Chromatographic analysis method of garlic sulfur-containing compound

Abstract

The invention provides a chromatographic analysis method of garlic sulfur-containing compounds, and particularly develops a preparation method of an instant reference substance solution, and creates a chromatographic analysis method capable of synchronously detecting various unstable garlic sulfur-containing compounds according to the preparation method. Various active ingredients in garlic and garlic products can be detected to the greatest extent. The method is easy and convenient to operate, good in durability, high in precision and suitable for real-time detection.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and more specifically relates to a chromatographic analysis method for sulfur-containing compounds in garlic. Background Art

[0002] Garlic is the bulb of Allium sativum L., a plant of the lily family. It is recognized worldwide as a plant for both medicinal and edible uses. Garlic and related products have been included in the pharmacopoeias of China, the United States, Europe, the United Kingdom, and Japan. The biologically active ingredients in garlic are mainly divided into two parts: one is the sulfur-containing amino acid component, mainly alliin; the other is the thiosulfinate produced by the alliinase reaction, such as allicin (diallyl thiosulfinate). Modern pharmacological studies have shown that thiosulfinates have good antioxidant, anti-inflammatory, anti-tumor, antibacterial, and antifungal pharmacological activities.

[0003] When garlic cells are damaged, the sulfoxide precursors in the cytoplasm or cytoplasmic vesicles are immediately converted by alliinase in the vacuoles of the vascular bundle sheath cells into extremely unstable sulfenic acid intermediates, pyruvic acid and ammonia; then, two molecules of sulfenic acid form thiosulfinates. The substrates of alliinase in garlic include alliin, methyl alliin and isoalliin, which react with alliinase to produce three sulfenic acids, and theoretically produce nine thiosulfinates.

[0004] At present, the detection methods for active substances in garlic mainly focus on the detection of alliin and allicin, and the methods are single. Therefore, it is urgent to develop a detection method that can simultaneously detect multiple sulfur compounds in garlic. Summary of the invention

[0005] The object of the present invention is to provide an analytical method for sulfur-containing compounds in garlic.

[0006] The first aspect of the present invention provides a method for preparing an instant reference substance for chromatographic analysis of sulfur-containing compounds in garlic, characterized in that the preparation method is as shown in Formula 1:

[0007] Formula 1: Alliinase substrate reference + alliinase → alliinase hydrolysis product reference

[0008] Among them, alliinase substrates include:

[0009] C1: Alliin; C2: Methylalliin; C3: Isolliin.

[0010] Alliinase hydrolysis products include:

[0011] Z1: diallyl thiosulfinate (garlicin); Z2: dimethyl thiosulfinate;

[0012] Z3: diallyl thiosulfinate; Z4: methyl allyl thiosulfinate;

[0013] Z5: allyl methyl thiosulfinate; Z6: methyl methacrylate thiosulfinate;

[0014] Z7: allyl methyl thiosulfinate; Z8: allyl allyl thiosulfinate;

[0015] Z9: Propylene allyl thiosulfinate.

[0016] In another preferred embodiment, the preparation method further comprises one or more steps selected from the following group:

[0017] (A) C1 + alliinase → Z1

[0018] (B) C2 + alliinase → Z2

[0019] (C) C3 + alliinase → Z3

[0020] (D) C1+C2+alliinase→Z1, Z2, Z4, Z5

[0021] (E) C2+C3+alliinase→Z2, Z3, Z6, Z7

[0022] (F) C1+C3+alliinase→Z1, Z3, Z8, Z9

[0023] (G) C1+C2+C3+alliinase→Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9.

[0024] In another preferred embodiment, the garlic sulfur-containing compounds include alliinase substrates and alliinase hydrolysis products.

[0025] In another preferred embodiment, the reaction time of the alliinase substrate and the alliinase to generate the enzymatic hydrolysis product is 10-15 minutes.

[0026] In another preferred embodiment, the instant reference solution is stable within 4-6 hours.

[0027] The second aspect of the present invention provides a chromatographic analysis method for sulfur compounds in garlic, comprising the steps of:

[0028] (a) providing a sample solution;

[0029] (b) preparing an instant reference solution using the method described in the first aspect of the present invention;

[0030] (c) The sample solution and the instant reference solution are subjected to liquid chromatography analysis.

[0031] In another preferred embodiment, the chromatographic analysis method is used to obtain a characteristic spectrum of garlic sulfur compounds, in which:

[0032] If Z1 is taken as the reference peak and its relative retention time is 1, then the relative retention time of Z2 is 0.65, the relative retention time of Z4 / Z5 is 0.77, and the relative retention time of Z8 / Z9 is 1.17.

[0033] In another preferred embodiment, by comparing the characteristic spectra of the test sample and the reference sample, the composition information of the sulfur-containing compounds in the test sample can be obtained.

[0034] In another preferred embodiment, the retention time of Z1 is 7.5 min.

[0035] In another preferred embodiment, the retention time of Z2 is 4.8 min.

[0036] In another preferred embodiment, Z4 and Z5 are isomers, and their peaks emerge at the same time during chromatographic analysis.

[0037] In another preferred embodiment, the retention time of Z4 / Z5 is 5.7 min.

[0038] In another preferred embodiment, the Z8 and Z9 are isomers and emit peaks at the same time during chromatographic analysis.

[0039] In another preferred embodiment, the retention time of Z8 / Z9 is 8.7 min.

[0040] In another preferred embodiment, the Z3 is derived into other substances, which show split peaks in liquid chromatography.

[0041] In another preferred embodiment, the mobile phase of the chromatographic analysis method is a mixed solution of methanol and formic acid.

[0042] In another preferred embodiment, in the mobile phase, the ratio of methanol:1% formic acid is 1:1-2:1.

[0043] In another preferred embodiment, the detection conditions of the chromatographic analysis method also include that the chromatographic column is a reverse phase C18 column.

[0044] In another preferred embodiment, the detection conditions of the chromatographic analysis method further include a column temperature of 30-40°C.

[0045] In another preferred embodiment, the detection conditions of the chromatographic analysis method also include a flow rate of 0.5-1.0 mL / min.

