Determination of phenylalanine and tyrosine using high performance liquid chromatography

In this research, hydrophilic interaction liquid chromatography (HILIC) with diode array detection was developed for the simultaneous determination of polar compounds namely phenylalanine (Phe), tyrosine (Tyr) and creatinine (Cre). Chromatographic separation was carried out using a HILIC column (dihydroxypropyl phase, 3.0 mm x 150 mm x 3 µm) with three detection wavelengths (210 nm for Phe and α-methyl phenylalanine (internal standard), 225 nm for Tyr and 234 nm for Cre). The mobile phase consisted of 84:16 v/v of acetonitrile and 50 mM ammonium formate pH 3.0 with isocratic elution at a flow rate of 0.8 mL min-1. The injection volume was 4 µL.The proposed method was applied to determine Phe, Tyr and Cre in urine for an alternative diagnosis and monitoring of phenylketonuria (PKU). The spot urine collected sample was diluted 10 times with 0.1 M hydrochloric acid. One milliliter of the diluted urine was introduced to condition strong cation exchange solid-phase extraction cartridge and then washed by 4.00 mL of methanol in order to elimination the matrices. The analytes were eluted with 4.00 mL of 0.5 M ammonia in ethanol. The eluate was evaporated to dryness under nitrogen at 60°C and reconstituted with 100 µL of mobile phase prior to injection into the HILIC system. The analytical performance was successfully validated. The calibration curves with the coefficient of determination higher than 0.999 ranged from 1 to 400 mg L-1, 0.5 to 200 mg L-1 and 3 to 3000 mg L-1 for Phe, Tyr and Cre, respectively. The proposed method provided satisfactory precision (%RSD < 5.58) and recoveries (88 – 108%). This method was applied to determine Phe, Tyr and Cre in human urine with various ages and genders.Additionally, this proposed HILIC method was also applied to determine Phe and Tyr in dietary supplements that necessary for PKU or tyrosinemia patient. The homogeneous powders of the dietary supplement were accurately weighed of 0.0250 g and extracted with 25.00 mL of 0.1 M hydrochloric acid by vortex for 3 minutes. One milliliter of the extracted solution was diluted to 10.00 mL with acetonitrile prior to injection into the HILIC system. The validation of the proposed HILIC and sample extraction method was achieved. The calibration curves of Phe and Tyr ranged from 1 to 500 mg L-1 with the coefficient of determination at 0.9999. The proposed method was optimized to obtain satisfactory precision (%RSD < 1.76) and recoveries (95 – 102%). The developed method was applied to simultaneously determine Phe and Tyr in single and multi-nutrients dietary supplements.