İtrakonazol ve Metabolitinin İnsan Plasmasından HPLC-Tandem Mass LC-MS/MS Spektrometresiyle Tayini
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.
Anahtar Kelimeler:
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
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,
___
- Suarez-Kurtz, G., Bozza, F.A., Vicente, F.L., Ponte, C. G., Struchiner, C. J. :Limited- sampling strategy models for Itraconazole and Hydroxy-Itraconazole based on data from a bioequivalence study. Antimicrob. Agents Chemother., 134-140, 43, (1) (1999)
- Bharathi, D.V., Hotha, K.K., Sagar, P.V.V., Kumar, S.S., Reddy, P.R., Naidu, A., Mul- langi, R.: Development and validation of a highly sensitive and robust LC-MS/MS with electrosray ionization method for simultaneous quantitation of itraconazole and hy- droxyitraconazole in human plasma: Application to a bioequivalence study. J. Chro- matograhy B, 1, 868(1-2), 70-6, (2008)
- Vogeser, M., Spöhrer, U., Schiel, X.: Determination of Itraconazole and Hydoxyitra- conazole in plasma by use of liquid chromatography-Tandem Mass Spectrometry with On-line solid-Phase extraction. Clin. Chem. Lab. Med., 41(7), 915-920, (2003)
- Choi, Y.W., Nam, D., Kang, K.H., Ha, K.W., Han, I.H., Chang, B. K., Yoon, M., Lee, J.: High-Performance Liquid Chromatographic-Tandem Mass Spectrometric Determina- tion of Itraconazole in Human Plasma for Bioavailability and Bioequivalence Studies. Bull. Korean Chem. Soc., 27, (2), 291-294, (2006)
- Sirvatsan, V., Dasgupta, A.K., Kale, P., Rama, R.D., Soni, D., Patel, M., Patel, R., Ma- vadhiya, C.: Simultaneus determination of itraconazole and hydoxyitraconazole in hu- man plasma by high-performance liquid chromatography.J. Chromatograhy, 1031, (1-2) 307-313, (2004)
- Wong, J.W., Nisar, U., Yuen, K.H.:Liquid chromatographic method for the determina- tion of plasma itraconazole and its hydroxy metabolite in pharmacokinetic/bioavail- ability studies.J. Chromatograhy B, 798, (2) 355-360, (2003)
- Guidance for Industry, Bioanalytical Method Validation, FDA, (2001)
- Hartmann C., Smeyers-Verbeke J., Massart D.L., McDowall, R.D.: Validation of bioana- lytical chromatographic methods. J. Pharm. Biomed. Anal., 17(2), 193-218, (1998)
- Yayın Aralığı: Yılda 4 Sayı
- Başlangıç: 1981
- Yayıncı: Hacettepe Üniversitesi Eczacılık Fakültesi Dekanlığı
Sayıdaki Diğer Makaleler
İnsülin Glarjin Kullanımının Toksikolojik Açıdan Değerlendirilmesi
Pınar ERKEKOĞLU, Belma GİRAY, Gönül ŞAHİN
Pharmacy Education and Practice in Pakistan: A Guide to Further Development
Ghulam MURTAZA, Mahmood AHMAD, Muhammad IQBAL, Shujaat Ali KHAN, Munazza EJAZ, Tehmina YASMİN
İtrakonazol ve Metabolitinin İnsan Plasmasından HPLC-Tandem Mass LC-MS/MS Spektrometresiyle Tayini