| Ginseng is known as the king of all herbs,with significant pharmacological effects such as invigorating and improving immunity.Ginsenosides and volatile oils are important indicators for the quality evaluation of ginseng products in national standards,past editions of the Pharmacopoeia of the People’s Republic of China,and ginseng research.Pesticide residues are important risk factors that affect the safety of food and medicine.This thesis is the first time that a liquid chromatography mass spectrometry(LC-MS/MS)has been used to conduct a methodological study on the standard "Determination of 21 ginsenosides in ginseng by liquid chromatography mass spectrometry".According to the requirements of GB/T20001.4-2015,a draft standard(referred to as the draft method)has been developed,providing a detection method for the efficient,rapid,high throughput,and accurate detection of ginsenoside content.This paper will clarify how to apply national standard methods and draft methods to analyze the total saponins,Re,Rg1,Rb1,21 ginsenosides,volatile oil,and pesticide residues of ginseng stored under natural conditions for 4 years,and analyze the quality and safety status of ginseng stored for different periods of time,as well as evaluate its edible and medicinal value and safety.1.Establishment of ultra high performance liquid chromatography-triple quadrupole mass spectrometry for the determination of 21 ginsenosides.The chromatographic column temperature and mobile phase of the established method were optimized,and the chromatographic conditions were determined as follows: the chromatographic column was Thermo Scientific Symmetry-C18,(100 × 2.1 mm,1.7μm);The mobile phase is 0.1% formic acid water(A): acetonitrile(B);Gradient elution,flow rate of 0.3m L/min;The column temperature is 30 ℃;The injection volume is 2 μL.The mass spectrometry conditions were optimized and the parent ion(DP)and daughter ion(CE)of 21 ginsenosides were determined.The methodological study showed that the 21 ginsenosides displayed a good linear relationship in the range of 0.0125-32 μg/m L,and the corresponding correlation coefficient(r)was greater than 0.99.The limit of detection(LOD)and limit of quantification(LOQ)of the method are in the range of 0.00319 ～ 0.0215 μg/m L and0.0102 ～ 0.0896 μg/m L respectively.The RSD values of the peak areas of each saponin control sample calculated are less than 3%,the instrument precision is good,and it is stable within 48 hours.The RSD values of the peak areas of 21 saponins in the test solution are less than 3%,and the repeatability is good.By comparing the ultrasonic extraction method with the homogenate extraction method,the homogenate extraction method of sample preparation solution is determined to be more convenient,stable and efficient.By optimizing the time and times of homogenate extraction,the homogenate extraction effect is determined to be more complete for 2 minutes and 2 times.The establishment of this method provides new technical support for the quality control of conventional and rare ginsenosides.2.Analysis of the changes of ginseng components in different storage periods:(1)Total ginsenosides: According to the provisions of GB/T18765-2015,the content of total ginsenosides is determined by ultraviolet vanillin sulfuric acid colorimetry.The content of total ginsenosides in different storage periods was analyzed,and there was no significant difference between total ginsenosides and storage life(P>0.05).After 3 or 4 years of storage,the average content of total ginsenosides in ginseng that meets the standard still reaches over 2.0%.Although it does not meet the national standard 2.5%(GB/T 22536-2018),and it does not meet the requirements of commodity quality,the content is still high,and it still has edible and medicinal value.(2)Ginseng monomer saponins: The content of three monomer saponins in ginseng stored naturally for 1 to 4 years was analyzed according to the method specified in Pharmacopoeia2020.The results showed that ginsenoside Rb1 represented a decreasing trend with the increase of storage life,and the change of Rgl+Re content was not positively correlated with storage life.The average change of relative content was between 0.04884 and 0.08805,with a relatively stable overall change;Using the draft method to detect the content of monomer ginsenosides in different storage periods,13 types of monomer ginsenosides were detected,including Rb1,Rb2,Rb3,Rc,Rd,Re,Rg1,Rg2,Rf,Rh1,Ra1,Ro,and Rg3.The results showed that ginseng stored under natural conditions for 1 to 4 years still has food and medicinal value.The content of Rg1,Re,and Rb1 detected by ultra high performance liquid chromatography triple quadrupole mass spectrometry is 0.01-0.06% higher than that detected by ultra high performance liquid chromatography combined with PDA,which proves that using ultra high performance liquid chromatography triple quadrupole combined homogenization method is more time-saving,rapid,and accurate.(3)Volatile oil: single factor experiment and response surface optimization were used to assist Soxhlet extraction of volatile oil.The ratio of material to liquid was determined to be 1 :40,the extraction time was 4 hours,the extraction temperature was 85 ℃,and the calculated average yield of volatile oil was 0.385%.The yield of volatile oil extracted from ginseng with different storage years by the optimized extraction method decreased continuously with the increase of storage years,which was 0.215%,0.135%,0.0539% and 0.031% respectively.A total of 100 components of the volatile oil were detected through the detection of the volatile oil of the gas quality and the search library,of which 60 were detected after 1 year of storage,56 after 2 years of storage,56 after 3 years of storage,and 43 after 4 years of storage.The content of the volatile oil was 99.4%,99.29%,97.78%,and 97.69% respectively.A total of 21 components were detected,of which the terpene compounds were the most detected,accounting for 60%-70%.(4)Through analysis and comparison of pesticide residues in ginseng samples from different storage years,the differences in the content of pentachloronitrobenzene and hexachlorobenzene were not significant with the increase of storage years.The change value of azoxystrobin stored for one year has a difference of about 0.006;According to the detection of humic acid,the variation value of humic acid after storage for 3 years is significantly different from that of the other 4 pesticides,because the half-life period of humic acid is very short,and it can degrade under the influence of air humidity and other conditions.As for the two pesticide residues,azoxystrobin and Pythicarb,they can help to extend the storage period to reach the daily dietary consumption.To sum up,liquid chromatography-mass spectrometry(LC-MS/MS)can effectively,rapidly,high-throughput,and accurately detect the content of ginsenosides.For ginseng stored for 1 to 4years,the content of total ginsenosides,monomer ginsenosides,and volatile oil has an impact,but still has edible and medicinal value.By selecting ginseng products with qualified pesticide residues,food safety can be ensured. |
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