[0046] In another preferred embodiment, the detection conditions of the chromatographic analysis method also include a detection wavelength of 240-270 nm.

[0047] In another preferred embodiment, the method is a quantitative, qualitative and / or impurity detection method.

[0048] In another preferred embodiment, the method further comprises: a liquid chromatography analysis method II for analyzing alliin, methyl alliin, isoalliin and γ-glutamine-S-allyl-L-cysteine ​​(GSAC), wherein the method II comprises the steps of:

[0049] (1) preparing sample solution and reference solution;

[0050] (2) subjecting the sample solution and the reference solution to liquid chromatography analysis;

[0051] Wherein, the chromatographic column used in step (2) is a strong cationic chromatographic column bonded with sulfonic acid groups.

[0052] In another preferred embodiment, the strong cation chromatography column is CAPCELL PAK SCX.

[0053] In another preferred embodiment, the method II is used to obtain characteristic spectra of alliin, methyl alliin, isoalliin and GSAC, in which:

[0054] If alliin is taken as the reference peak and its relative retention time is 1, the relative retention time of methyl alliin is 0.86, the relative retention time of isoalliin is 1.10, and the relative retention time of GSAC is 1.96.

[0055] In another preferred embodiment, the retention time of alliin is 5.94 min.

[0056] In another preferred embodiment, the retention time of methyl alliin is 5.14 min.

[0057] In another preferred embodiment, the retention time of isoalliin is 6.54 min.

[0058] In another preferred embodiment, the retention time of the GSAC is 11.62 min.

[0059] In another preferred embodiment, the mobile phase of method II is potassium dihydrogen phosphate solution.

[0060] In another preferred embodiment, the concentration of the potassium dihydrogen phosphate solution is 5-15 mM.

[0061] In another preferred embodiment, the pH of the potassium dihydrogen phosphate solution is 2-4.

[0062] In another preferred embodiment, the detection conditions of method II also include a column temperature of 40-50°C.

[0063] In another preferred embodiment, the detection conditions of method II also include a flow rate of 0.5-1.0 mL / min.

[0064] In another preferred embodiment, the detection conditions of method II also include a detection wavelength of 210-230 nm.

[0065] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 shows: the HPLC chromatogram of the allicin control solution after alliinase reaction (1a); the HPLC chromatogram of the alliin control solution before alliinase reaction (1b); and the HPLC chromatogram of the alliinase extract (1c).

[0067] FIG. 2 shows: HPLC chromatogram of dimethyl thiosulfinate reference solution after alliinase reaction ( 2a ); HPLC chromatogram of methyl alliin reference solution before alliinase reaction ( 2b ).

[0068] FIG3 shows: HPLC chromatogram of diallylthiosulfinate reference solution after alliinase reaction (3a); HPLC chromatogram of isoalliin reference solution before alliinase reaction (3b).

[0069] FIG4 shows: HPLC chromatogram of AM control solution after alliinase reaction (4a); HPLC chromatogram of alliin and methylalliin mixed control solution before alliinase reaction (4b).

[0070] FIG5 shows: HPLC chromatogram of AP control solution after alliinase reaction (5a); HPLC chromatogram of alliin and isoalliin mixed control solution before alliinase reaction (5b).

[0071] Figure 6 Shown is the HPLC chromatogram of the MP control solution after alliinase reaction.

[0072] FIG. 7 shows: the total ion current and extracted ion chromatogram of the garlic powder test solution (7a); the total ion current and extracted ion chromatogram of the garlic test solution (7b).

[0073] Figure 8 The HPLC profile of the thiosulfinate esters is shown.

[0074] Fig. 9 The HPLC overlay spectra (S1-S16, i.e., G1-G16) and the reference spectra (R) of thiosulfinates in 16 batches of garlic are shown.

[0075] Fig.10 The HPLC overlay spectra (S1-S31, ie, P1-P31) and the reference spectra (R) of thiosulfinates in 31 batches of garlic powder are shown.

[0076] Fig.11 Liquid chromatograms of four alliinase substrate reference solutions are shown: from left to right: methylalliin, alliin, isoalliin, and GSAC.

[0077] Fig.12 Shown is a liquid chromatogram of the alliinase substrate of fresh garlic in one embodiment.

[0078] Fig.13 Shown is a liquid chromatogram of the alliinase substrate of garlic powder in one embodiment.

[0079] Fig.14 Shown is an overlay of alliinase substrate profiles from 16 batches of garlic powder. DETAILED DESCRIPTION

[0080] Due to the instability of some sulfur-containing compounds in garlic, no analytical identification method for its unstable products has been developed in the prior art. The inventors have developed a method for preparing an instant reference solution through extensive and in-depth research, and based on this, have created an analytical method that can simultaneously detect a variety of unstable sulfur-containing compounds in garlic, which can detect a variety of active ingredients in garlic and garlic products to the greatest extent, and the method is simple to operate, has good durability, and is suitable for real-time detection. On this basis, the present invention was completed.

[0081] the term

[0082] In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0083] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0084] In the present invention, the garlic sulfur-containing compound includes an alliinase substrate and an enzymolysis product. After the alliinase substrate is catalyzed by the alliinase, an enzymolysis product is generated.

[0085] As used herein, the term "alliinase substrate" refers to sulfoxide-containing precursor compounds in garlic, including:

[0086] C1: S-allyl-L-cysteine ​​sulfoxide (alliin);

[0087] C2: S-methyl-L-cysteine ​​sulfoxide (methylalliin);

[0088] C3: S-allyl-L-cysteine ​​sulfoxide (isollylin).

[0089] As used herein, the term "enzyme hydrolysate" refers to the thiosulfinate generated by the alliinase reaction of the alliinase substrate, including:

[0090] Z1: diallyl thiosulfinate (garlicin); Z2: dimethyl thiosulfinate;

[0091] Z3: diallyl thiosulfinate; Z4: methyl allyl thiosulfinate;

[0092] Z5: allyl methyl thiosulfinate; Z6: methyl methacrylate thiosulfinate;

[0093] Z7: allyl methyl thiosulfinate; Z8: allyl allyl thiosulfinate;

[0094] Z9: Propylene allyl thiosulfinate.

