Use of chemometrics for development and validation of an RP-HPLC method for simultaneous determination of haloperidol and related compounds

Use of chemometrics for development and validation of an RP-HPLC method for simultaneous determination of haloperidol and related compounds A rapid resolution reversed-phase high performance liquid chromatographic (RR RP-HPLC) method has been developed and validated for simultaneous determination of haloperidol and six related compounds. Investigated matrix was a laboratory mixture of a therapeutic active substance haloperidol and its six related compounds in concentration ratio 300:1. Experimental design was used during method optimization (full factorial 23 design) and robustness testing (Central Composite Circumscribed design). Three factors: organic phase variation during gradient elution, flow rate and gradient rise time were independent variables. To estimate the system response during the optimization procedure and robustness testing, resolution (Rs) and a chromatographic response function (CRF) were used. Chromatography was performed with the mobile phase containing phosphate buffer pH 6.5 and acetonitrile as organic modifier. Separation was achieved using gradient elution (organic phase fraction changed linearly from 20 to 72 %) over 7 min. A Zorbax Eclipse XDB C18 Rapid Resolution HT 4.6 mm x 50 mm, 1.8 μm particle size, column was used at 25 °C at a flow rate of 1.5 mL min-1. UV detection was performed at 230 nm. The total time for chromatographic separation was 5.5 min with a total analysis time of 7.0 min. The method was validated for its linearity, precision, modal recovery and robustness. Uporaba kemometrije za razvoj i validaciju RP-HPLC metode za simultano određivanje haloperidola i srodnih spojeva Razvijena je i validirana metoda reverzno-fazne tekućinske kromatografije visoke učinkovitosti i brze rezolucije (RR RP-HPLC) za simultano određivanje haloperidola i srodnih spojeva. U tu svrhu ispitivana je smjesa ljekovite tvari haloperidola i šest srodnih spojeva u omjeru 300:1. Za optimiranje metode korišten je eksperimentalni dizajn (23 faktorijalni dizajn) i testiranje robustnosti (Central Composite Circumscribed design). Tri faktora: variranje organske faze za eluaciju, brzina protoka i vrijeme uspostave gradijenta eluensa bile su nezavisne varijable. Za procjenu odgovora sustava za vrijeme optimizacije i testiranje robustnosti, korištene su razlučivanje (Rs) i funkcija kromatografskog odziva (CRF). Mobilna faza tijekom kromatografije bila je fosfatni pufer pH 6,5 i acetonitril kao organska faza. Razdvajanje je postignuto pomoću gradijenta eluacije (udio organske faze linearno se mijenjao od 20 do 72%) tijekom 7 min. Za rad je upotrebljena kolona Zorbax Eclipse XDB C18 Rapid Resolution HT kolona, dimenzije 4,6 mm x 50 mm, veličine čestica 1,8 μm. Kromatografija je provedena pri 25 °C, uz protok eluensa 1,5 mL min-1 i UV detekciju na 230 nm. Vrijeme kromatografskog razdvajanja bilo je 5,5 min, a ukupno vrijeme potrebno za kromatografiju 7,0 min. Metoda je u potpunosti validirana.