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

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 yolaçmaktadır. Ayrıca hayvan yemindeki penisilin kalıntılarının varlığı, insanların et ve süt ürünlerini tüketmesi ile ağır alerjik tepki gibihalk sağlığı üzerinde olumsuz etkiler yaratabilmektedir. Bu nedenle bu bileşiklerin tayinleri için analitik metotlarda bir gelişme veya bubileşiklerin özellikleri ile ilgili bir bilgi büyük katkı sağlayacaktır. İlaç formunda yer alan (tablet, kapsül vb.) etken maddenintanımlanmasında, klinik çalışmalar sırasında plazma ve serum gibi fizyolojik sıvılardaki ilaç ve olası metabolitlerin tayini için HPLCyöntemi kullanılmaktadır. Bu çalışmada penisilin grubu 4 bileşiğin (metisilin, penisilin V, amoksasilin, ampisilin) ayrımı ve farmasötikve 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 20µL, kolon sıcaklığı 30 ${}^oC$, dedeksiyon dalga boyu 200 nm’dir. Geliştirilen metot için sistem uygunluk parametreleri çalışılmış ve kabuledilebilir sonuçlar elde edilmiştir. Doğrusallık, seçicilik, dedeksiyon limiti (LOD), kantitatif tayin limiti (LOQ), gün içi ve günler arasıkesinlik parametreleri belirlenerek metot validasyonu gerçekleştirilmiştir. Penisilin V için kalibrasyon eğrisi 2-20 mg/L konsantrasyonaralığında doğrusaldır ($R^2$=0,9998) ve LOD ve LOQ sırasıyla 0,203 mg/L ve 0,679 mg/L olarak bulunmuştur. Bu metot çeşitli farmasötikformülasyonlar ve insan idrar numunelerinde penisilin V tayinine uygulanmıştır.

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

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 lasma 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.

PDF

___

Babington, R., Matas, S., Marco, M.P., Galve, R. (2012), Current bioanalytical methods for detection of penicillins, Analytical and Bioanalytical Chemistry, 403, 1549–1566.
Becker, M., Zittlau, E., Petz, M. (2004), Esidue analysis of 15 penicillins and cephalosporins in bovine muscle, kidney and milk by liquid chromatography-tandem mass spectrometry, Analytical Chemistry Acta, 520, 19–32.
Berthod, A., Garcia-Alvarez-Coque, M.C. (2000), Micellar Liquid Chromatography, 622 p. Marcel Dekker Inc. Newyork.
Cámara, M., Gallego-Picó, A., Garcimuño, R.M., Fernández-Hernando, P., Durand-Alegría, J.S., Sánchez, P.J. (2013), An HPLC-DAD method for the simultaneous determination of nine β-lactam antibiotics in ewe milk, Food Chemistry, 141, 829–834.
Dural, E.A.Ö., (2012), Farmakoloji, İstanbul: Nobel Tıp Kitapevleri.
El-Shaheny, R.N., El-Maghrabey, M.H., Belal, F.F. (2015), Micellar liquid chromatography from green analysis perspective, Open Chemistry, 13, 877–892.
Evaggelopoulou, E. N., Samanidou, V. F., (2013), Development and validation of an HPLC method for the determination of six penicillin and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue according to the European Union Decision 2002/657/EC, Food Chemistry, 136, 1322-1329.
Fagerquist, C.K., Lightfield, A.R. (2003), Confirmatory analysis of β-lactam antibiotics in kidney tissue by liquid chromatography/electrospray ionization selective reaction monitoring ion trap tandem mass spectrometry, Rapid Communications in Mass Spectrometry, 17, 660–671.
Joshi, S., (2002), HPLC separation of antibiotics present in formulated and unformulated samples, Journal of Pharmaceutical and Biomedical Analysis, 28, 795–809
Pryde, A., Gilbert, M.T. (1999), Applications of High Performance Liquid Chromatography, Mishawaka: Better Word Books.
Ramezani, A.M., Absalan, G., Ahmadi, R. (2018), Green-modified micellar liquid chromatography for isocratic isolation of some cardiovascular drugs with different polarities through experimental design approach, Analytical Chemistry Acta, 1010, 76–85.
Riediker, S., Rytz, A., Stadler, S.H. (2004), Cold-temperature stability of five β‐lactam antibiotics in bovine milk and milk extracts prepared for liquid chromatography-electrospray ion- ization tandem mass spectrometry analysis, Journal of Chromatography A, 1054, 359–363.
Ruiz-Angel, M.J., Caballero, R.D., Simo-Alfonso, E.F., Garcia-Alvarez-Coque, M.C. (2002), Micellar liquid chromatography: suitable technique for screening analysis, Journal of Chromatography A 947(1): 31-45.
Samanidou, V. F., Evaggelopoulou, E. N. (2007), Analytical strategies to determine antibiotic residues in fish, Journal of Separation Science, 30, 2549–2569.
Samanidou, V. F., Evaggelopoulou, E. N., Papadoyannis, I. N. (2006), Chromatographic analysis of penicillins in pharmaceutical formulations and biological fluids, Journal of Separation Science, 29, 1879–1908.
Schugerl, K. (2001), Progress in Monitoring, Modeling and Control of Bioprocesses During the Last 20 Years, Journal of Biotechnology, 85, 149-173.
Song, E., Yu, M., Wang, Y., Hu, W., Cheng, D., Swihart, M.T., Song, Y. (2015), Multi-color quantum dot-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk, Biosensors and Bioelectronics, 72, 320–325.
Tong, L., Wang, Y.X., Hermo, M.P., Barrón, D., Barbosa, J. (2014), Simultaneous determination and toxicological assessment of penicillins in different water matrices, Ecotoxicology 23, 2005–20.