[0095] Alliinase substrates, enzymatic hydrolysis products and reaction relationships

[0096] Table 1 Alliinase substrates and enzymatic hydrolysis products

[0097]

[0098]

[0099] Reaction relationship:

[0100] 1. Alliin + Alliinase → Allicin

[0101] 2. Methyl alliin + alliinase → dimethyl thiosulfinate

[0102] 3. Isollyin + alliinase → diallyl thiosulfinate

[0103] 4. Methyl alliin + alliin + alliinase → allicin, dimethyl thiosulfinate, methyl allyl thiosulfinate, allyl methyl thiosulfinate

[0104] 5. Methyl alliin + isoalliin + alliinase → dimethyl thiosulfinate, diallyl thiosulfinate, methyl allyl thiosulfinate, allyl methyl thiosulfinate

[0105] 6. Alliin + isoalliin + alliinase → allicin, diallyl thiosulfinate, allyl thiosulfinate, allyl allyl thiosulfinate

[0106] 7. Methyl alliin + alliin + isoalliin + alliinase → allicin, dimethyl thiosulfinate, diallyl thiosulfinate, methyl allyl thiosulfinate, allyl methyl thiosulfinate, methyl allyl thiosulfinate, allyl methyl thiosulfinate, allyl methyl thiosulfinate, allyl methyl thiosulfinate, allyl allyl thiosulfinate, allyl allyl thiosulfinate

[0107] CAPCELL PAK SCX

[0108] CAPCELL PAK SCX is a strong cation exchange column bonded with sulfonic acid groups. It can achieve highly sensitive quantitative HPLC analysis of a variety of amino acids under simple liquid phase conditions without the need for derivatization or the addition of ion pair reagents. It can analyze a variety of sulfur-containing amino acids.

[0109] The cation exchange chromatography column used in the embodiment of the present invention is CAPCELL PAK SCX UG (5 μm, 250mm×4.6mm).

[0110] Liquid chromatography analysis

[0111] The liquid chromatography analysis of the present invention refers to the qualitative or quantitative analysis of various garlic sulfur compounds using liquid chromatography. Alliinase exists in garlic, which causes some compounds to generate enzymatic hydrolysis products through enzyme catalysis. Since these enzymatic hydrolysis products are unstable and easy to transform, a method for preparing instant reference substances for these unstable substances is developed based on this, and a characteristic fingerprint spectrum of garlic sulfide is constructed, which can simultaneously analyze various garlic active ingredients.

[0112] Compared with the prior art, the advantages of the present invention are as follows:

[0113] 1. The present invention develops for the first time a method for preparing an instant reference substance for unstable sulfur-containing compounds in garlic, and thereby creates a method for analyzing and identifying unstable sulfur-containing compounds in garlic, thus making up for the deficiencies of the prior art.

[0114] 2. The analytical method of the present invention can simultaneously detect a variety of garlic sulfur compounds, further improving the analysis of various active ingredients in garlic.

[0115] 3. CAPCELL PAK SCX is a strong cation exchange column bonded with sulfonic acid groups, which can realize highly sensitive quantitative HPLC analysis of various sulfur-containing amino acids in garlic under simple liquid phase conditions without the need for derivatization or the addition of ion pair reagents.

[0116] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the following examples without specifying specific conditions are usually based on the conditions described in the normal conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0117] Example 1. Identification of enzymatic hydrolysis products - thiosulfinates (HPLC and UPLC-MS / MS)

[0118] 1. Experimental instruments

[0119] High performance liquid chromatograph (LC-2050C, LC-2030 3D Plus, Shimadzu Corporation); ultra-high performance liquid phase triple quadrupole mass spectrometer (ACQUITY UPLC-Class / Waters TQ-S, Waters Technology (Shanghai) Co., Ltd.); analytical balance (ME204E / 02, AB135-S, Mettler-Toledo International Trading (Shanghai) Co., Ltd.); ultrapure water analyzer (Excees-E-UV, Chengdu Tang Corning Technology Development Co., Ltd.); high-speed low-temperature centrifuge (TGL-20bR, Shanghai Anting Scientific Instrument Factory); small high-speed low-temperature refrigerated centrifuge (5424R, Aibende (Shanghai Trading Company)); CNC ultrasonic cleaner (KQ3200DE, Kunshan Ultrasonic Instrument Co., Ltd.); homogenizer (HTY-761, Zhejiang Tailin Biotechnology Co., Ltd.), ultraviolet detector (SJ-1031A, Shimadzu).

[0120] 2. Experimental Reagents

[0121] Alliin reference substance (99%, Xinjiang Elexin Pharmaceutical Co., Ltd.); methyl alliin reference substance (99%, LKT Laboratories); isoalliin reference substance (98.0%, MTAR Research Chemicals Inc.); alliinase extract (>10000 U·g -1 , Xinjiang Garlic Research Institute (Co., Ltd.)); butylparaben reference substance (99%, China Food and Drug Inspection Institute); methanol (≥99.9%, SIGMA-ALDRICH), formic acid, anhydrous methanol, n-hexane, isopropanol, ethyl acetate, and sulfuric acid were all of analytical grade.

[0122] Garlic test sample: purchased from different origins, see Table 2; freeze-dried powder test sample: garlic from different origins was purchased and freeze-dried by Xinjiang Garlic Research Institute (Co., Ltd.) to obtain freeze-dried powder test sample.

[0123] Table 2 Source of test products

[0124]

[0125] Note: G stands for garlic; P stands for garlic powder

[0126] 3. Solution Preparation

[0127] Alliin reference solution: Take an appropriate amount of alliin reference, weigh accurately, and add water to make a solution containing 1 mg per 1 ml.

[0128] Methyl alliin reference solution: Take an appropriate amount of methyl alliin reference, weigh accurately, and add water to make a solution containing 0.85 mg per 1 ml.

[0129] Isolialiin reference solution: Take an appropriate amount of isolialiin reference, weigh accurately, and add water to make a solution containing 1 mg per 1 ml.

