Publication: Film kaplı siprofloksazin hidroklorür tabletlerinde miktar tayini metodu ve safsızlık metodu geliştirilmesi ve analitik validasyonları
Abstract
Bu çalışmada siprofloksazin hidroklorür ve safsızlıkları için Avrupa Farmakopesi Ek 4.6 ve İngiliz Farmakopesi 99 referans alınıp hammadde siprofloksazin hidroklorür için belirtilen miktar tayini ve safsızlık tayin yönteminin film kaplı tabletlerde uygunluğunun araştırılması ve analitik yöntem validasyonu yapılması amaçlanmıştır. Çalışmada hareketli faz olarak ortofosforik asidin sudaki çözeltisi (pH 3.0) / ACN (87:13 h/ h), ? = 278 nm UV detektör kullanılmıştır. Bu kromatografik şartlarda sabit fazın belirlenmesi için yapılan denemelerin sonucunda siprofloksazin hidroklorür'ün yapısına uygun, etken madde ile etkileşime girmeyen, metal safsızlıkları bulunmayan Inertsil ODS3 4.6 x 250 mm, 5 µm özellikte kolon seçilmiştir. Miktar tayini için geri kazanım %95 güven düzeyinde %100.30 ± 0.44 olarak saptanmıştır. Doğrusallık 0.25 - 0.75 mg/ ml konsantrasyon aralığında bulunmuştur. Gün içinde tekrarlanabilirlik testinde siprofloksazin hidroklorür pik alanı değerlerinin bağıl standart sapması 0.2041 olarak bulunmuştur. Miktar tayini sonuçları farklı analizci ve farklı cihaz kullanılarak tekrar edildiğinde %99 güven düzeyinde birbirinden farklı olmadığı bulunmuştur. Sağlamlık testinde yöntemin yapılan değişimlerden anlamlı bir şekilde etkilenmediği gözlenmiştir. Sistem uygunluk testleri için etken maddenin 0,5 mg/ ml konsantrasyonda hazırlanan çözeltisinin 6 kez enjeksiyonu ile elde edilen bağıl standart sapma 0.0668 olarak bulunmuştur. Safsızlık tayin yönteminde safsızlık B için geri kazanım %95 güven düzeyinde %100.0 ± 1.13, safsızlık C için %102.81 ± 1.36, safsızlık D için %102.08 ± 1.60, safsızlık E için %100.88 ± 2.82, kloro analog için %102.75 ± 0.95 olarak saptanmıştır. Tüm safsızlıklar için 0.0005 - 0.0015 mg/ ml konsantrasyon aralığında doğrusallık bulunmuştur. Gün içinde tekrarlanabilirlik testinde safsızlık B, safsızlık C, safsızlık D, safsızlık E ve kloro analog pik alanı değerlerinin bağıl standart sapması sırasıyla 0.4516, 0.5605, 0.5783, 0.3266 ve 0.4574 olarak tespit edilmiştir. Safsızlık tayin yöntemi sonuçları farklı analizci ve farklı cihaz kullanılarak tekrar edildiğinde %99 güven düzeyinde sonuçların birbirinden farklı olmadığı bulunmuştur. Sağlamlık testinde yöntemin yapılan değişimlerden anlamlı bir şekilde etkilenmediği gözlenmiştir. Sistem uygunluk testleri için safsızlık B ve safsızlık C pikleri arasındaki ayırım gücü 3.025 olarak bulunup, yöntemin uygunluğu kanıtlanmıştır. Hızlandırılmış bozundurma çalışmalarında siprofloksazin hidroklorür'ün bozunma ürünü olarak bilinen safsızlık C değerinde artış gözlenmiş fakat artışın farmakopede belirtilen sınır değerlerinin içinde kaldığı, böylece yöntemin siprofloksazin hidroklorür ve safsızlıkları için seçici olduğu saptanmıştır.
