Berberine gives precipitation with most of the alkaloidal reagents. Some specific tests for identification of berberine are listed below:

1. To a few crystals of berberine dissolved in water, add a drop of bromine water. A bright red colour is produced.
2. To a small quantity of berberine add 0.5 ml of nitric acid. A reddish-brown colour is obtained.
3. To a small quantity of berberine add 0.5 ml of sulphuric acid. An orange-yellow solution is obtained changing to an olive green colour on warming.
4. To a solution of berberine in sulphuric acid add a few crystals of potassium dichromate. A black colour changing to brownish-violet is produced.
5. To 1 ml of a solution of berberine in water add Froehde’s reagent (0.5 g molybdic acid or sodium vanadate in 100 ml hot sulphuric acid). A brownish-green colour is produced.
6. To 1 ml of a solution of berberine in water add Mandelin’s reagent (1 g ammonium vanadate in 100 ml sulphuric acid). A violet colour is produced.

Estimation

Estimation of berberine in traditional medicine formulations and conventional dosage forms may be undertaken by several spectrophotometric, chromatographic and other methods.

A. Method I (Gravimetric method)

1. Extract to exhaustion 5 g of powdered plant material with 200 ml ethanol in a Soxhlet apparatus.
2. Evaporate the extract to dryness on a water bath and add 50 ml hot water to the residue.
3. Separate the precipitated resin by filtration and concentrate the filtrate by evaporation on a hot plate to about 20 ml.
4. Add 10 ml of 15% sodium hydroxide and add 5 ml of a 2% solution of gum tragacanth and extract with 5 × 20 ml portions of ether.
5. Collect the ether layers into a 100 ml standard flask and make up to volume if necessary.
6. Transfer 50 ml of the ether solution to a beaker and add a saturated solution of picrolonic acid in ether in small amounts until precipitation is complete.
7. Allow to settle and filter carefully over a tared sintered funnel. Wash the residue on the filter with ether to remove excess picrolonic acid. Dry the product at 105°C and note its weight.
8. The yield of berberine multiplied by the factor 0.561 gives the equivalent amount of berberine. Calculate the percentage purity from the known weight of the plant material.

B. Method II (Spectrophotometric assay)

1. Weigh accurately 50 mg of berberine and prepare a stock solution of 100 µg/ml in water.
2. Transfer 5 ml to a 10 ml standard flask and make up to volume with water.
3. Determine the absorbance of the solution at 345 nm.
4. Calculate the percentage purity of berberine using 722 as the specific absorptivity of berberine at 345 nm.