[0130] Alliinase extract solution: Take an appropriate amount of alliinase extract, weigh it accurately, and add water to make a solution containing 5 mg of alliinase extract per 1 ml.

[0131] Allicin control solution: Accurately pipette 1 ml of alliin reference solution and alliinase extract solution respectively, react for 10 min, transfer to a 10 ml volumetric flask, dilute to the mark with 50% methanol, shake well, transfer to a centrifuge tube, centrifuge at low temperature (4°C) (12,000 rpm) for 5 min, and take the supernatant for later use.

[0132] Dimethyl thiosulfinate reference solution: Accurately pipette 1 ml of methyl alliin reference solution and alliinase extract solution respectively, prepare in the same way, and take the supernatant for later use.

[0133] Diallyl thiosulfinate reference solution: Accurately pipette 1 ml of isoalliin reference solution and alliinase extract solution respectively, prepare in the same way, and take the supernatant for later use.

[0134] Mixed reference solution of allicin, dimethyl thiosulfinate and methyl allyl (allyl methyl) thiosulfinate (AM): accurately pipette 1 ml of alliin reference solution and 1 ml of methyl alliin reference solution with 2 ml of alliinase extract solution respectively, mix and react, prepare in the same way, and take the supernatant for later use.

[0135] Mixed reference solution of allicin, diallyl thiosulfinate, and allyl propylene (propylene allyl) thiosulfinate (AP): Accurately pipette 1 ml of alliin reference solution, 1 ml of isoalliin reference solution, and 2 ml of alliinase extract solution, respectively, mix and react, prepare in the same way, and take the supernatant for later use.

[0136] Mixed reference solution of dimethyl thiosulfinate, diallyl thiosulfinate, and methyl allyl (allyl methyl) thiosulfinate (MP): Accurately pipette 1 ml of methyl alliin reference solution, 1 ml of isoalliin reference solution, and 2 ml of alliinase extract solution, respectively, mix and react, prepare in the same way, and take the supernatant for later use.

[0137] Garlic test solution: take about 50g of the product, accurately weigh, add 200ml of water, homogenize (20000 rpm) for 1 minute, transfer to a 500ml volumetric flask, let stand for 30 minutes, dilute to the mark with water, shake well; transfer to a centrifuge tube, centrifuge at low temperature (4℃) (8000 rpm) for 15 minutes, accurately transfer 5ml of supernatant and place in a 25ml volumetric flask, dilute to the mark with mobile phase (methanol: 1% formic acid = 60:40), shake well, transfer to a centrifuge tube, centrifuge at low temperature (4℃) (8000 rpm) for 5 minutes, take the supernatant for use; filter through a 0.22μm microporous filter membrane, and analyze by HPLC.

[0138] Garlic powder test solution: take about 0.2g of the product, accurately weigh, accurately add 20ml of water, ultrasonically treat (power 100W) for 10 minutes, let it stand for 30 minutes and then transfer to a centrifuge tube, centrifuge at low temperature (4℃) (8000 revolutions per minute) for 10 minutes, accurately transfer 10ml of supernatant and place it in a 25ml volumetric flask, add mobile phase (methanol: 1% formic acid = 60:40) to the scale, shake well, transfer to a centrifuge tube, centrifuge at low temperature (4℃) (8000 revolutions per minute) for 5 minutes, take the supernatant for later use; filter through a 0.22μm microporous filter membrane, and analyze by HPLC.

[0139] 4. Identification of Thiosulfinates (HPLC)

[0140] Chromatographic conditions:

[0141] Column: GL Sciences Inertsil TM (ODS-SP, 4.6×250mm, 5μm); mobile phase: methanol: 1% formic acid (60:40); flow rate: 0.8mL / min; column temperature: 25℃; detection wavelength: 254nm; injection volume: 10μL; analysis time: 30min.

[0142] Injection method:

[0143] The allicin control solution, dimethyl thiosulfinate control solution, dipropylene thiosulfinate control solution, AM control solution, AP control solution, and MP control solution were filtered through a 0.22 μm microporous filter membrane, analyzed by HPLC, and the chromatograms were recorded.

[0144] HPLC test results:

[0145] Figure 1 shows that Z1 is generated after enzymatic reaction of C1. The chromatogram of allicin control solution shows a main peak with an elution time of 7.5 min ( Figure 1a ), and at the same time, the chromatogram of the allicin control solution did not show the peak of the alliin control substance at 6.4min ( Figure 1b ), and the chromatogram of the garlic enzyme extract solution also showed no peak ( Figure 1c ), it can be shown that C1 is completely reacted to produce Z1, which is the main peak in the chromatogram with a retention time of 7.5 min and good separation.

[0146] Figure 2 shows that: C2 generates Z2 after enzymatic reaction, and the chromatogram of the dimethylthiosulfinate reference solution shows a main peak with an elution time of 4.8 min ( Figure 2a ), and no peak of methyl alliin appeared at 5.4min ( Figure 2b ), it can be shown that C2 is completely reacted to produce Z2, which is the main peak in the chromatogram with a retention time of 4.8 min and good separation under the chromatographic conditions.

[0147] Figure 3 shows that: C3 is converted into Z3 after enzymatic reaction. The chromatogram of the diallylthiosulfinate reference solution shows three main peaks, one of which is a split peak. The elution times are 8.3, 9.4, 9.6, and 10.2 min, respectively, which should be a derivative of Z3 ( Figure 3a ), and at the same time, no peak of isoalliin appeared at 7.0min ( Figure 3b ), indicating that the reaction of C3 was complete, but since the product Z3 was unstable, it might have been converted into other substances, so the chromatographic peak appeared as multiple mixture peaks.

[0148] Figure 4 shows that when C1 and C2 are mixed substrates, Z1, Z2, Z4, and Z5 are generated through enzymatic reaction. The chromatogram results show that there are three main peaks, and the peak times are 4.8, 5.7, and 7.5 min, respectively. Figure 4a ), while the chromatogram of the C1 and C2 mixed solution without enzyme reaction only showed 5.4 and 6.4min ( Figure 4b ), indicating that in the absence of alliinase, C1 and C2 will not react.

