Simultaneous Determination of Acyclovir, Metoprolol and Phenol Red by a RP-HPLC Method for Intestinal Perfusion Studies

Year 2015, Issue: 2, 146 - 161, 01.06.2015

Müge Ateş Mustafa Sinan Kaynak Selma Şahin

Abstract

Barsak perfüzyon tekniği, bir ilacın barsaklardan permeabilitesini tayin etmek amacıyla en çok kullanılan tekniklerden biridir. Perfüzyon çalışmalarında incelenen maddenin bu çalışmada asiklovir permeabilite katsayısını değerlendirmek amacıyla metoprolol ve fenol kırmızısı referans maddeler olarak sıklıkla kullanılır. Bu çalışmanın amacı, barsak perfüzyonu çalışmalarında kullanılmak için asiklovir, metoprolol ve fenol kırmızısının eş zamanlı olarak tayin edileceği bir ters faz sıvı kromatografisi yöntemi geliştirmek ve valide etmektir. Analiz bir C18 kolonunda 4.6 mm x 250 mm, 5 μm metanol: 0.0125 M potasyum dihidrojen fosfat tampon 55:45, h/h; pH 7.0 ’ dan oluşan mobil faz kullanılarak gerçekleştirilmiştir. Yöntem FDA kılavuzuna göre seçicilik, duyarlılık, doğrusallık, kesinlik, doğruluk ve stabilite için valide edilmiştir. Tüm kalibrasyon eğrileri doğrusaldır r2> 0.999 . Kantitatif olarak tayin edilebilir en küçük değer asiklovir için 0.04 μg/mL, metoprolol için 0.02 μg/mL, fenol kırmızısı için 0.01 μg/mL’dir. Saptayabilme sınırı asiklovir için 0.01 μg/mL, metoprolol için 0.002 μg/mL, fenol kırmızısı için 0.003 μg/mL’dir. Kesinlik ve doğruluk sonuçları talep edilen sınırları karşılamıştır. Yeni geliştirilen ve valide edilen bu yöntem in situ barsak permeabilite deneylerinde rutin olarak kolayca kullanılabilir.

Keywords

Asiklovir, metoprolol, fenol kırmızısı, HPLC, in situ barsak perfüzyonu

Simultaneous Determination of Acyclovir, Metoprolol and Phenol Red by a RP-HPLC Method for Intestinal Perfusion Studies

Abstract

Intestinal perfusion SPIP technique is one of the most commonly used techniques to determine the intestinal permeability of a drug. In perfusion studies, metoprolol as tartrate and phenol red are widely used as a reference compounds to evaluate the permeability coefficient of the compound of interest acyclovir in this study . The aim of our study was to develop and validate a reversed-phase liquid chromatographic method for the simultaneous determination of acyclovir, metoprolol and phenol red for use in intestinal perfusion studies. The analysis was performed on a C18 column 4.6 mm x 250 mm, 5 μm using a mobile phase consisting of methanol:0.0125 M potassium dihydrogen phosphate buffer 55:45, v/v; pH 7.0 . Method was validated according to the FDA guidelines for selectivity, sensitivity, linearity, precision, accuracy, stability. All calibration curves were linear r2> 0.999 . Lower limit of quantitation was 0.04 μg/mL for acyclovir, 0.02 μg/mL for metoprolol, 0.01 μg/mL for phenol red. Detection limit was 0.01 μg/mL for acyclovir 0.002 μg/mL for metoprolol, 0.003 μg/mL for phenol red. Precision and accuracy results of the method fulfilled the required limits. This newly developed and validated method can be readily used on a routine basis for the standardization of in situ intestinal permeability experiments.

Keywords

Acyclovir, metoprolol, phenol red, HPLC, in situ intestinal perfusion

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