İtrakonazol ve Metabolitinin İnsan Plasmasından HPLC-Tandem Mass LC-MS/MS Spektrometresiyle Tayini

Year 2010, Issue: 2, 125 - 138, 01.06.2010

Zeynep İrem Diler Durişehvar Özer Ünal Dilek Demir Erol

Abstract

İtrakonazol ITR , triazol türevi bir antifungal ilaçtır. Öncelikli olarak mantar hücre membranlarının gerekli bileşeni olan ergosterol sentezini inhibe etmede rol oynar. Çeşitli sistemik mikozların tedavisinde önemli yer tutar. Oral absorbsiyonun arkasından, yan zincir hidroksillenme CYP3A4 ile ile yaygın bir şekilde metabolize olarak hidroksi-itrakonazole HITR dönüşür. ITR ve metabolitinin insan plasmasından HPLC-MS sistemiyle tayininde basit ve hassas bir biyoanalitik metod geliştirildi ve valide edildi. İtrakonazol ve metaboliti, plazmadan sıvı-sıvı ekstraksiyonla ekstrakte edildi. Ekstraksiyon solventi olarak hekzan: isoamilalkol 97:3 karışımı kullanıldı. X-Terra RP18, 3.5 μm, 50 x 4.6 mm HPLC kolonu ve [ asetonitril: 0.066 % ammonia solution 80:20 v/v ] mobil fazı kullanılarak ITR, metaboliti ve ketakonazolün IS kromatografik ayrımı sağlandı. Mobil faz akış hızı 0.4 mL/dak, enjeksiyon hacmi 15μL’dir. Kütle spektrometresine ait parametreler birim rezolüsyonda maksimum hassasiyet sağlayacak şekilde optimize edildi. API-ES pozitif modda çalışıldı. Datalar MRM multiple reaction monitoring olarak toplandı. ITR için m/z 705.30>392.30, HITR için m/z 721.25 >408.30, IS için m/z 531.15 >489.20 kütleleri üzerinden miktar tayini yapıldı. ITR için 1-600 ng/mL, HITR için 2-600 ng/mL derişim aralığında doğrusal kalibrasyon eğrisi oluşturuldu. LOQ değerleri ITR için 1 ng/mL, HITR için 2 ng/mL’dir. Çeşitli koşullarda stabilite sonuçları değerlendirildi ve analiz sonuçları uygun bulundu. Geliştirilen bu metod bioeşdeğerlik ve farmakokinetik çalışmalarda kullanılabilir.

Keywords

İtrakonazol, Hidroksi-itrakonazol, Ketakonazol, insan plazması, LC-MS/MS, bioanalitik metod validasyonu

Determination of Itraconazole and its Metabolite From Human Plasma by High Performance Liquid Chromatography- Tandem Mass LC-MS/MS Spectrometry

Abstract

Itraconazole is a triazole antifungal agent with a broad spectrum of activity. It acts primary by inhibiting the biosynthesis of ergosterol, an essential component of fungal cell membrans. It is used in the treatment of a variety of fungal infections. Following oral absorption, it is extensively metabolized by side chain hydroxylation by CYP3A4 to form hydroxy-itraconazole. A simple and sensitive high performance liquid chromatographic method with MS detection HPLC-MS for the determination of itraconazole and its metabolite from plasma was developed and validated. Liquid-liquid extraction was used for extracting itraconazole and its metabolite from plasma. hexan:isoamylalcohol 97:3 was used as extraction solvent. The chromatographic separation of itraconazole, metabolite and ketoconazole IS was carried out using reverse phase X-Terra RP18, 3.5 μm, 50 x 4.6 mm with mobile phase of [ Acetonitrile: 0.066 % ammonia solution 80:20 v/v ]. The flow rate of mobile phase was 0.4 mL/min, injection volume was 15 μL. The mass spectrometric parameters were optimized to obtain maximum sensitivity at unit resolution. Atmospheric pressure ionization-electrospray mode API-ES was used at positive ionization. Data was collected by multiple reaction monitoring MRM . The ions used to quantify were selected as m/z 705.30>392.30 for itraconazole, m/z 721.25 >408.30 for hydroxy-itraconazole, and m/z 531.15 >489.20 for IS. The calibration curve was linear within the concentration range 1-600 ng/mL for itraconazole and 2-600 ng/mL for hydroxy- itraconazole. The limit of quantification was 1 ng/mL and 2 ng/mL itraconazole, hydroxy-itraconazole, respectively, with good accuracy and precision. The stability was assessed under a variety of conditions and found that appropriate for the quantification. The method developed can be used for bioequivalence and pharmacokinetic studies.

Keywords

Itraconazole, Hydroxy-itraconazole, ketoconazole, human plasma, LC-MS/MS, bioanalytical method validation

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