Determination of tryptophan in bee pollen and royal jelly by high-performance liquid chromatography with fluorescence detection.

A method for tryptophan analysis in bee pollen and royal jelly was developed using HPLC with fluorescence detection. To determine the free tryptophan in bee pollen and royal jelly, ultrasonic extraction was performed using water (pH 6.3)-acetonitrile (10:1, v/v) as extraction solvent. While determining the total tryptophan in these bee products, the method involves alkaline hydrolysis of the proteins with 4 mol/L sodium hydroxide at 110 degrees C for 20 h under anaerobic conditions. The operating conditions for the HPLC analysis were: Symmetry C(18) column (4.6 x 250 mm, 5 microm), 0.1% trifluoroacetic acid-methanol (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min at 30 degrees C. The fluorescence detector was operated at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. A linear response (r(2 )> 0.9998) was obtained in the range 0.0625-5.0 microg/mL. The method was successfully applied to the determination of the free and total tryptophan contents in bee pollens, which were 0.069 +/- 0.003 and 2.693 +/- 0.476 mg/g, respectively, while only the total tryptophan was detected in royal jelly, with a content of 1.743 +/- 0.066 mg/g.