the value of LLOQ for doxazosin and its enantiomers in biological samples is above 0.1 ng·mL-1.The present study aims to establish a new LC-MS/MS method for the quantification of(-)doxazosin at very low concentration in rat plasma after intravenous administration of(-)doxazosin(3.0 mg·kg-1).Methods: The plasma samples containing prizosin as an internal standard were extracted by solid-phase extraction(SPE)and separated on ACQUITY BEH C18(50 mm × 2.1 mm,1.7 μm)column under alkaline conditions of the mobile phase at 0.45 mL·min-1.(-)Doxazosin was monitored under positive ionization condition by multiple reaction monitoring(MRM)with an ESI source.Results: The retention time of(-)doxazosin and the internal standard was 0.47 and 0.35 min,respectively.The chromatograms of(-)doxazosin and the internal standard were also inspected visually for interfering endogenous substances from chromatographic peaks.The good linear range of(-)doxazosin varied from 10.4 pg·mL-1 to 13 ng·mL-1(r=0.9922),and the lowest limit of quantification was 10.4 pg·mL-1.An assessment of the matrix effect using post-extraction spiking method and post-column infusion method revealed that co-eluting matrix components did not significantly influenced the ionization of(-)doxazosin and IS.Conclusion: The new method we used led to accurately measured(-)doxazosin concentration at 48 h after intravenous administration to the rats,and the concentration was 0.0344 ± 0.0102 ng·mL-1.The method is specific,sensitive,and applicable for determining(-)doxazosin at very low concentration in rat plasma samples,which provides new insights into the study of other clinical drugs.Part two Pharmacokinetics and metabolites study of(±)doxazosin in rats after intravenous administrationObjective: To explore pharmacokinetics of(±)doxazosin in rats after intravenous administration,and to study the metabolites of(±)doxazosin in rats by establishing a LC-MSn method.Methods: Six Sprague-Dawley male rats were intravenously administrated with(±)doxazosin at a dosage of 0.686 mg·kg-1.Blood samples were subsequently collected from fundus oculi venous plexus at different time points and blood samples were collected at 72 h into heparinized capillary tube after intravenous administration.The plasma samples were pretreated by liquid-liquid extraction method,in which extraction solvent was a mixture solution composed of hexane and ethyl acetate at the ratio of 1:1(v/v).The plasma concentration of(±)doxazosin in every rat at different time points was assayed by UPLC-MS/MS.The chromatographic separation of plasma samples containing prizosin as an internal standard was achieved on Hypersil Gold C18(100 mm × 2.1 mm,1.9 μm)column with gradient elution,and the column temperature was set at 35 ℃.The mobile phase was acetonitrile-0.1% formic acid and the flow rate was 0.35 mL·min-1.Because plasma concentration-time data were most appropriately fitted to two compartmental model,so the main pharmacokinetic parameters of(±)doxazosin after single intravenous administration were calculated by two compartmental model for IV-bolus input using WinNonlin 5.1 software.The metabolites of(±)doxazosin in rat were identified by the high resolution orbitrap fusion mass spectrometry.Results: The main pharmacokinetic parameters of Dox were caculated using the two-compartment model.The main pharmacokinetic parameters t1/2β and AUC0-t of(±)doxazosin after single intravenous administration were(1.44 ± 0.27)h and(403.63 ± 50.88)h·ng·mL-1,respectively.Of them,the pharmacokinetic parameter t1/2β was in accordance with the previous finding(1.41 ± 0.15)h in our laboratory.We indentified a total of 9 metabolites,including 3 monohydroxylated products,1 oxygen demethyl product,1 product of piperazine ring after hydroxylation and dehydration,M4(m/z 451.1612),M5(m/z 470.2034),M6(m/z 466.1721)and M7(m/z 422.1459).Of them,hydroxylation,oxygen demethylation,piperazine ring hydroxylation after dehydration products have been reported;M4-M7 were identified for the first time.In the process of piperazine ring hydroxylation,the original drug DOX underwent hydroxylation,dehydration,and deamination reaction to produce M4;benzodioxane ring undergoing hydroxylation was to produce M5;piperazine ring underwent hydroxylation and dehydration to produce M6;piperazine ring underwent hydroxylation and dehydration as well as oxygen desmethyl reaction to produce M7.Conclusion: When plasma samples were collected at 48 h,the half-life of(±)doxazosin in rats was consistent with that reported in the medical literatures.In the meantime,we indentify 4 metabolites(M4-M7)of(±)doxazosin in rats which have not been reported,and the metabolic modifications are still mainly C-hydroxylation,and their metabolic modifications process for(±)doxazosin is more complex than the known metabolites.