[0149] This shows that 4.8min is Z2, 7.5min is Z1, and 5.7min is Z4 / Z5. Z4 / Z5 are isomers and cannot be separated, but under this chromatographic condition, the separation degree of this pair of isomer peaks is good.

[0150] Figure 5 shows that when C1 and C3 are mixed substrates, Z1, Z3, Z8, and Z9 are generated by enzymatic reaction. The chromatogram results show that there are 7 peaks at 7.5, 8.3, 8.7, 9.4, 9.6, 10.2, and 12.2 min ( Figure 5a), while the chromatogram of the C1 and C3 mixed solution without enzyme reaction only showed 7.0 and 6.4 min ( Figure 5b ), indicating that in the absence of alliinase, C1 and C3 will not react.

[0151] It can be explained that 7.5min in the figure is Z1, 8.3, 9.4, 9.6, 10.2min are derivatives of Z3, and 8.7min is inferred to be Z8 / Z9, and 12.2min may be a derivative of Z8 / Z9. Z8 / Z9 are isomers and cannot be separated, but under this chromatographic condition, the separation of this pair of isomer peaks is good, and only a small part is derived into other substances.

[0152] Figure 6 It shows that when C2 and C3 are mixed substrates, Z2, Z3, Z6 and Z7 are generated through enzymatic reaction. The chromatogram results show multiple peaks, among which 4.8min is Z2, 8.3, 9.4, 9.6 and 10.2min are Z3 derivatives, and 5.9, 6.1 and 6.3min may be Z6 / Z7 derivatives. The separation degree is not high.

[0153] In summary: under the chromatographic conditions, the separation of Z1, Z2, Z4 / Z5, and Z8 / Z9 was good.

[0154] 5. Identification of Thiosulfinates (UPLC-MS / MS)

[0155] Chromatographic conditions:

[0156] Chromatographic column: Water ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm); mobile phase: 0.1% formic acid-methanol, gradient elution according to Table 3; column temperature: 30°C; flow rate: 0.25 mL / min; injection volume: 5 μL; analysis time: 8 min.

[0157] Table 3 Gradient elution program

[0158]

[0159] Mass spectrometry conditions:

[0160] Mass spectrometry ionization mode: electrospray ionization (ESI); ion polarity: positive ion; capillary voltage: 3KV; cone voltage: 60V; source temperature: 150°C; desolvation temperature: 350°C; argon flow rate: 50L / Hr; nitrogen flow rate: 500L / Hr.

[0161] Injection method:

[0162] The Z1 control solution, Z2 control solution, AM control solution, and AP control solution were diluted with 50% methanol to a concentration of 0.1 mg / ml, respectively, centrifuged at low temperature (4°C) (14,000 rpm) for 20 min, and the supernatant was collected for UPLC-MS / MS analysis.

[0163] The garlic powder test solution and the garlic test solution were diluted 100 times with 50% methanol, centrifuged (14,000 rpm) at low temperature (4°C) for 20 min, and the supernatant was taken for UPLC-MS / MS analysis.

[0164] UPLC-MS / MS test results:

[0165] Table 4 Compound information

[0166]

[0167] like Figure 7a , 7b As shown in Table 4, the total ion current of the garlic powder test solution ( Figure 7a G) shows that under this chromatographic condition, three peaks appeared with retention times of 1.97 min, 2.17 min, and 2.24 min, among which the peak at 2.17 min had a stronger abundance, which was Z1 in the solution, the peak at 1.97 min was Z4 / Z5 in the solution, and the peak at 2.24 min was Z8 / Z9 in the solution.

[0168] The extracted ion chromatogram shows that when the parent ion is m / z 163 and the daughter ion is m / z 73 ( Figure 7a A) A single peak appeared with a retention time of 2.17 min (the compounds characterized were Z1, Z8 / Z9).

[0169] When the parent ion is m / z 111 and the daughter ion is m / z 63 ( Figure 7a B) A single peak appeared with a retention time of 1.67 min (the characterized compound was Z2).

[0170] When the parent ion is m / z 137 and the daughter ion is m / z 73 ( Figure 7a C) Two peaks appeared with retention times of 1.97 min and 2.05 min (the characterized compound was Z5).

[0171] When the parent ion is m / z 137 and the daughter ion is m / z 89 ( Figure 7a D) Two peaks appeared with retention times of 1.97 min and 2.04 min (the characterized compound was Z4).

[0172] Z4, Z5, Z6 and Z7 have the same molecular weight, and all peak at the parent ion m / z 137. Z4 / Z5 has a high content, so the abundance is relatively high, and the retention time is 1.97 min, so it is estimated that 2.05 min / 2.04 min is Z6 / Z7.

[0173] When the parent ion is m / z 163 and the daughter ion is m / z 87 ( Figure 7a E) also showed two peaks with retention times of 2.17 min and 2.24 min (the characterized compounds were Z1 and Z9).

[0174] When the parent ion is m / z 163 and the daughter ion is m / z 121 ( Figure 7a F) also showed two peaks with retention times of 2.17 min and 2.24 min (the characterized compounds were Z1 and Z8).

[0175] The results showed that the total ion current of the garlic powder sample showed three peaks at 1.97, 2.17 and 2.24 min. The extracted ion chromatogram showed that the characteristic sub-ions of Z1 (2.17 min), Z2 (1.67 min), Z4 / Z5 (1.97 min), Z6 / Z7 (2.05 min) and Z8 / Z9 (2.24 min) appeared in the garlic powder. Among them, Z1 at 2.17 min had the highest abundance and content, which was consistent with the chromatographic results.

[0176] In addition, the total ion current and extracted ion chromatogram of the garlic test solution ( Figure 7b ) showed that the components in the garlic test solution were consistent with the test results in the garlic powder test solution.

[0177] Example 2. Establishment of HPLC characteristic spectrum of thiosulfinate

[0178] Chromatographic method: as described in Example 1.

[0179] Sampling method: Garlic test solution and garlic powder test solution were filtered through a 0.22 μm microporous filter membrane, analyzed by HPLC, and the chromatograms were recorded.

