Safety And Quality Control Of Mycotoxins And Pyrrolizidine Alkaloids In Xiaoyao Tablets

Purpose: Xiaoyao Tablets are based on the addition and subtraction of the classic name Xiaoyao Pills.The effect is to soothe the liver and invigorate the spleen,nourish blood and regulate menstruation.It is used for depression,uncomfortable menstruation,irregular menstruation and other symptoms caused by liver depression and spleen deficiency,and has antidepressant properties effect.Xiaoyao tablets are composed of BUPLEURI RADIX,GLYCYRRHIZAE RADIX ET RHIZOMA PRAEPARATA CUM MELLE,PAEONIAE RADIX ALBA,ATRACTYLODIS MACROCEPHALAE RHIZOMA,ANGELICAE SINENSIS RADIX.It is made into extract 1 and extract 2.At the same time,as a project of Tasly Pharmaceutical Group Co.,Ltd.applied for the European Medicines Agency,Xiaoyao tablets must not only ensure its quality and safety,but also meet the standards and regulations of the European Pharmacopoeia.This subject establishes the testing standards for the safety and quality control of Xiaoyao tablets,which mainly include the detection of ochratoxin A and aflatoxins B1,B2,G1,G2 and 32 pyrrolizidine alkaloids in mycotoxins,which can be used as traditional Chinese medicine.The establishment of quality control of preparations provides references and methods,also lays a solid foundation for Chinese medicine to go abroad,and contributes to the internationalization of Chinese medicine.Methods: 1.Samples were extracted with 80% methanol solution ultrasonically,centrifuged,enriched by immunoaffinity column,UPLC-FLD analysis and detection,Acquity UPLC BEH C18 column separation,mobile phase A was 0.5% glacial acetic acid solution(p H=2.97),mobile phase B is acetonitrile,gradient elution.The excitation wavelength is 310 nm,the emission wavelength is 465 nm,the column temperature is 30 ℃,the volume flow rate is 0.4m L/min,and the injection volume is 2 μL.2.The sample was ultrasonically extracted with 70% methanol solution,centrifuged,enriched by immunoaffinity column,and concentrated by rotary evaporation.UPLC-FLD was used to analyze and quantify aflatoxin B1B2G1G2,and establish a detection method.Mobile phase A is ultrapure water,mobile phase B is methanol-acetonitrile(1:1),gradient elution.The excitation wavelength is 365 nm,the emission wavelength is 456 nm,the column temperature is 30 ℃,the volume flow rate is 0.4 m L/min,and the injection volume is 2 μL.3.The sample was ultrasonically extracted with 1% formic acid solution,centrifuged,purified by PCX solid phase extraction column,analyzed and detected by UPLC-MS/MS,separated by Thermo Hypersil Gold C18 column,mobile phase A was 0.05% formic acid aqueous solution,mobile phase B is methanol-0.05% formic acid solution,column temperature 40°C,flow rate 0.4 m L/min,injection volume 2 μL,multi-reaction monitoring mode determination,external standard Method was used to quantitatively separate 32 kinds of pyrrolizidine alkaloids with similar structures at the same time.Results: 1.OTA showed a good linear relationship between 1.5-37.5 ng/m L,and the correlation coefficient r=0.999.The recovery rate of OTA addition is 92.4%-119.9%,and the RSD is 2.5%-9.1%.The sample is stable within 48 h.A total of 42 batches of samples were tested and 33 batches were positive.Bupleurum and licorice were positive.Based on this result,18 batches of Bupleurum were purchased from the market,15 batches were positive,2 batches exceeded the EP limit of 20 μg/kg,and the highest level reached 101.97 μg/kg.2.The aflatoxin B1B2G1G2 of extract 1,extract 2 has a good linear relationship,the correlation coefficient r=0.999,the recovery rate of 4 kinds of AFT is between 90.9%-128.4%,and the repeatability RSD% is 2.6%-9.9% In the meantime,the samples are stable within 48 hours,and the methodological items meet the requirements.A total of 24 batches of samples were tested,and no aflatoxin was detected.3.The 32 kinds of PAs detection methods established have a good linear relationship,and the correlation coefficient r is greater than 0.994;the recovery rate at different levels of 10 μg/kg-2000 μg/kg is between 73.3% and 118.5%,and the RSD% is 2.1%-15.4%.The LOD of Xiaoyao tablets is 0.13 μg/kg-21.39 μg/kg,and the LOQ of all matrices is 7.61 μg/kg-114.40 μg/kg.The sample is stable within 48 hours.Both have good repeatability and accuracy,the recovery rate is within the acceptable range,and the repeatability is good.A total of 24 batches,none of the 32 PAs were detected.Conclusion: 1.This study established an IAC-UPLC-FLD method,which can be used for the detection of mycotoxins in traditional Chinese medicines and their extracts.The established method is fast and accurate.It meets the requirements of trace detection in samples and supplements the intermediate extraction.The blank of mycotoxin test.Surfactants are used in the pretreatment to reduce matrix interference;the special antibody filler of the IAC specifically binds to mycotoxins,reducing the occurrence of false positive results;UPLCFLD detection avoids post-column derivatization and shortens the detection time.This method needs to be further tested whether it meets a wider range of testing of traditional Chinese medicines and related extracts.2.This article uses 1% formic acid solution extraction,PCX solid phase extraction column purification,LC-MS/MS detection,external standard method quantification,and simultaneous analysis and determination of 32 kinds of PAs in medicinal materials,extracts and preparations.The current methods for detecting PAs have fewer types,usually 7 or 8 alkaloids,and most of them are concentrated on honey,traditional Chinese medicine,and plants.This paper establishes the simultaneous separation and quantification of 32 PAs,which is suitable for traditional Chinese medicine preparations with complex matrices.This method has high sensitivity and simple operation,provides a reference for detecting samples with complex matrix,and is of great significance for ensuring the quality and safety of drugs.3.Based on the production process of the medicinal material-extract-preparation of Xiaoyao Tablets,from the perspective of the entire industry chain,establish a safety and quality control strategy for Xiaoyao Tablets.According to relevant regulations,the European Pharmacopoeia0277 licorice stipulates OTA≤20 μg/kg.EP stipulates that AFB1≤2 μg/kg and AFT≤4 μg/kg.The Dutch Food and Drug Administration requires that the maximum daily dose of the controlled drug PAs and its nitrogen oxides is 0.35 μg/day/50 kg.In the safety control strategy of Xiaoyao Tablets,OTA is controlled in medicinal materials and extracts.Bupleurum and Glycyrrhiza,extract 1and 2 are strictly controlled within 2 μg/kg.If exceeded,each sample is≤20 μg/kg and the average value ≤10 μg/kg are allowed to be used.Aflatoxin B1B2G1G1,controlled in the extract,needs to meet AFB1≤2 μg/kg and AFT≤4 μg/kg.Pyrolizidine alkaloids are controlled in Xiaoyao tablets.A total of 32 PAs need to be controlled 350 μg/kg.