| Garlic has important research value as a medicinal and edible plant.The purpose of this study is to establish a method for the separation and analysis of allicin,diallyl trisulfide,diallyl disulfide and other components by using ultra-high performance liquid chromatography,high performance liquid chromatography and ultra-high performance liquid chromatography-mass spectrometry,and apply it to food and drug analysis,to provide technical support for the efficient completion of allicin analysis tasks,and to provide consumers with a reasonable choice of garlic.To provide scientific basis for eating mode and effective purchasing garlic products,and to provide practical reference for further improving garlic quality analysis and monitoring system and rational medication of garlic compound preparations.The main research contents are as follows:（1）Taking hydroxyphenylethyl ester as a substitute reference substance,a method for rapidly determining allicin in fresh garlic was established based on UPLC.The column:Waters Acquity BEH C18（2.1 mm×100 mm,1.7μm）,detection wavelength:242 nm,flow rate:0.3 m L/min,injection volume:1μL,column Temperature:30℃,mobile phase:acetonitrile-0.1%aqueous formic acid（30:70）.The results showed that under optimized chromatographic conditions,the separation of allicin and hydroxyphenylethyl ester was achieved within 4 min,showing a good linear relationship（r≥0.9998）within a certain mass concentration range,and the relative correction factor is well reproduced under different conditions.The recovery rate of standard addition is 98.8-100.9%,and the lower limit of quantification is1.73～2.03 mg/kg.Compared with HPLC method,the analysis time is obviously shortened and the analysis efficiency is higher.The external standard method and the substitute reference method have no obvious difference（P>0.05）.It provides new technical support for the development and utilization of medicinal garlic plant resources and the improvement of medicinal garlic quality analysis system.（2）Based on HPLC,a method for the determination of three components of allicin,diallyl trisulfide and diallyl disulfide in garlic and its products was established using ethyl hydroxybenzoate as a substitute for control.Reversed-phase C18column（250 mm×4.6 mm,5μm）was used,acetonitrile-0.02%phosphoric acid aqueous solution was used as mobile phase with gradient elution,detection wavelength was 242 nm,flow rate was 1.0 m L/min,column temperature was 24℃.The results showed that four substances including ethyl hydroxybenzoate were baseline-separated within 64 minutes by optimizing the parameters such as mobile phase composition,elution mode,detection wavelength and column temperature,and showed a good linear relationship within a certain concentration range（r≥0.9996）.Relative correction factor was reproducible under different conditions.The recovery was 98.5%～103.3%and the lower limit of quantitation was 2.5～10.0 mg/kg.For the first time,this study established an alternative reference method based on HPLC for simultaneous detection of three components of using ethyl hydroxybenzoate as a substitute for control.in garlic and its products.The method is accurate and reliable,and is suitable for the detection of various garlic products.（3）Based on HPLC and UPLC-MS/MS to study the correlation between allicin and diallyl disulfide,diallyl trisulfide in vivo and the possibility and conversion rate of alliin in the enteric pellets to produce allicin in the body.HPLC method,column:C18（250 mm×4.6 mm×5μm）,mobile phase:acetonitrile-water（53:47）,（65:35）,column temperature:25℃,flow rate:1 m L/min,1.2 m L/min;UPLC-MS/MS method,column:C18（Waters ICQUITY UPLCBEH,100×2.1 mm,1.7μm）,column temperature:40℃,flow rate:0.15 m L/min,injection volume:2μl,Mobile phase:0.1%formic acid（A）-acetonitrile（B）,gradient elution;mass spectrometry ionization:ESI+,ion polarity:positive ion;capillary voltage:2 KV;cone voltage:22 V;source temperature:150℃;Desolvation temperature:350℃;argon flow rate:50 L/Hr;nitrogen flow rate:600 L/Hr.The results showed that the presence of diallyl disulfide and diallyl trisulfide was not detected in rat plasma after intragastric administration of allicin;The pharmacokinetic parameters of alliin in rats were determined by intragastric administration of the same alliin content of alliin solution and garlic enteric pellet solution.The results showed that about 34%of alliin was directly absorbed in the enteric pellets,and about 66%of alliin was converted in the body.When the allicin solution or the garlic enteric pellet was administered,no significant increase in hydrogen sulfide was observed in the plasma relative to endogenous hydrogen sulfide.After further administration for 24 hours,the hydrogen sulfide level in the rat tissue was measured and found to be administered.The level of hydrogen sulfide in the tissues of the group was generally higher than that in the blank group.The overall trend of hydrogen sulfide levels in each group was:garlic enteric pellet group>alliin solution group>blank group.This study established HPLC method for qualitative determination of diallyl disulfide and diallyl trisulfide in rat plasma and UPLC-MS/MS method for quantitative determination of alliin and hydrogen sulfide in plasma and tissues in vivo.The in vivo metabolic process of allicin could not be studied indirectly by tracking the content of diallyl disulfide and diallyl trisulfide;The conversion process of 66%of alliin in enteric pellets may be firstly catalyzed by garlic enzyme to produce allicin,and then metabolized to hydrogen sulfide through a series of reactions. |
