High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate

A new, simple, and reproducible isocratic high-performance liquid chromatography (HPLC) method has been developed for the determination of free and total tyrosine and tryptophan in a protein concentrate. To determine total amino acids, the method involves alkaline hydrolysis of the proteins with sodium hydroxide at 120 °C for 4 h in the absence of air. Best results were achieved with a SS Exil ODS column 5 μm (25 cm × 0.46 cm i.d.), with an eluent of methanol: 40 mM sodium acetate buffer (adjusted to pH 4.5 with acetic acid; 20:80, v/v), a flow rate of 0.80 mL/min at 26 °C, and with programmable fluorescence detection. Under optimum conditions excellent linearity was obtained, and the overall recovery was 90.5, and 95.9% for total tryptophan and tyrosine, respectively. The precision results showed that the relative standard deviation of the repeatability and reproducibility were ≤4.78 and ≤4.65, respectively. This method was used to quantify the cited analytes in the protein concentrate obtained during the lactic acid fermentation of shrimp waste.