[0180] Results: Figure 8 As shown in the figure, the HPLC characteristic spectrum of thiosulfinates in garlic and garlic powder was established. Taking allicin as the reference peak (7.5min), its relative retention time was set as 1.00, then the relative retention time of Z2 was 0.65; the relative retention time of Z4 / Z5 was 0.77; and the relative retention time of Z8 / Z9 was 1.17.

[0181] Example 3. HPLC analysis of thiosulfinates in different batches of garlic and garlic powder

[0182] like Fig. 9 and Fig.10 As shown, all HPLC chromatograms of garlic and garlic powder contain dimethyl thiosulfinate (peak 1), methyl allyl (allylmethyl) thiosulfinate (peak 2), allicin (peak 3), and allyl propenyl (propenylallyl) thiosulfinate (peak 4).

[0183] The results showed that this chromatographic method can effectively detect dimethyl thiosulfinate (peak 1), methyl allyl (allyl methyl) thiosulfinate (peak 2), allicin (peak 3), and allyl propenyl (propenyl allyl) thiosulfinate (peak 4) in each batch of garlic and garlic powder.

[0184] Example 4. Methodological validation of thiosulfinate HPLC analysis method

[0185] (1) Linearity and range

[0186] Accurately measure 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 ml of allicin control solution in a 10 ml volumetric flask, accurately add 0.5 ml of internal standard solution, dilute to scale with mobile phase, shake well, and obtain allicin series standard solutions with concentrations of 15.28, 30.55, 61.11, 91.66, 122.22, 152.77, 183.32 μg / ml, respectively, and determine according to the chromatographic conditions described in Example 1. Linear regression is performed with the ratio of the peak area of ​​allicin to the peak area of ​​the internal standard butyl hydroxybenzoate solution Y as the ordinate and the mass concentration C of the allicin series standard solution as the abscissa.

[0187] Dimethyl thiosulfinate series standard solutions with concentrations of 0.17, 0.34, 1.70, 3.41, 8.52, 17.03, 25.55, 34.06, 42.58, 51.09, and 68.12 μg / ml were prepared, respectively, and the results were determined according to the chromatographic conditions described in Example 1. Linear regression was performed with the ratio of the peak area of ​​dimethyl thiosulfinate to the peak area of ​​butylparaben solution Y as the ordinate and the mass concentration C of the dimethyl thiosulfinate series standard solutions as the abscissa.

[0188] Table 5 Regression equation, correlation coefficient, linear range and limit of quantification (n=7)

[0189]

[0190] (2) Precision

[0191] The garlic powder test sample (P1) solution was taken and HPLC analysis was performed according to the chromatographic conditions in Example 1. The sample was injected 6 times continuously and the chromatogram and peak area were recorded. The results showed that the relative retention time RSD of each thiosulfinate was 0.01% to 0.02% (n=6), indicating that the precision of the instrument was good.

[0192] Table 6 Precision test relative retention time results (n = 6)

[0193]

[0194] (3) Repeatability

[0195] Take garlic powder test sample (P1) solution, prepare 6 test sample solutions in parallel according to the method, perform HPLC analysis according to the chromatographic conditions in Example 1, and record the chromatogram and peak area. Results The relative retention time RSD of each thiosulfinate was 0.004% to 0.022% (n=6), indicating that this method has good repeatability.

[0196] Table 7 Precision test relative retention time results (n = 6)

[0197]

[0198]

[0199] (4) Stability test

[0200] The garlic powder test sample (P1) solution was taken and HPLC analysis was performed according to the chromatographic conditions in Example 1. The solution was placed at room temperature and 0°C-4°C for 0, 2, 4, 8, 12, and 24 hours, respectively, and the chromatogram and peak area were recorded. The results showed that the relative retention time RSDs of the thiosulfinates were 0.02%-0.08% and 0.02%-0.06%, respectively, indicating that the stability of the test solution within 24 hours was good.

[0201] Table 8 Relative retention time results at room temperature (n=3)

[0202]

[0203] Table 9 Relative retention time results at 0-4°C (n=3)

[0204]

[0205]

[0206] (5) Durability

[0207] Take the garlic powder test sample (P1) solution, use the mobile phase ratio methanol: 1% formic acid = 65:35, 60:40, 55:45 as the mobile phase, column temperature 20 ℃, 25 ℃, 30 ℃, flow rate 0.6ml / min, 0.8ml / min, 1ml / min, perform HPLC analysis according to the chromatographic conditions in Example 1 and record the chromatogram. The results show that the relative retention time content RSD of thiosulfinate under different chromatographic conditions meets the requirements, indicating that this method has good durability.

[0208] Table 10 Relative retention time results of different fluidity ratios (n=3)

[0209]

[0210] Table 11 Relative retention time results at different column temperatures (n=3)

[0211]

[0212]

[0213] Table 12 Relative retention time results at different flow rates (n=3)

[0214]

[0215] (6) Sample recovery rate

[0216] Take garlic powder test sample (P1), prepare 9 test sample solutions in parallel, add 0.6, 0.8, 1.0 ml of allicin control solution into three groups, which is equivalent to 80%, 100%, 120% of allicin in the test sample, analyze according to the chromatographic condition 1 in Example 1 and record the chromatogram. Calculate the recovery rate and RSD. The results show that the recovery rate of allicin added is 100.7% to 105.1%, and the RSD value is 0.58% to 1.6%. It shows that the recovery rate of this method is good; the recovery rates of dimethyl thiosulfinate are 103.0%, 103.3%, and 103.0%, respectively, and the RSD is less than 2%, indicating that the recovery rate of the method is good.

[0217] 6.1 Determination of recovery rate of allicin

[0218] Accurately weigh 12 portions of the test sample, 0.2 g each, and divide them into 4 groups. One group is used as the background, and the other three groups are added with appropriate amounts of allicin control solution (peak 3) of known concentration, sampled and measured, and the recovery rate is calculated.

