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Safinamid mesilat safsızlıklarının ve izomerlerinin HPLC ile analizi

Analysis of safinamide mesylate impurities and isomers by HPLC

  1. Tez No: 944713
  2. Yazar: NİHAL DOLAPCI
  3. Danışmanlar: DOÇ. DR. GÜLÇİN YILMAZ
  4. Tez Türü: Yüksek Lisans
  5. Konular: Kimya, Chemistry
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2025
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Lisansüstü Eğitim Enstitüsü
  11. Ana Bilim Dalı: Kimya Ana Bilim Dalı
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 134

Özet

Parkinson hastalığı, istemsiz ve kontrolsüz hareketlerle karakterize edilen nörolojik bir bozukluktur. Hastalar, günlük aktivitelerini gerçekleştirirken titreme ve koordinasyon güçlüğü yaşarlar. Safinamid gibi parkinson hastalığının tedavisinde kullanılan ilaçlar, titreme ve diğer motor semptomların kontrol altına alınmasına yardımcı olur. Safinamid, seçici bir Monoamin oksidaz-B inhibitörü olarak görev yapar ve beyindeki dopamin düzeylerinin artmasını sağlar. İlaçlardaki safsızlıkların kontrolü, hasta güvenliği açısından büyük önem taşır. Farmasötik ürünlerdeki safsızlıkların tespiti, tanımlanması ve nicelendirilmesi amacıyla genellikle Yüksek Performanslı Sıvı Kromatografisi (HPLC) gibi analitik teknikler kullanılır. HPLC, yüksek duyarlılığı, hızlı analiz imkanı ve tekrarlanabilirliği sayesinde diğer analitik metotlara kıyasla önemli avantajlar sunmaktadır. Safsızlıklar gibi numunede düşük miktarlarda bile çok tehlikeli sonuçlar doğurabilen maddelerin analizinin HPLC ile gerçekleştirilmesi yüksek doğruluk sağlar ve bu da hasta güvenliği açısından hayati önem taşır. İzomerik safsızlıkların HPLC'de ters faz kromatografisi ile tespit edilmesi zordur; bu nedenle izomer ayrımı ve nicelendirmesi genellikle normal faz kromatografi ile yapılır. Ancak bu çalışma, safinamid mesilat izomerlerinin belirlenmesi için ters faz kromatografisi kullanan basit bir yöntemi ele almaktadır. Bu tez çalışmasında ICH Q2 (R1) – Analitik Prosedürlerin Validasyonu için yayınlanan klavuzda belirtilen parametrelerle valide edilen yöntem kapsamında, sabit faz olarak XBridge Shield RP18 (250 x 4,6 mm, 3,5 µm) kolon kullanılmış; hareketli faz A 25 mM K2HPO4 tamponu ve metanol hacimce 47,5:52,5 oranında birleştirilerek kullanılmıştır. Yöntemin kromatografik koşulları; 0,8 mL/dakika gradient akış hızı, 25 °C kolon sıcaklığı ve 224 nm dalga boyunda dedeksiyon şeklinde belirlenmiştir. Yöntemin doğrusal çalışma aralığı; Safinamid Mesilat için 0,29–3,95 µg/mL, Safinamid para izomer için 0,20–2,97 µg/mL ve Safinamid orto izomer için 0,21–3,07 µg/mL aralığında saptanmıştır. Valide edilen HPLC yöntemi, ilgili safsızlıkları ve bozunma ürünlerini herhangi bir girişim olmadan tespit edebilmiştir. Geçerliliği sağlanan bu analitik yöntem, safinamid mesilat etkin maddesinin ve tabletlerinin asidik, bazik, oksidatif ve termal bozundurma gibi zorlayıcı koşullar altındaki bozunma davranışlarını da inceleyerek kapsamlı bir safsızlık analizi olarak gerçekleştirilmiştir. Geliştirilen yöntem, safsızlık analizlerinde sağlayacağı basitlik sayesinde, farmasötik endüstride kullanıma son derece uygundur.

Özet (Çeviri)

