Study On Distribution And Change Rules Of Alkaloids In Bee Products

Technical barriers to trade(TBT) has become a main obstacle to export trade in every country. For years, as exposure cases of alkaloids in bee products occured frequently, alkaloids as an emerging risk factor on bee products quality and safety has become a new research hotspot to scientists in recent years. However, we have not yet conducted risk assessment and monitoring of alkaloids in bee products which include domestically produced products and imported products from other countries. Consequently, domestic consumers are confronted with a potential threat to human health from bee products containing alkaloids and exported bee products are possibly subject to TBT. Therefore, this research aims to develop detection methods for pyrrolizidine alkaloids and isoquinoline alkaloids in honey and bee pollen, to investigate the distribution laws of two alkaloids in domestic honey, bee pollen and imported honey, and to explore their change rules in the storage after collection, process of honey processing and storage, to finally provides theoretical basis for monitor-control and early-warning of our bee products market and perfect risk factors detection system for bee product quality and safety.This thesis mainly divided into three parts:In the first part, a method for determination of pyrrolizidine alkaloids and isoquinoline alkaloids was separately developed in honey and bee pollen by dispersive solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry(QuEChERS-HPLC-MS/MS). For the detection of pyrrolizidine alkaloids, the sample needed to be reduced with zinc dust firstly, then extracted with acetonitrile solution and cleaned up with PSA absorbent, finally analyzed by HPLC-MS/MS. While the samples can be directly pretreated using QuEChERS without reduced procedure for the determination of isoquinoline alkaloids. Matrix-matched calibration curves with good correlation coefficients(R2> 0.99) were obtained in the concentration range of 0.1-100 μg/L. The recoveries of the spiked samples at 1-100 μg/kg were in the range of 70% to 110% with the RSD of intra-day and inter-day lower than 15% and 20%, respectively. The limits of detection(LOD) and limits of quantification(LOQ) for all alkaloids were 0.3 μg/kg and 1.0 μg/kg, respectively. This method was successfully applied to the determination of pyrrolizidine alkaloids and isoquinoline alkaloids for quantification and confirmation in honey and bee pollen samples.In the second part, the alkaloids concentration in major types of domestic honey, bee pollen and imported honey were analyzed. The results showed that the detection rate and concentration of pyrrolizidine alkaloids and isoquinoline alkaloids in domestic bee products were low; nevertheless, the detection rate and concentration from imported honey were significantly higher. And the highest detection rate and the highest amount among alkaloids were lycopsamine and echimidine, which were 46%,40%, and 177.0 μg/kg, 177.6 μg/kg, respectively. Moreover, there were at most 4 alkaloids types in one sample, and the total amount found in highest levels of pyrrolizidine alkaloids summed up to 226.1 μg/kg. From the honey type point, we could obtain that detection risk of manuka honey and multiflorous honey were higher. In the aspect of region, the detection rate and concentration of alkaloids from New Zealand were highest. This study proved that domestic bee products were safer than imported sample in the aspect of pyrrolizidine alkaloids and isoquinoline alkaloids.In the third part, it mainly studied the change rules of pyrrolizidine alkaloids and isoquinoline alkaloids in honey during three different process of storage after collection, processing and storage. The study results indicated that pyrrolizidine alkaloids and isoquinoline alkaloids were stable at storage process in the open air temperature of 40 ℃ for 12 weeks, and were not changed obviously at processing condition of 50 ℃ for 24 hours. Moreover, in the storage experiments, it was found that the concentration of the two alkaloids maintained almost unchanged in 25 ℃ for 6 months. Thus, relevant regulators need to do well for positive samples screening and monitoring to make sure health and safety of consumers.