Effect of drying methods on the free and conjugated bufadienolide content in toad venom determined by ultra-performance liquid chromatography-triple quadrupole mass spectrometry coupled with a pattern recognition approach

• The level of 36 bufadienolides in dried toad venom was determined using LC–MS/MS.
• Vacuum-drying at 60 °C produced the highest quality of dried toad venom.
• Air-drying at room temperature (RT) caused the loss in bufadienolide content.
• Cinobufagin and its derivatives decreased following traditional air-drying at RT.

Drying is a useful technique for extending the shelf-life of biological products and enabling long-term storage; however, improper drying can reduce the chemical quality of the products. In this study, we used ultra-performance liquid chromatography–triple quadrupole/mass spectrometry (LC–MS/MS) and multivariate statistical analysis to investigate the effects of four drying methods (V: vacuum-drying at 60 °C, F: freeze-drying, H: air-drying at 60 °C and R: air-drying at room temperature) on the levels of 36 bufadienolides in toad venom. Vacuum-drying at 60 °C produced the highest quality dried toad venom in terms of total bufadienolide content, whereas traditional air-drying at room temperature (RT) to dehydrate the toad venom led to a dramatic loss in free and conjugated bufadienolides, reaching up to 60% and 70%, respectively. Assaying for free bufadienolides ranked the drying methods as V ≈ F > H > R, whereas assaying for conjugated bufadienolides slightly changed the order to V > F ≈ H > R. Furthermore, we identified 21 bufadienolides as biomarkers responsible for the decline in the quality of dried toad venom, whose loss varied from 1.5-fold to 100-fold. Of these biomarkers, group I bufadienolides that contain 16-OAc (e.g., cinobufagin and its hydroxyl or arginine ester derivatives) were characteristic components and were reduced to trace levels (loss of more than 10-fold) following traditional air-drying at RT. This might be attributed to the fact that most enzyme-sensitive bufadienolides were biotransformed or degraded at room temperature but were retained using other drying methods.

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