1. Ferric chloride test: To a small quantity of hesperidin taken in a test tube add 3 ml of 5% ferric chloride solution. Formation of a wine red colour indicates the presence of hesperidin.
2. Shinoda test: To a few milligrams of hesperidin taken in a test tube add 2 ml methanol and a few bits of magnesium ribbon. Add concentrated hydrochloric acid dropwise. Formation of a bright violet colour indicates the presence of hesperidin.
3. Solution of hesperidin in 5% sodium hydroxide exhibits an orange red colour.
4. To 0.1 g of hesperidin add 10 ml hydrochloric acid, boil on a water bath, cool and filter. Neutralize with 20% sodium hydroxide solution and add 1 ml each of Fehling’s solution A and B. Formation of brick red colour indicates the presence of a reducing sugar in the hydrolysis mixture.

Estimation

Hesperidin estimation is reported in literature based on spectrophotometric and HPLC methods, with the latter being the method of choice for its determination in orange juice, body fluids and in herbal dosage forms.

A. Method I (Spectrophotometric assay)

1. Hesperidin sample is dried at 105°C for 3 h, 50 mg is accurately weighed and transferred to a 10 ml standard flask.
2. Dissolve in 0.01 N sodium hydroxide solution and make up to volume.
3. Transfer 2 ml of this solution into a 50 ml standard flask and make up to volume with 0.01 N potassium hydroxide solution. This is the test solution.
4. Determine the absorbance of the test solution at 286 nm and estimate the percentage purity of hesperidin taking 251.7 as the standard extinction of hesperidin.

B. Method II (HPLC method)

1. Shake 10 g air-dried powdered peel with 20 ml dimethyl sulphoxide (DMSO) and filter.
2. Wash the residue with the same solvent and collect the filtrate and the washings in a 25 ml standard flask. Make up to volume with DMSO.
3. Prepare a standard solution (5 mg dissolved in 0.05 ml of DMSO) using reference sample of hesperidin.
4. Perform HPLC analysis of both the test and standard samples using a Spherisorb ODS1 column with mobile phase of 2% acetic acid (A) and acetonitrile (B) used in gradient mode (0–15 min: A 100%; 15–45 min: A 100-70, B0-30; 45–50 min: A 70%, B 30%; 50–55 min: B 100%; 55–60 min: A 0-100, B-100-0; 60–90 min: A 100%).
5. Volume of injection is 20 µl and mobile phase flow rate 1 ml/min, with oven temperature at 40°C and the wavelength of detection 285 nm.
6. Calculate the hesperidin content of orange peel based on the peak area versus concentration data for the standard at comparable retention times for the test and the standard.