HPLC analysis of sennosides A and B in Sennae folium and fructus. A proposal for the European Pharmacopeia monographs

The current monographs in the European Pharmacopoeia (Ph. Eur. 7.3) for Senna pods and Senna leaves describe a photometric assay based on the Borntraeger reaction to determine hydroxyanthracene glycosides, calculated as sennoside B. The method is time consuming, unspecific for sennosides and the precision is not adequate for a modern assay. Its adapation to herbal medicinal products is complex and the method is not suitable for process validation. According to references [1] about 70% of the total anthraquinone content is due to sennosides A and B. These substances are therefore suitable for the standardisation of Senna products. The Chinese Pharmacopoeia (PPRC) and the Japanese Pharmacopoeia (JP) already describe HPLC methods to determine sennoside A and B in the respective monographs. The JP uses ion-pair-chromatography with tetraheptylammonium bromide. The runtime is 70min and even a time-optimised version according to Seidlitz [2] still takes 62min. The adapted and validated method [3] based on Yamasaki [4] uses solid phase extraction (SPE) with an anion exchange matrix, which allows a selective sample preparation. The runtime is 10min, the chromatogram shows two peaks due to sennosides A and B and two additional smaller peaks. One of them is rhein-8-O-glucoside.

The procedure has been successfully validated according to the ICH guidelines. Up to now six batches (more will follow and presented on the poster) of Senna were analysed. The pods (Senna angustifolia) showed a total content of sennoside A and B of 1.74 to 2.76% and the content of the leaves was of 1.07 to 1.19%.

As stationary phase a RP C18 column Tosh TSKgel ODS-80TS (4.6mm x 150mm, 5µm) and as mobile phase a mixture of acetonitrile, water and phosphoric acid (200:800:1 V/V/V) were used. The flow rate was 1.2ml/min, the column temperature 40°C, the detection wavelength 380nm, and the injection volume 20µl.

We propose to include the method into the senna monographs of the European Pharmacopeia.

Acknowledgement: We thank SWISSMEDIC, Swiss Agency for Therapeutic Products, Pharmacopoeia division, for the financial support.

References: [1] Grimmiger W, Witthohn K. Analytics of Senna drugs with regard to the toxicological discussion of anthranoids. Pharmacology 1993; 47 (Suppl. 1): 98–109

[2] Seidlitz M. Optimierung der Bestimmungsmethode für Sennosid A und B gemäss Japanischer Pharmakopöe mit HPLC [Internal document]. Wädenswil: Zurich University of Applied Science; 2011

[3] Seidlitz M. Adaption von HPLC-Methoden zur Bestimmung von Anthrachinonglykosiden in Aloe und Senna im Hinblick auf die Erneuerung der entsprechenden Arzneibuchmonografien [Bachelor thesis]. Wädenswil: Zurich University of Applied Science; 2011

[4] Yamasaki K, Kawaguchi M, Tagami T et al. Simple and rapid analysis of sennoside A and sennoside B contained in crude drugs and crude drug products by solid-phase extraction and high-performance liquid chromatography. J Nat Med 2010; 64: 126–132