[0219] Table 13 Allicin sample recovery test (n=3)

[0220]

[0221] 6.2 Determination of recovery rate of dimethyl thiosulfinate

[0222] Accurately weigh 12 portions of the test sample, 0.2 g each, and divide them into 4 groups. One group is used as the background, and the other three groups are added with appropriate amounts of dimethyl thiosulfinate reference solution (peak 1) of known concentration, injected and measured, and the recovery rate is calculated.

[0223] Table 14 dimethyl thiosulfinate sample addition recovery test

[0224]

[0225] Example 5. HPLC analysis of alliinase substrate

[0226] 1. Experimental instruments

[0227] Chromatograph: Shimadzu LC2030C 3D chromatography system, including quaternary gradient infusion pump, autosampler, column oven, diode array UV detector, LC solution chromatography workstation;

[0228] Electronic analytical balance: METTLER TOLEDO AB135-S (1 / 100,000), ME 204E (1 / 10,000);

[0229] Ultrasonic instrument: KQ-200KDE Kunshan Ultrasonic Instrument Co., Ltd.

[0230] Centrifuge: TGL-20bR Shanghai Anting Scientific Instrument Factory.

[0231] 2. Test Reagents

[0232] Methanol, formic acid, phosphoric acid, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate were of chromatographic grade, and water was ultrapure water; other reagents were of analytical grade.

[0233] Alliin standard sample: purchased from Xinjiang Elexin Pharmaceutical Co., Ltd., standard sample number: GSB 11-3079-F01-2024, purity: 99.0%.

[0234] The sources of garlic and garlic powder samples are as follows:

[0235] Table 15 Garlic batch information

[0236]

[0237]

[0238] Table 16 Garlic powder batch information

[0239]

[0240] Note: G stands for garlic; P stands for garlic powder

[0241] 3. Solution Preparation

[0242] Accurately weigh appropriate amounts of alliin, methyl alliin, isoalliin and γ-glutamine-S-allyl-L-cysteine ​​(GSAC) reference substances, dissolve in 50% methanol and dilute to scale to prepare mixed reference substance solutions 1# with mass concentrations of 0.416, 0.124, 0.0829 and 0.117 mg / ml, respectively. Take the 1# mixed reference substance solution and dilute it stepwise for 6 times, each time by 2 times, to obtain a series of mixed reference substance solutions 2# to 7#.

[0243] Garlic test solution: Take about 50g of fresh garlic, weigh accurately, place in a 500ml beaker, add 200ml of water, boil in water to inactivate enzyme for 10min, homogenize for 1min, transfer the slurry to a 500ml volumetric flask, dilute to scale with water, centrifuge (8000 rpm) for 15min, take 2.0ml of supernatant, place in a 10ml volumetric flask, dilute to 10ml with 100mM potassium dihydrogen phosphate (pH 4.0), filter with a 0.22μm filter membrane, and store until tested (<10℃).

[0244] Garlic powder test solution: Take about 0.5g of garlic powder, weigh accurately, place in a 25ml volumetric flask, add methanol-water-formic acid (50:50:1, V / V / V) to dissolve and make up to volume. Ultrasonic extraction for 20min, centrifuge (8000 rpm) for 5min. Accurately pipette 2mL and dilute to 10ml with 100mM potassium dihydrogen phosphate (pH4.0). Filter with a 0.22μm filter membrane and store for testing (<10℃).

[0245] 4.HPLC analysis

[0246] Chromatographic conditions:

[0247] Chromatographic column: CAPCELL PAKSCX (4.6×250mm, 5μm); mobile phase: 10mM potassium dihydrogen phosphate (pH 2.5); flow rate: 1.0ml / min; detection wavelength: 210nm; column temperature: 45℃; injection volume: 10μl; analysis time: 60min. The theoretical plate number calculated based on the alliin peak should not be less than 3000.

[0248] Injection method: Accurately pipette 10 μl of the control solution and the test solution respectively and inject them into the liquid chromatograph.

[0249] Calculation formula:

[0250]

[0251] Where:

[0252] AX——peak area of ​​each amino acid in the test sample;

[0253] CR——Concentration of each amino acid in the reference substance, mg / ml;

[0254] AR——peak area of ​​each amino acid in the reference substance;

[0255] D——dilution multiple;

[0256] m——the weight of the test sample, g.

[0257] result:

[0258] Fig.11 It shows that under this chromatographic condition, the four reference solutions: alliin, methyl alliin, isoalliin and GSAC have a good separation effect, among which the retention time of alliin is 5.94min; the retention time of methyl alliin is 5.14min; the retention time of isoalliin is 6.54min; and the retention time of GSAC is 11.62min.

[0259] Fig.12 It shows that under this chromatographic condition, alliin, methyl alliin, isoalliin and GSAC in fresh garlic sample have good separation effect.

[0260] Fig.13 It shows that under this chromatographic condition, alliin, methyl alliin, isoalliin and GSAC in the garlic powder sample have a good separation effect.

[0261] Fig.14 It shows that under this chromatographic condition, alliin, methyl alliin, isoalliin and GSAC in 16 batches of garlic powder have good separation effect, which shows that the chromatographic condition is very stable.

[0262] 5. Methodological Validation

[0263] (1) Linearity and range

[0264] The series of concentration mixed reference solution 2#~7# in Example 5 were respectively taken, and the determination was carried out according to the method described in Example 5, with the peak area of ​​the reference substance as the ordinate (y), and the concentration of the reference substance (mg / ml) as the abscissa (x). The least squares method was used for linear regression analysis to obtain the regression equation, correlation coefficient and linear range of each reference substance, and the corresponding concentration of each component when the signal-to-noise ratio (S / N) was 3 was used as the detection limit, and the corresponding concentration when the signal-to-noise ratio (S / N) was 10 was used as the quantification limit.

[0265] Table 17 Regression equation, correlation coefficient, linear range and quantification limit (n = 7)

[0266]

[0267] (2) Repeatability

[0268] According to the method described in Example 5, the same batch of garlic powder test solutions (n=6) were prepared and liquid chromatography was performed. The chromatographic peak area of ​​each amino acid was recorded, and the RSDs of the alliin, methyl alliin, isoalliin and GSAC contents were calculated to be 0.23%, 1.8%, 1.6% and 1.4%, respectively, indicating that the method had good repeatability.

