Development of an on-line lab-on-valve micro-solid phase extraction system coupled to liquid chromatography for the determination of flavonoids in citrus juices.

An on-line lab-on-valve system for renewable micro-solid phase extraction coupled with high performance liquid chromatography with photodiode array detection was developed for the determination of hesperidin and diosmin in natural and commercial citrus samples. The method is based on the automatic loading of 8 mL of sample into the C18 resin, followed by matrix clean-up and elution of the analytes with 0.27 mL of acetonitrile. Next, 0.02 mL of the extract were injected into the chromatographic system. Separation was performed by using a Symmetry® C18 column (250 × 3.0 mm x 5.0 µm) with a mobile phase consisting in a mixture of methanol, acetonitrile and acidic water (pH = 2.5) in gradient mode at 0.58 mL min-1. The detection wavelength was chosen as 285 nm. Limits of detection, quantification and relative standard deviations were lower than 0.1 µg mL-1, 0.3 µg mL-1 and 4.1%, respectively.

Use of multiresponse statistical techniques to optimize the separation of diosmin, hesperidin, diosmetin and hesperitin in different pharmaceutical preparations by high performance liquid chromatography with UV-DAD.

A new method for the separation and determination of four flavonoids: hesperidin (HES), diosmin (DIO), hesperitin (HTIN), and diosmetin (DTIN) in pure form and pharmaceutical formulations has been developed by using high performance liquid chromatography (HPLC) with UV-DAD detection. Multivariate statistics (2k full factorial and Box Behnken Designs) has been used for the multiresponse optimization of the chromatographic separation, which was completed in 22min, and carried out on a symmetry® C18 column (250×3mm; 5µm) as stationary phase. Separation was conducted by gradient elution mode using a mixture of methanol, acetonitrile and water pH: 2.5 (CH3COOH), as mobile phase. Analytes were separated setting the column at 22°C, with a flow rate of 0.58mLmin-1 and detected at 285nm. Under the optimized conditions, the flavonoids showed retention times of: 8.62, 11.53, 18.55 and 19.94min for HES, DIO, HTIN and DTIN, respectively. Limits of detection and quantification were <0.0156µgmL-1 and <0.100µgmL-1, respectively. Linearity was achieved with good correlation coefficients values (r2=0.999; n=5). Intra-day and inter-day precision were found to be less than 3.44% (n=7). Finally, the proposed method was successfully applied to determine the target flavonoids in pharmaceutical preparations with satisfactory recoveries (between 95.2% and 107.9%), demonstrating that should also find application in the quality control, as well as in the pharmacokinetic studies of these drugs.