TEZ KONU BAŞLIĞI Ciprofloxacin Hydrochloride Assay and Impurity Method Development and Validation in Film Coated Tablets SUMMARY In the present work, the aim is search appropriateness of quantitative method for ciprofloxacin hydrochloride and it's impurities, which was taken reference from European Pharmacopeia Supplement 4.6 and British Pharmacopeia' 99, for film coated tablets and do method validation. Mobile phase was aqua solvent of phosphoric acide (pH 3.0) / ACN (87:13 v/ v), the detector wavelength was UV at ? = 278 nm. At this chromatographic conditions, several stationary phases were tried. Finally Inertsil ODS3 4.6 x 250 mm, 5 µm properties column was selected because of it's suitable structure with ciprofloxacin hydrochloride. There was no interaction between column and active substance and there had no metal impurities in this column. For quantitative method, recovery was 100.30 ± 0.44 % with 95% confidence limits. Linearity was obtained in the concentration range of 0.25 - 0.75 mg/ ml. Repeatability was calculated from the relative standart deviation of peak area values of ciprofloxacin hydrochloride, RSD is 0.2041. Quantitative determination was repeated by different analyst and different instrument tests and the results were statistically not different from each other at 99% confidence level. In robustness test it was observed that, the method was not influenced from variations. For system suitability test active substance's solution (c = 0.5 mg/ ml) was injected to HPLC at 6 times and relative standart deviation (RSD) of its peak area was 0.0668. In impurity method, recovery was 100.0 ± 1.13 for impurity B, 102.81 ± 1.36 for impurity C, 102.08 ± 1.60 for impurity D, 100.88 ± 2.82 for impurity E and 102.75 ± 0.95 for chloro analog with 95% confidence limits. Linearity was obtained in the concentration range of 0.0005 - 0.0015 mg/ ml for all impurities. Repeatability was calculated from the RSD of peak area values of impurity B was 0.4516, impurity C was 0.5605, impurity D was 0.5783, impurity E was 0.3266 and chloro analog was 0.4574. Impurity method was repeated by different analyst and different instrument tests and the results were not different from each other at 99% confidence level. In robustness test it was observed that, the method was not influenced from small variations. For system suitability the resolution between impurity B and impurity C must be minimum 1.3 and it was reached 3.025. In accelerated degradation studies, impurity C which is known as degradation product of ciprofloxacin hydrochloride increased but it remained in the limit of pharmacopeia. So it observed that this was selective method for ciprofloxacin hydrochloride and it's impurities.
TEZ KONU BAŞLIĞI Ciprofloxacin Hydrochloride Assay and Impurity Method Development and Validation in Film Coated Tablets SUMMARY In the present work, the aim is search appropriateness of quantitative method for ciprofloxacin hydrochloride and it's impurities, which was taken reference from European Pharmacopeia Supplement 4.6 and British Pharmacopeia' 99, for film coated tablets and do method validation. Mobile phase was aqua solvent of phosphoric acide (pH 3.0) / ACN (87:13 v/ v), the detector wavelength was UV at ? = 278 nm. At this chromatographic conditions, several stationary phases were tried. Finally Inertsil ODS3 4.6 x 250 mm, 5 µm properties column was selected because of it's suitable structure with ciprofloxacin hydrochloride. There was no interaction between column and active substance and there had no metal impurities in this column. For quantitative method, recovery was 100.30 ± 0.44 % with 95% confidence limits. Linearity was obtained in the concentration range of 0.25 - 0.75 mg/ ml. Repeatability was calculated from the relative standart deviation of peak area values of ciprofloxacin hydrochloride, RSD is 0.2041. Quantitative determination was repeated by different analyst and different instrument tests and the results were statistically not different from each other at 99% confidence level. In robustness test it was observed that, the method was not influenced from variations. For system suitability test active substance's solution (c = 0.5 mg/ ml) was injected to HPLC at 6 times and relative standart deviation (RSD) of its peak area was 0.0668. In impurity method, recovery was 100.0 ± 1.13 for impurity B, 102.81 ± 1.36 for impurity C, 102.08 ± 1.60 for impurity D, 100.88 ± 2.82 for impurity E and 102.75 ± 0.95 for chloro analog with 95% confidence limits. Linearity was obtained in the concentration range of 0.0005 - 0.0015 mg/ ml for all impurities. Repeatability was calculated from the RSD of peak area values of impurity B was 0.4516, impurity C was 0.5605, impurity D was 0.5783, impurity E was 0.3266 and chloro analog was 0.4574. Impurity method was repeated by different analyst and different instrument tests and the results were not different from each other at 99% confidence level. In robustness test it was observed that, the method was not influenced from small variations. For system suitability the resolution between impurity B and impurity C must be minimum 1.3 and it was reached 3.025. In accelerated degradation studies, impurity C which is known as degradation product of ciprofloxacin hydrochloride increased but it remained in the limit of pharmacopeia. So it observed that this was selective method for ciprofloxacin hydrochloride and it's impurities.