Prednosti tekućinske kromatografije ultra visoke učinkovitosti u razvoju stabilitetno-indikativne metode za kontrolu kakvoće nepafenaka

CILJ ISTRAŽIVANJA Cilj istraživanja je analiza studije o razvoju i validaciji stabilitetno-indikativne UHPLC metode za određivanje kontrole kakvoće nepafenaka razvijene u Pliva Hrvatska d.o.o te usporedba s postojećom internom HPLC metodom s naglaskom na prednosti korištenja UHPLC metoda i tehnike masene spektrometrije (LC-MS/MS). Obradit će se i sve literaturno poznate kromatografske metode razvijene i korištene za analitičko ispitivanje nepafenaka u farmaceutskim formulacijama. MATERIJAL/METODE Istraživanja provedena u okviru ovog specijalističkog rada obuhvaćaju pregled svih dostupnih publikacija koristeći dostupne bibliografske baze podataka i servise (Medline/PubMed, ScienceDirect, EMBASE i drugih), uz primjenu ključnih riječi poput: nepafenac, quality control, UHPLC, HPLC, Stability indicating study, forced degradation i sl. Napravljena je detaljna studija razvijene UHPLC metode za stabilitetna ispitivanja aktivne farmaceutske supstancije nepafenaka, te prednosti korištenja iste naspram postojeće HPLC metode također razvijene u odjelu Istraživanja i razvoja TAPI R&D Pliva Hrvatska d.o.o. Za potrebe razvoja stabilitetno-indikativne metode uvjeti prisilne razgradnje su preuzeti iz ICH Q1A i ICH Q1B vodiča za farmaceutsku industriju: kisela i bazična razgradnja, oksidacija, povišena temperatura, fotoliza te povišena vlažnost. Korišten je Waters UHPLC H-Class s PDA detektorom i gradijentnom pumpom. Optimalna kromatografska kolona je Waters Acquity BEH C18, dimenzija 100 mm x 2,1 mm i promjera čestica 1,7 μm. Za evaluaciju podataka korišteno je Waters Empower 2 CDS upravljačko sučelje. Detekcija nepoznatih onečišćenja je izvršena tehnikom masene spektroskopije, uz korištenje Agilent 6490 LC-MS/MS sistema spregnutog s Agilent 1290 UHPLC te Agilent MassHunter 2003-2007 Data Acquisition for Triple Qual B.01.04 (B84) upravljačko sučelje. REZULTATI Aktivna farmaceutska supstancija nepafenak je podvrgnuta studiji prisilne razgradnje (kisela i bazična hidroliza, oksidacija, povišena vlažnost i temperatura, utjecaj svjetlosti) pod instrumentalnim uvjetima novorazvijene UHPLC metode za određivanje sadržaja i onečišćenja da bi se ispitala prikladnost metode za stabilitetna ispitivanja. Gradijentna UHPLC-UV metoda odjeljuje nepafenak i sva onečišćenja (2-aminobenzofenon, Cl-tionepafenak, tionepafenak, Cl-nepafenak, hidroksi-nepafenak i ciklički nepafenak) koristeći kromatografsku kolonu Waters Acquity BEH C18. Valna duljina detekcije je 235 nm, protok pokretne faze je 0,6 mL/min. Razlučivanje između pikova nepafenaka i detektiranih onečišćenja je veće od 2. Metoda je dokazano selektivna, linearna, precizna, točna, robusna, a određene su granice detekcije i kvantifikacije. Linearnost je dokazana visokim faktorom korelacije između površina pikova i koncentracija analita koji je iznosio ≥0,999 za sve analite. Granica detekcije je potvrđena za sve komponente i iznosi 0,005%, relativno u odnosu na koncentraciju ispitivane otopine uzorka od 1,0 mg/mL. Značajna razgradnja je zabilježena u kiseloj, bazičnoj i oksidativnoj razgradnji. Razgradni produkti su identificirani tehnikom masene spektrometrije, dva su identificirana kao poznata procesna onečišćenja (hidroksi-nepafenak i ciklički nepafenak), a identificirana su i četiri nova razgradna produkta. ZAKLJUČAK Ukazana je prednost razvoja metode za tekućinsku kromatografiju ultra visoke učinkovitosti nad metodama za tekućinsku kromatografiju visoke učinkovitosti na primjeru nepafenaka. Studija uvelike doprinosi prikupljanju informacija o stabilnosti same supstancije (ubrzana razgradnja), procesnim onečišćenjima