New HPLC – method to determine Frangulin A and B as well as Glucofrangulin A and B in Frangulae cortex

Introduction: The current monograph in the European Pharmacopeia for Frangulae cortex describes a photometric assay based on an adapted bornträger reaction to determine hydroxyanthracene glycosides, calculated as frangulin B. The method is time consuming, unspecific for frangulines and the precision is not adequate for a modern assay.

Aim: The photometric method shall therefore be replaced by a modern HPLC-method. There is no HPLC method published in the literature that allows the determination of frangulin A/B and glucofrangulin A/B in Frangulae cortex.

Method: About 300 mg of freshly milled drug are extracted for 15 min with ultrasound. The extraction solution consists of acetonitrile/water 50:50 v/v and 2 g/L NaHCO₃. A conventional RP C₁₈ Nucleodur (4 mm x 125 mm), 3 µm was used as stationary phase. Mobile phase A consists of water (pH of 2.0, adjusted with phosphoric acid). Mobile phase B consists acetonitrile/methanol 20:80 v/v. The flow rate is 1.0 mL/min, the detection wavelength 435nm, the column temperature is 50 °C, and the injection volume 20µL. The gradient is shown in table 1.

Tab. 1: Gradient table

Time (min)	Mobile phase A (%)	Mobile phase B (%)
0	66	34
15.0	66	34
16.0	50	50
26.0	24	76
26.5	0	100
28.5	0	100
29.0	66	34
45.0	66	34

Results: The mobile phase separates the four frangulins sufficiently. Results of several samples will be presented on the poster. A chromatogram from a Frangulae cortex sample is shown in figure 1.

Fig. 1: Chromatogram from a Frangulae cortex sample.

Conclusion: The method we developed is simple, robust and precise. It is a reasonable option for pharmacopeia applications to replace the outdated photometric assay.