Analysis of hydrocortisone acetate in eye ointment applying reversed-phase high performance liquid chromatographic method

Abstract

In this paper the reversed-phase high performance liquid chromatographic method (RP-HPLC) was applied for the determination of hydrocortisone acetate in eye ointment. Analysis were performed on the chromatographic system Hewlett Packard 1100 which consisted of a HP 1100 pump, HP 1100 UV-VIS Detector and HP ChemStation integrator. Separations were performed on a Bio-Sil C18 250 mm x 4,6 mm, 5µm particle size column at 30.5 °C. The samples were introduced through a Rheodyne injector valve with a 20 µL sample loop. Methanol - water (75:25 V/V) was used as a mobile phase, at flow rate 1.15 mL/min and pH was adjusted to 4.0 with ortophosphoric acid. UV detection was performed at 254 nm. Mometasone furoate was used as an internal standard. Developed RP-HPLC method was validated and all the validation parameters of the method are given. The proposed method is rapid, accurate, selective and because of its sensitivity and reproducibility, it may be used in routine control of pharmaceutical dosage forms which contain hydrocortisone acetate.

Postavljena je metoda reverzno-fazne tečne hromatografije pod visokim pritiskom (RP-HPLC) za analizu hidrokortizon-acetata u mastima za oči. Analiza je izvršena na hromatografskom sistemu Hewlett Packard 1100 koji čine HP 1100 binarna pumpa, HP 1100 UV-VIS detektor i HP ChemStation za automatsku obradu podataka. Postupak je obuhvatio izbor odgovarajućih hromatografskih uslova, izbor pogodnog postupka ekstrakcije hidrokortizon-acetata iz masti, kao i validaciju metode. Za hromatografsku analizu odabrana je kolona Bio-Sil C18 250 mm x 4,6 mm, 5µm veličine čestica, termostatirana na 30,5°C. Volumen injiciranja bio je 20 µL. Mobilnu fazu činila je smeša metanol?voda (75 : 25 V/V) čiji je pH podešen na 4,0 sa 85%-tnom ortofosfatnom kiselinom. Protok mobilne faze bio je 1,15 ml/min., a talasna dužina detekcije 254 nm. Kao interni standard korišćen je mometazon-furoat. Postavljena RP-HPLC metoda je validirana, tj. ispitana je robusnost, selektivnost, linearnost, tačnost, preciznost - ponovljivost i određeni su limit detekcije i limit kvantifikacije. Kako je predložena metoda pouzdana i robusna može se koristiti u rutinskoj analizi doziranih oblika koji sadrže hidrokortizon-acetat.