Determination of insulin in rabbit plasma using high-performance liquid chromatography

Bu çalışmanın amacı tavşan plazmasında insülinin tespit edilmesi için yüksek basınçlı sıvı kromatografisine (YBSK) dayalı bir yöntem geliştirmek ve geçerlilik testi yapmaktır. İnsülinin ayırımı dalga boyu λ=206 nm olan diyot array dedektör (DAD) kullanılarak Ace C18 kolon (5 μm, 250×4.6 mm i.d.) ile yapılmıştır. Hareketli faz 0.2 M sülfat tamponu (pH 2.4)-asetonitril (75:25, h/h) den oluşmuştur. Analiz 1.2 mL/dk akış hızı ile 15 dakikadan daha az sürede yapılmıştır. 0.15 ve 10 μg/mL arasında çok iyi doğrusallık bulunmuştur. Plazmada insülin için gün içi ve günler arası kesinlik değerleri %10.2’den küçüktü ve doğruluk (bağıl hata) %12.4’den daha iyiydi. Bütün örnekler için geri kazanım >%87.3 idi. İnsülinin belirlenebilen (LOD) ve ölçülebilen (LOQ) en düşük değeri sırasıyla 0.10 and 0.15 μg/mL’dir. Tanımlanan YBSK yöntemi tavşanlarda insülinin farmakokinetiğini incelemek için yeterli duyarlılığa ve özgüllüğe sahiptir ve klinik farmakokinetik çalışmalara da uyarlanabilir.

Anahtar Kelimeler:

doğruluk, analitik yöntemler, kan plazması, saptama, HPLC, insülin, metodoloji, farmakokinetik, tavşan

Yüksek performanslı sıvı kromatografisi kullanarak tavşan plazmasında insülinin tayini

The aim of the present study was to develop and validate a procedure based on high-performance liquid chromatography (HPLC) for determination of insulin in rabbit plasma. Separation of insulin was achieved on an Ace C18 column (5 μm, 250×4.6 mm i.d.) using diode array detection (DAD) with λ=206 nm. The mobile phase consisted of 0.2 M sulfate buffer (pH 2.4)-acetonitrile (75:25, v/v). The analysis was performed in less than 15 min with a flow rate of 1.2 mL/min. Excellent linearity was found between 0.15 and 10 μg/mL. Intra- and inter-day precision values for insulin in plasma were less than 10.2, and accuracy (relative error) was better than 12.4%. The recoveries for all samples were >86.7%. The limits of detection (LOD) and quantification (LOQ) of insulin were 0.10 and 0.15 μg/mL, respectively. The described HPLC method has adequate sensitivity and specificity to study pharmacokinetics of insulin in rabbits, and could be adapted also to clinical pharmacokinetic study.

Keywords:

accuracy, analytical methods, blood plasma, detection, HPLC, insulin, methodology, pharmacokinetics, rabbits,

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