Fluorometric determination of proline in honey by high-performance liquid chromatography after pre-column derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F)

An optimization method of NBD-F derivatization has been developed for the determination of proline in honey using high performance liquid chromatography (HPLC) with fluorescence detection. To determine the free proline in different samples of honey, ultrasound extraction was performed using 0.1 M borate buffer solution as the extraction solvent. Chromatographic separation was performed on a XDB C18 column (150 × 4.6 mm i.d.) using 0.1 M sodium acetate buffer solution (pH 7.2) : methanol : tetrahydrofuran (900 : 95 : 5, respectively, v/v/v) as mobile phase A and methanol as mobile phase B at a flow rate of 1.0 mL min−1 with a run time of 10 min. The assay was linear over a range of 0.15 μg mL−1 to 100.00 μg mL−1 with a lower limit of detection of 3.00 mg kg−1, and recoveries were greater than 90%. The intra-day and inter-day precision was less than 4%. This method was successful in determining free proline in honey, ranging from 80.5 mg kg−1 to 426.4 mg kg−1 in different honey samples.