Quantitation of limonin in Iranian orange juice concentrates using high-performance liquid chromatography and spectrophotometric methods

Bitterness is a major problem in the citrus industry worldwide, and is mostly due to the presence of limonoid compounds, especially limonin. In this study, the limonin content of orange juice concentrates from three active factories in west Mazandaran province, after reconstitution, was assayed using high-performance liquid chromatography (HPLC) and spectrophotometric methods. With HPLC, employing acetonitrile/water (v/v) (32:68) with a flow rate of 0.9 ml/min at lambda=207 nm, the limonin content was in the range 11.7-23.3 mg/ml with a mean +/- standard deviation (SD) of 18.5 +/- 3.6 mg/ml. Spectrophotometric measurement of limonin at 503 nm with Burnham reagent gave a range of 9.9-21.8 mg/ml with a mean +/- SD of 17.16 +/- 4.1 mu g/ml. The correlation coefficients of the detector linear response for the limonin standard (0-50 mg/ml) for HPLC and spectrophotometry were 0.995 and 0.989, respectively. The percentages of limonin recovery with and without sample addition were 101.02-111.40 and 93.0-100.8, respectively, for HPLC. The percentages of limonin recovery for the spectrophotometric method resulted in 95.6-101.8 for limonin-added samples. The precision factors for the two assay methods were +/- 2.10% and +/- 1.69, respectively. The correlation coefficient of the two methods was 0.84. Sensory evaluation of the reconstituted samples from the view point of bitterness confirmed 85% of the results of the analytical methods. This study recommends spectrophotometric analysis as a useful and suitable method for quantitation of limonin in the quality control laboratories of orange juice plants.