With the steady rise in the global elderly population, addressing age-associated neurodegenerative disorders such as Parkinson's disease has become an increasingly urgent public health priority. Parkinson's disease is a chronic and progressive neurological disorder that primarily impairs motor function and substantially reduces quality of life. The hallmark symptoms include involuntary and uncontrollable movements, muscle rigidity, tremors, and deteriorating coordination. These symptoms intensify as the disease advances, eventually limiting patients' ability to carry out everyday tasks independently. In response to these challenges, a wide range of therapeutic strategies have been developed over the years to manage symptoms, prolong functional capacity, and enhance life expectancy in affected individuals. Among the pharmacological treatments available, safinamide has gained attention as an effective adjunct therapy, particularly beneficial for managing“off”episodes— intervals in which conventional medications lose their efficacy and motor symptoms return. Safinamide plays an essential role in the symptomatic management of Parkinson's disease due to its dual mechanism of action. It functions as a highly selective monoamine oxidase B (MAO-B) inhibitor, thereby increasing dopamine levels in the brain, and also exerts modulatory effects on glutamatergic transmission. Although there is currently no definitive cure for Parkinson's disease, pharmacological therapies like safinamide can significantly slow disease progression and improve patients' overall quality of life by enhancing motor control and reducing symptom fluctuations. The evaluation of impurity profiles in newly developed pharmaceutical compounds, including safinamide, is vital to ensuring therapeutic efficacy and long-term safety. Impurities, even in trace amounts, may compromise the effectiveness of treatment or pose serious health risks. Impurity analyses serve as a valuable guide in various areas, ranging from the selection of appropriate formulations and packaging to the determination of optimal storage conditions. Subjecting active pharmaceutical ingredients and pharmaceutical preparations to harsh environmental conditions beyond those of accelerated testing allows for the identification of potential degradation pathways and products, which is of great significance for structural characterization. Therefore, comprehensive analytical methods are necessary to monitor and control these impurities throughout the drug development and manufacturing processes. To this end, various analytical approaches have been devised to ensure the quality and reliability of safinamide-containing pharmaceutical products, particularly during the production of the active pharmaceutical ingredient (API) and in the final dosage form such as tablets.High-Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques in the pharmaceutical industry for the detection, identification, and quantification of impurities. Its popularity is attributed to its high sensitivity, reproducibility, and suitability for analyzing complex mixtures in a relatively short time. HPLC is particularly effective for impurity profiling because it allows for precise quantification of low-level impurities. This level of analytical rigor is indispensable for ensuring patient safety and maintaining regulatory compliance. Isomeric impurities, which are structurally similar to the active compound, often present a significant challenge in impurity profiling. These types of impurities are typically difficult to separate using conventional reversed-phase chromatographic methods, and normal-phase chromatography is usually the preferred technique for their resolution. However, the current study introduces a novel and simplified reversed-phase HPLC method capable of separating and quantifying the para-isomer and ortho-isomer of safinamide mesylate effectively. The reversed-phase C18 column, also known as RP18, enabled separation based on hydrophobic interactions, which are particularly effective for distinguishing structurally similar isomers. In the case of safinamide isomers, such hydrophobic interactions can reveal significant differences between closely related molecules. For instance, structural variations between para and ortho isomers alter the surface area in contact with the stationary phase, leading to differences in retention times. As a result of these differences, distinct peaks were observed in the chromatogram. The RP-HPLC method was developed and validated according to the guidelines specified in ICH Q2(R1) for analytical method validation. The chromatographic analysis was carried out using an XBridge Shield RP18 column (250 mm × 4,6 mm, 3,5 µm), which enabled efficient separation of safinamide mesylate and its isomers. The mobile phase consisted of 25 mM dipotassium hydrogen phosphate (K₂HPO₄) buffer and methanol in a 47,5:52,5 (v/v) ratio. The optimized chromatographic conditions included a flow rate of 0,8 mL/min with a gradient flow, a column temperature maintained at 25 °C, and detection at a wavelength of 224 nm. A full wavelength scan was conducted between 190 and 400 nm to determine the optimal wavelength for analysis. The wavelength of 224 nm was selected as it provided the highest sensitivity or maximum absorbance. In the method developed within this thesis, safinamide para and ortho isomers could not be adequately separated at a column temperature of 40 °C. The resulting peak overlap led to interference, which in turn caused falsely elevated quantification values. To achieve a clearer resolution of the isomers, the column temperature was therefore adjusted to 25 °C. Although this modification increased the overall analysis time, it significantly enhanced the specificity and accuracy of the method. Furthermore, the composition of the mobile phase used in the study—specifically, the volumetric percentage of methanol—was found to be critical for the selective separation of the safinamide para and ortho isomers. This optimized ratio not only ensured successful resolution but also provided optimal peak shape for both isomers. The method demonstrated excellent linearity in the concentration ranges of 0,29–3,95 µg/mL for safinamide mesylate, 0,20–2,97 µg/mL for the para-isomer, and 0,21–3,07 µg/mL for the ortho-isomer. The precision of the method was verified through interday and inter-analyst repeatability assessments, with consistent results confirming its robustness. Accuracy was further established via recovery studies at three spiking levels (%LOQ, 100%, and 150%) using placebo matrices that simulated the tablet formulation. The average recovery rates were found to be 100,30% for safinamide mesylate (RSD: 0,73%), 100,82% for the para-isomer (RSD: 0,77%), and 100,76% for the ortho-isomer (RSD: 0,55%), indicating the method's suitability for quantitative analysis across a wide concentration range. Stability studies further affirmed the method's reliability by showing that standard and sample solutions of safinamide remained chemically stable for at least 48 hours under normal laboratory and fridge conditions. This stability profile is essential for practical implementation in routine quality control environments where delays in analysis may occur. Additionally, forced degradation studies were conducted to assess the chemical stability of both the API and its tablet formulation under various stress conditions, including acidic, basic, oxidative, thermal, and photolytic environments. The findings revealed significant degradation of the tablet formulation under basic conditions, while degradation under acidic and oxidative stress was more limited. Under thermal and light-induced stress, the formulation exhibited high stability, underscoring the physical and chemical robustness of the developed formulation. In summary, this research contributes a novel, validated RP-HPLC method tailored for the comprehensive analysis of safinamide mesylate impurities and its isomeric impurities in active pharmaceutical ingredient and finished pharmaceutical forms. The method's simplicity, reliability, and broad applicability make it a valuable tool for routine use in the pharmaceutical industry, particularly in quality control laboratories where accurate impurity profiling is essential for ensuring the safety and efficacy of therapeutic products.

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