Discovery of peptide quality markers for quality control and identification of toad venom using LC-MS/MS and label-free methods

Toad venom, a secretion from the parotoid glands of toads such as Bufo bufo gargarizans Cantor, is a traditional Chinese medicine renowned for its cardiotonic, anti-inflammatory, and antitumor properties. However, due to the rising price of toad venom, its quality control faces significant challenges from widespread adulteration with substances like toad skin and dried toads, which compromise its authenticity and therapeutic efficacy. Currently, no comprehensive method exists for the simultaneous detection of these adulterants. In this study, dried toads were subdivided into skin, flesh, and bone and compared with toad venom from Bufo bufo gargarizans Cantor using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with multivariate statistical analysis. This is the first study to propose a peptide-based method for simultaneously identifying and quantifying adulterants such as toad skin, flesh, and bone in toad venom, offering a promising approach to improve quality control techniques for toad venom. Twenty-seven peptide quality markers were identified, including eight for toad venom, six for toad skin, five for toad flesh, and eight for toad bone. These markers were validated via bioinformatics analysis and HPLC-Triple Quadrupole MS, enabling the establishment of a novel and reliable quality control strategy. A method for determining toad venom content was established using the peptide quality marker NNAYDINEER. The method demonstrated high specificity, excellent linearity (R² > 0.9977), a limit of quantification (LOQ) of 1 ng/mL, and outstanding repeatability, precision, and stability. Analysis of five batches of toad venom samples revealed their content ranged from 0.0387 % to 0.0963 %. This study offers an innovative approach for both qualitative identification and quantitative determination, providing critical technical and theoretical support for ensuring the quality and authenticity of toad venom-based traditional medicines and related formulations.