Comparison of two RP-HPLC methods for determination of recombinant human thrombin in pharmaceutical formulations. / Porovnanie dvoch RP-HPLC metód pre stanovenie rekombinantného ľudského trombínu vo farmaceutických formuláciách

Abstract

Abstract Two reversed-phase high performance liquid chromatography analytical methods (Method I and Method II) for determination of assay of recombinant human thrombin in pharmaceutical formulations were developed and validated. Analysis was performed on chromatographic system Agilent 1200 series SL with diode array detection and mass selective detection. Method I was intended for faster determination of thrombin assay. Gradient programme was optimised to achieve sufficient separation and acceptable runtime. Chromatographic analysis was performed on analytical column Grace Vydac, C4 250 × 4.6 mm, 5 mm. Method II is Method I adapted to use the mass selective detector. Chromatographic separation was performed on analytical column Zorbax 300SB-C8 SolvSaver Plus, 150 × 3 mm, 3.5 mm. Both analytical methods were validated with respect to specificity, linearity, precision and accuracy. The response of thrombin was a linear function of concentration over the range 0.1-1.0 mg/ml. Precision and accuracy of thrombin was evaluated at three concentration levels low (0.2 mg/ml), medium (0.4 mg/ml) and high (0.8 mg/ml). Both validated methods have been successfully applied for determination of assay and thrombin degradation products in pharmaceutical formulations. Slovak abstract Na stanovenie obsahu rekombinantneho ľudskeho trombinu boli vyvinute a validovane dve analyticke metody (Metoda I a Metoda II). Analyzy boli uskutočnene na chromatografickom systeme Agilent 1200 series SL s DAD a MS detektorom. Metoda I je určena pre rychlejšiu analyzu obsahu trombinu. Pre dosiahnutie lepšej separacie a prijateľneho času analyzy bol optimalizovany gradientovy program. Chromatograficka separacia sa uskutočnila na analytickej kolone Grace Vydac, C4 250x4.6mm, 5mm. Metoda II bola odvodena od Metody I tak aby bola použiteľna v kombinacii s hmotnostnym detektorom. Chromatograficka separacia sa uskutočnila na analytickej kolone Zorbax 300SB-C8 Solv Saver Plus, 150x3mm, 3.5mm. U oboch metod boli validovane nasledujuce parametre: špecificita, linearita, presnosť a spravnosť. Odozva trombinu bola linearnou funkciou koncentracie v rozsahu od 0,1 do 1,0 mg/ml. Presnosť a spravnosť trombinu bola hodnotena pri troch koncentračnych hladinach: nizkej (0,2 mg/ml), strednej (0,4 mg/ml) a vysokej (0,8 mg/ml). Robustne validovane metody boli uspešne použite pri stanoveni obsahu a čistoty trombinu počas formulačneho vyvoja liekovej formy.