Razvoj in validacija analiznega postopka za določanje vsebnosti atorvastatina in njegovih razpadnih produktov

Jančan, Tatjana (author), Sollner Dolenc, Marija (mentor), Mitrović, Bojan (comentor)

Razvoj analiznih metod za določanje zdravilne učinkovine je eden od odločilnih korakov v farmacevtskem razvoju, še posebno takrat, kadar se za izbrani produkt pojavijo novi proizvajalci in s tem nove nečistote na trgu. Namen magistrske naloge je bil razviti primerno analizno metodo za kvantitativno določanje vsebnosti zdravilne učinkovine, ter vsebnosti njenih nečistot v atorvastatin kalciju. Za analizno metodo smo izbrali tekočinsko kromatografijo visoke ločljivosti (HPLC). Pri tem smo upoštevali načela, da je nova analizna metoda čimbolj podobna že znanim metodam, da je selektivna za čim več nečistot atorvastatina in da omogoča spremljanje kakovosti vseh obstoječih proizvajalcev te učinkovine. Metodo smo razvili na osnovi že obstoječe monografije za atorvastatin kalcij trihidrat v Evropski farmakopeji (PhEur) in jo optimizirali za potrebe spremljanja vseh znanih nečistot na trgu v Evropi. Po obstoječi farmakopejski metodi ni mogoče ločevati vseh znanih nečistot atorvastatina in čas analize je predolg. Raziskali smo vpliv različnih kromatografskih pogojev na izvedbo analize: izbira topila, sestava mobilne faze, izbira separacijske kolone, temperature kolone in spreminjanja gradienta. Razvito metodo smo v nadaljevanju tudi validirali v skladu z ICH smernicami v farmacevtski industriji. Za preizkušanje v laboratorijski fazi bi bila celotna validacija predolga, zato smo se odločili za preizkušanje ključnih validacijskih parametrov (specifičnost, linearnost, meja zaznave in določanja, natančnost, točnost, delovno območje in stabilnost standardne raztopine in vzorca). Vsi preizkušeni validacijski parametri so ustrezali kriterijem, s čimer smo dokazali, da je metoda točna, natančna, specifična in linearna. Prav tako smo izračunali mejo zaznavnosti in določljivosti posamezne nečistote in potrdili stabilnost standardne raztopine (76 ur) in raztopine vzorca (52 ur) za določanje vsebnosti, če sta shranjeni v temnih HPLC vialah v avtomatskem vzorčevalniku v temi pri 4 °C. Medtem, ko je vzorce za določanje razpadnih produktov potrebno pripravljati sproti. Analizna metoda, ki smo jo v okviru te magistrske naloge razvili oziroma vsi pridobljeni rezultati bodo uporabljeni za pripravo in objavo revidirane monografije za aktivno učinkovino atorvastatin kalcij v Evropski farmakopeji.

• Language: Slovenian
• Keywords: atorvastatin, tekočinska kromatografija visoke ločljivosti, vsebnost, nečistote, validacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-125049
• Publication date in RUL: 03.03.2021

JANČAN, Tatjana, 2021, Razvoj in validacija analiznega postopka za določanje vsebnosti atorvastatina in njegovih razpadnih produktov [online]. Master’s thesis. [Accessed 24 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=125049

Secondary language

• Language: English
• Title: Development and validation of ananalytical procedure for determination of atorvastatin and its degradation products

Abstract

The development of analytical methods for active pharmaceutical ingredients is one of the decisive steps in pharmaceutical development, especially when new manufacturers and thus new impurities appear on the market for the selected product. The purpose of the master's thesis was to develop an appropriate analytical method for the quantitative determination of the content of active substance and the content of related substances and degradation products in atorvastatin calcium. High performance liquid chromatography (HPLC) was chosen for the analytical method. We considered principles: that the new analytical method is as similar as possible to the already known methods, that it is selective for as many impurities of atorvastatin as possible, and that it allows monitoring the quality of all existing manufacturers of this active ingredient. The method was developed on the basis of the already existing monograph for the atorvastatin calcium trihydrate in the European Pharmacopoeia (PhEur) and was optimized to monitor all known impurities on the European market. According to the existing pharmacopoeial method, it is not possible to separate all known impurities of atorvastatin and the analysis time is too long. We investigated the influence of different chromatographic conditions on the analysis: solvent selection, mobile phase composition, separation column selection, column temperature, and gradient changes. The developed method was then validated according to ICH guidelines in the pharmaceutical industry. The full validation would be too long for testing in the laboratory phase, so we decided to test the key validation parameters: specificity, linearity, limit of detection and determination, accuracy, precision, working range and stability of standard solution and sample solution. All tested validation parameters met the criteria and thus proved that the method is accurate, precise, specific and linear. We also calculated the limit of detection and detectability of each impurity and confirmed the stability of the standard solution (76 hours) and the sample solution (52 hours) for determination of content when stored in dark HPLC vials in an automated sampler in the dark at 4 °C. Samples for the determination of degradation products must be prepared on an ongoing basis. The analytical method developed in the master's thesis, or all the results obtained, will be used to prepare and publish a revised monograph for the active substance atorvastatin calcium in the European Pharmacopoeia.

• Keywords: atorvastatin, high resolution liquid chromatography, assay, impurities, validation