Establishment And Application Of Analytical Method For Phenolic Compounds In Propolis

Propolis is rich in flavonoids and phenolic acids,which has strong anti-oxidation and anti-cancer effects.At present,the evaluation method of propolis quality in China is relatively simple,mainly based on the content of total flavonoids.Poplar gum is mixed with flavonoids to pretend to be propolis because of the shortage of propolis products in the market.It is difficult to make a fine evaluation of the quality of propolis according to the content of flavonoids.In addition,the functional activity of propolis is closely related to the presence of the contained monomeric phenolic compounds.In this paper,a method for the detection of phenolic compounds in propolis by high performance liquid chromatography（HPLC）was established.The main results are as follows:（1）Two methodologies for simultaneous determination of 20 phenolic compounds in propolis by HPLC-UV and HPLC-MS/MS were developed.The sample extraction and chromatographic separation conditions,and ultraviolet detection wavelength,as well as mass spectrometry parameters were selected and optimized.Under the optimized conditions,good linearities（R²≥0.998）were obtained for all analytes.The limits of quantification（LOQs）of the proposed methods by HPLC-UV and HPLC-MS/MS for all analytes were in the range of 0.02～0.1 mg/g and 0.05～0.2 mg/g,respectively,and the recoveries were in the range of 95.4%～109.5%and 94.0%～106.8%with the relative standard deviations（RSDs）in the range of 1.1%～7.5%and 0.4%～11.1%,respectively.The proposed methods were applied for the determination of 20 phenolic compounds in 13propolis samples collected from China and Brazil.The detection results of the two methods are almost consistent with each other,indicating that both methods are applicable,and the inter-confirmation of the two methods can improve the accuracy of the determination of phenolic components in propolis.（2）High-throughput and rapid detection of 31 phenolic compounds in propolis was achieved by high performance liquid chromatography-tandem mass spectrometry.Propolis samples were extracted with ethanol and water,separated by Atlantis^TM T3 column,detected by electrospray ionization（ESI）and multiple reaction monitoring（MRM）mode,and quantified by external standard method.The linear relationships of 31 compounds were good（R²≥0.99）in their respective linear ranges.The recovery was 80.5%～109.6%,and the relative standard deviation（RSD）was 0.2%～12%.This method is suitable for the determination of 31 phenolic compounds in propolis samples.（3）The established method was used to detect 31 kinds of phenolic compounds in propolis capsules and propolis liquid.The results showed that most of the compounds in propolis products were similar to those in propolis.This method can be effectively used to detect phenolic compounds in propolis products,providing some reference for maintaining the quality stability of propolis products.