SIMULTANEOUS DETERMINATION TRACE LEVELS OF VITAMIN B1 AND VITAMIN B9 IN HUMAN SAMPLES BY ULTRASOUND-ASSISTED DISPERSIVE LIQUID-LIQUID MICROEXTRACTION COUPLED WITH HPLC-UV

Ultrasound-assisted dispersive liquid-liquid micro extraction (UA-DLLME) was proposed for simultaneous determination of trace levels of Vitamin B₁ (thiamine) and Vitamin B₉ (folic acid) in the human urine and serum samples. High performance liquid chromatography (HPLC) coupled with chemometrics method was employed to analysis the extraxtants. Objective: The optimal conditions of the extraction recovery, including pH of sample solution, type and volume of extraction solvent, disperser solvent type and volume, temperature and time of ultrasound, centrifugation time and ionic strength were were studied step-by-step via one-factor-at-a time procedure. Then significant variables were chosen and considered to further optimize using the hybrid Box–Behnken design -genetic algorithm. UA-DLLME method with HPLC-UV and utilizing hybrid Box–Behnken design -genetic algorithm to optimize the extraction process was presented for the first time in this study. Under the optimized extractions, relative standard deviations (RSD) of the analyses less than 4% (n= 3) and detection limit of 3.09-11.15 ng mL. Recoveries for both thiamine and folic acid were in the ranges of 88–107%. As the result, the UA-DLLME – HPLC coupled hybrid Box–Behnken design -genetic algorithm was successfully applied for the simultaneous determination trace levels of thiamine and folic acid in human urine and serum samples.