i potencijalnim razgradnim produktima (spregnuta tehnika LC-MS/MS). Razvijena UHPLC metoda ima prednost nad svim literaturno poznatim, zbog separacije svih sastavnica u vremenu od 5 minuta.OBJECTIVES The objective of this research is an analysis of study regarding development and validation of stability-indicating UHPLC method used for quality control of nepafenac. The method was developed in Pliva Croatia and it was compared with already existing in-house HPLC method, focusing on benefits of using UHPLC method and mass-spectroscopy technique (LC-MS/MS). The available literature regarding chromatographic methods used for analytical quality testing of nepafenac in pharmaceutical finished forms will also be reviewed. MATERIAL/METHODS This specialist study contains an overview of available publications using bibliographic databases (Medline/PubMed, ScienceDirect, EMBASE etc.) and keywords search (nepafenac, quality control, UHPLC, HPLC, Stability indicating study, forced degradation etc.). The investigation includes detail study regarding developed UHPLC stability-indicating method for quality control of active pharmaceutical substance nepafenac, and benefits of using UHPLC methods over already existing HPLC method also developed in TAPI R&D Pliva Croatia. ICH Q1A and ICH Q1B conditions of forced degradation are used in order to develop stability indicating method (SIM): acid and base hydrolysis, oxidation, humidity, heat and light. Used instrument was Waters UHPLC H-Class with PDA detector and gradient pump. Optimal chromatographic column was found during method development (Waters Acquity BEH C18, 100 mm x 2.1 mm, 1.7 μm). Used software for raw data evaluation was Waters Empower 2 CDS. For detection of unknown impurities using mass spectroscopy technique, Agilent 6490 LC-MS/MS system coupled with Agilent 1290 UHPLC instrument was used, along with Agilent MassHunter 2003-2007 Data Acquisition for Triple Qual B.01.04 (B84) software. RESULTS To test stability of active pharmaceutical ingredient nepafenac, it was subjected to forced degradation under different stress conditions (acid and base hydrolysis, oxidation, humidity, heat and light) according to instrumental parameters taken from novel UHPLC method for quantification of nepafenac assay and impurities. Gradient UHPLC-UV method separates nepafenac and potential impurities (2-aminobenzophenon, Cl-thionepafenac, thionepafenac, Cl-nepafenac, hydroxy-nepafenac and cyclic-nepafenac), using Waters Acquity BEH C18 chromatographic column. Detection wavelength was 235 nm at a flow rate of 0.6 mL/min. The resolution between nepafenac and six potential impurities is found to be greater than 2.0. Method is selective, precise, linear, accurate, and robust with established LOD and LOQ level. Linearity is proven with r2 ≥0.999 for all components. Limit of detection is 0.005%, with respect to test concentration of 1.0 mg/mL. Significant degradation is observed in acid, base and oxidative degradation. Degradation products are identified using mass spectrometry technique, two of them were found to be known process related impurities (hydroxy- nepafenac and cyclic-nepafenac), whereas four degradation products were identified as new degradation impurities. CONCLUSION Benefits of using UHPLC method for quality control of nepafenac over HPLC method was shown. This study provides information regarding the stability of substance (forced degradation), process impurities and potential degradation products (coupled technique LC-MS/MS). Developed UHPLC method has the ability to separate all components of interest within 5 min, and therefore is more applicable for quality testing of nepafenac than any other known analytical HPLC method in literature