Evaluation and optimization of HPTLC and assay methods for Aurantii amari flos and Aurantii dulcis flos

Introduction The European Pharmacopoeia contains a monograph for bitter-orange flowers [1] but the TLC test on sweet-orange flowers as well as the photometric assay method are not satisfactory in respect of specificity and reproducibility.

Aims The current work focuses on the optimization of the identification and test method by HPTLC for bitter- and sweet-orange flowers. Additionally, a new assay method by U/HPLC for the main flavanone-glycosides in both drugs was developed.

Methods The mobile phase of the existing HPTLC method was slightly improved to formic acid : water : ethyl acetate (15:20:65 v/v/v) and naringin and rutin were introduced as intensity markers. For the assay, a new U/HPLC gradient method on a BEH C18 column provided satisfactory separation. For both methods, the samples were extracted with methanol 50% v/v.

Results Based on their chromatographic profiles, the samples could be divided into three groups. All bitter-orange flowers showed a similar fingerprint, whereas the sweet-orange flowers formed two groups, correlated with their geographic origin. Nevertheless, the differences in the HPTLC pattern between bitter- and sweet-orange flowers are quite small and make it hard to detected adulterations of bitter-orange flowers ([Fig. 1]).

Fig. 1 U/HPLC (upper three) and HPTLC (lower two) chromatograms of bitter and sweet orange flowers.

The results of the assay showed a total flavonoid content of 12.84 ± 2.16 % w/w (calculated as naringin, mean ± s) for the bitter-orange flowers and 1.18 ± 0.05 % w/w for the sweet-orange flowers.

Conclusions The content of total flavonoids in the bitter-orange flowers was about 13 times higher, compared to sweet-orange flowers. Rutin was found only in sweet-orange flowers and may allow the detection of a possible adulteration of bitter- with sweet-orange flowers.

Acknowledgements We thank SWISSMEDIC, Swiss Agency for Therapeutic Products, Pharmacopoeia division, and DIXA AG for the financial support.

Publication History

Article published online:
13 June 2022

• References

• 1 EDQM. European Pharmacopoeia, 10th Edition (2020), Bitter orange flower, Monograph 1810