[0269] Table 18 Repeatability test results of the test solution

[0270]

[0271]

[0272] (4) Intermediate precision

[0273] According to the method described in Example 5, different personnel prepared 6 samples of the same batch of garlic powder on different dates, and performed liquid chromatography determination. The peak area of ​​each amino acid was recorded, and the RSDs of the alliin, methyl alliin, isoalliin and GSAC contents were calculated to be 0.41%, 0.86%, 0.68% and 0.66%, respectively, indicating that this method has good precision.

[0274] Table 19 Intermediate precision

[0275]

[0276] (5) Accuracy (sample recovery rate)

[0277] Take 4 kinds of garlic powder test samples with known amino acid contents, prepare 9 portions according to the method described in Example 5, add each reference solution according to the ratio of each amino acid content in the test sample to the reference content (0.8:1, 1:1, 1.2:1), and perform liquid chromatography determination. The average sample recovery rates and RSDs of alliin, methyl alliin, isoalliin, and GSAC are calculated to meet the recovery limit requirements.

[0278] Table 20: Recovery of amino acids in garlic powder (n=3)

[0279]

[0280]

[0281] (6) Durability

[0282] Different flow rates:

[0283] Take 2# reference solution and inject it at different flow rates (0.9, 1.0, 1.1 ml / min) according to the method described in Example 5 to detect the retention time of each substance. The results showed that the relative retention time RSD% was 0.03-1.90%, indicating that this method has good durability for different flow rates.

[0284] Table 21 Relative retention time of each substance at different flow rates (n=3)

[0285]

[0286] Different column temperatures:

[0287] Take 2# reference solution, inject at different column temperatures (40, 45, 50°C) according to the method described in Example 5, and detect the retention time of each substance. The results show that the relative retention time RSD% is 0.74-1.92%, indicating that the method has good durability at different column temperatures.

[0288] Table 22 Relative retention time of each substance at different column temperatures (n=3)

[0289]

[0290] Different instruments:

[0291] The retention time of each substance was detected by injecting samples on three chromatographs: Shimadzu LC 2030C plus, Shimadzu LC2050C, and Agilent LC 1220 using CAPCELL PAKSCX chromatographic column. The results showed that the relative retention time RSD% was 0.04-1.91%, indicating that the method has good durability on different instruments.

[0292] Table 23 Relative retention time of each substance under different instruments (n=3)

[0293]

[0294] Different pH values:

[0295] Take 2# reference solution and inject it under different pH mobile phases (2.3, 2.5, 2.7) according to the method described in Example 5. The results show that the RSD% of the relative retention time is 0.27-1.48%, indicating that this method has good durability for mobile phases with different pH values.

[0296] Table 24 Content of each substance at different pH (n=3)

[0297]

[0298] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing an instant reference substance for chromatographic analysis of sulfur-containing compounds in garlic, characterized in that: The preparation method is shown in Formula 1: Formula 1: Alliinase substrate reference substance + alliinase alliinase enzymatic hydrolysis product reference substance, wherein the alliinase substrate includes: C1: Alliin C2: Methylalliin C3: Isolialiin Alliinase hydrolysis products include: Z1: diallyl thiosulfinate (garlicin); Z2: dimethyl thiosulfinate; Z3: diallyl thiosulfinate; Z4: methyl allyl thiosulfinate; Z5: allyl methyl thiosulfinate; Z6: methyl methacrylate thiosulfinate; Z7: allyl methyl thiosulfinate; Z8: allyl allyl thiosulfinate; Z9: Propylene allyl thiosulfinate.

2. The method according to claim 1, characterized in that The preparation method further comprises one or more steps selected from the group consisting of: (A) C1 + alliinase → Z1 (B) C2 + alliinase → Z2 (C) C3 + alliinase → Z3 (D) C1+C2+alliinase→Z1, Z2, Z4, Z5 (E) C2+C3+alliinase→Z2, Z3, Z6, Z7 (F) C1+C3+alliinase→Z1, Z3, Z8, Z9 (G) C1+C2+C3+alliinase→Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9.

3. The method according to claim 1, characterized in that The reaction time of the alliinase substrate and the alliinase to generate the enzymolysis product is 10-15 minutes.

4. The method according to claim 1, characterized in that The instant reference solution is stable for 4-6 hours.

5. A chromatographic analysis method for sulfur compounds in garlic, characterized in that: Includes steps: (a) providing a sample solution; (b) preparing an instant reference solution using the method as claimed in claim 1; (c) The sample solution and the instant reference solution are subjected to liquid chromatography analysis.

6. The method according to claim 5, characterized in that Using the chromatographic analysis method, a characteristic spectrum of garlic sulfur compounds is obtained, in which: If Z1 is taken as the reference peak and its relative retention time is 1, then the relative retention time of Z2 is 0.65, the relative retention time of Z4 / Z5 is 0.77, and the relative retention time of Z8 / Z9 is 1.

17.

7. The method according to claim 5, characterized in that The mobile phase of the method is a mixed solution of methanol and formic acid.

8. The method according to claim 5, characterized in that In the mobile phase, the ratio of methanol to 1% formic acid is 1:1-2:

1.

9. The method according to claim 5, characterized in that The method further comprises: a liquid chromatography analysis method II for analyzing alliin, methyl alliin, isoalliin and γ-glutamine-S-allyl-L-cysteine ​​(GSAC), wherein the method II comprises the steps of: (1) preparing sample solution and reference solution; (2) subjecting the sample solution and the reference solution to liquid chromatography analysis; Wherein, the chromatographic column used in step (2) is a strong cationic chromatographic column bonded with sulfonic acid groups.

10. Method II according to claim 9, characterized in that Using the method II, characteristic spectra of alliin, methyl alliin, isoalliin and GSAC were obtained, in which: If alliin is taken as the reference peak and its relative retention time is 1, then the relative retention time of methyl alliin is 0.86, the relative retention time of isoalliin is 1.10, and the relative retention time of GSAC is 1.96.

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