Development and Validation of Micellar Liquid Chromatography Method for Separation of Penicillin Group Antibiotics and Analysis of Penicillin V in Pharmaceutical and Urine Samples

Abstract

Penicillin group antibiotics are the most commonly used antibiotics group due to the have less side effects and bactericide. Despite the positive effects of these drugs, the presence of food or aqueous media after consumption leads to serious health problems. Furthermore, in the presence of penicillin residues in animal feed, due to the consumption of meat and dairy products may have a negative impact on public health such as severe allergic reaction. Therefore, an improvement in analytic methods for determination of such compounds or an information about the characteristics of these compounds, will make great contributions. HPLC methods is used for idendification of the active substance in the form of pharmaceutical (tablets, capsules, etc.) and to determine the drug and possible metabolites in plasma and serum physiological fluids during clinical trials. In this study, micellar liquid chromatography (MLC) method has been developed and validated for the separation of the penicillin group 4 compounds (methicillin, penicillin V, amoxacillin, ampicillin) and quantitative determination of penicillin V from pharmaceutical and urine samples. Analyzes were performed on the X-Terra RP18 (250 mm x 4,6 mm I.D., 5 μm) column. The mobile phase is 0.01 phosphate (pH 3.5) buffer containing 0.05 M sodium dodecylsulfate in 5 % (v/v) 1-butanol-water. Flow rate is 1.3 mL min-1, injection volume is 20 µL, column temperature is 30 oC, detection wavelength is 200 nm. For the developed method, system suitability parameters were studied and acceptable results were obtained. Method was validated by the determination of linearity, selectivity, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision parameters. The calibration curve for penicillin V is linear in the concentration range of 2-20 mg/L. LOD and LOQ were found to be 0.203 mg/L and 0.679 mg/L, respectively. This method has been applied to the determination of penicillin V in various pharmaceutical formulations and human urine samples.

Keywords

• Micellar liquid chromatography
• Beta lactam antibiotics
• Penicillin V
• Method validation

Penisilin Grubu Antibiyotiklerin Ayrımı ve Penisilin V’nin Farmasötik ve İdrar Numunelerinde Analizi İçin Miseller Sıvı Kromatografi Yönteminin Geliştirilmesi ve Validasyonu

Abstract

Penisilin grubu antibiyotikler, yan etkilerinin azlığı ve bakterisid olmaları nedeniyle günümüzde en sık kullanılan antibiyotik grubudur. Bu ilaçların olumlu etkilerine rağmen tüketiminden sonra yiyecek veya sulu ortamlarda bulunması ciddi sağlık sorunlarına yol açmaktadır. Ayrıca hayvan yemindeki penisilin kalıntılarının varlığı, insanların et ve süt ürünlerini tüketmesi ile ağır alerjik tepki gibi halk sağlığı üzerinde olumsuz etkiler yaratabilmektedir. Bu nedenle bu bileşiklerin tayinleri için analitik metotlarda bir gelişme veya bu bileşiklerin özellikleri ile ilgili bir bilgi büyük katkı sağlayacaktır. İlaç formunda yer alan (tablet, kapsül vb.) etken maddenin tanımlanmasında, klinik çalışmalar sırasında plazma ve serum gibi fizyolojik sıvılardaki ilaç ve olası metabolitlerin tayini için HPLC yöntemi kullanılmaktadır. Bu çalışmada penisilin grubu 4 bileşiğin (metisilin, penisilin V, amoksasilin, ampisilin) ayrımı ve farmasötik ve idrar numunelerinden Penisilin V’nin kantitatif tayini için miseller sıvı kromatografi (MLC) metodu geliştirilmiş ve valide edilmiştir. Analizler X-Terra RP18 (250 mm x 4,6 mm I.D., 5 μm) kolununda gerçekleştirilmiştir. Hareketli faz olarak 1- bütanol-su (% 5 (h/h)) ortamında 0,05 M sodyum dodesil sülfat içeren 0,01 fosfat (pH 3,5) tamponu kullanılmıştır. Akış hızı 1,3 mL dk-1, enjeksiyon hacmi 20 µL, kolon sıcaklığı 30 0C, dedeksiyon dalga boyu 200 nm’dir. Geliştirilen metot için sistem uygunluk parametreleri çalışılmış ve kabul edilebilir sonuçlar elde edilmiştir. Doğrusallık, seçicilik, dedeksiyon limiti (LOD), kantitatif tayin limiti (LOQ), gün içi ve günler arası kesinlik parametreleri belirlenerek metot validasyonu gerçekleştirilmiştir. Penisilin V için kalibrasyon eğrisi 2-20 mg/L konsantrasyon aralığında doğrusaldır (R2=0,9998) ve LOD ve LOQ sırasıyla 0,203 mg/L ve 0,679 mg/L olarak bulunmuştur. Bu metot çeşitli farmasötik formülasyonlar ve insan idrar numunelerinde penisilin V tayinine uygulanmıştır.

Keywords

• Miseller sıvı kromatografi
• Beta-laktam antibiyotikler
• Metot validasyonu
• Penisilin V

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