Liquid chromatography-tandem mass spectrometry detection method of garlic flavor precursor

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method of a garlic flavor precursor, and belongs to the technical field of garlic flavor precursor detection.The method comprises the following steps that a sample is treated with microwaves and crushed, and then extraction is conducted with dipping and ultrasonic methods; liquid chromatography is adopted for separation, and multi-reaction monitoring mode mass spectrometry is adopted for detection. According to the method, the advantages of microwave enzyme deactivation and liquid chromatography-tandem mass spectrometry are combined, synchronous detection of five flavor precursors including alliin, isoalliin, methyl alliin, propyl alliin and S-allyl-L-cysteine is achieved, sample treatment is simple, the using amount of organic reagents is small, accuracy is high, reproducibility is good, and the method is suitable for industrial production. And an effective technical means is provided for quality evaluation of the garlic.

Description

Technical Field

[0001] The invention belongs to the technical field of garlic flavor precursor detection, and particularly relates to a liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors. Background Art

[0002] Garlic (Allium sativum L.) belongs to the genus Allium in the Liliaceae family. It is a vegetable with bulbs (garlic heads), flower stems (garlic scapes) and young leaves as the main edible organs. Originated from the Western Regions, Zhang Qian brought garlic back to China. Since then, garlic has become an indispensable part of people's lives, and people began to cultivate garlic. To date, my country has become a major producer and consumer of garlic, and occupies an important position in the world in terms of planting area, production volume, and export volume. my country's climate and soil are diverse, and the unique water and soil have cultivated a variety of garlic varieties, with different quality characteristics and functional components. At present, there is a lack of systematic analysis and evaluation of garlic functional components.

[0003] Garlic occupies an important position in the seasoning industry. Depending on the conditions of use, it has a variety of applicable characteristics. It can be used in meat dishes to cover the fishy smell of meat products, and play a role in balancing the taste and seasoning. It plays a role in sterilization and flavor enhancement in cold dishes. It can also be made into pickled garlic products for daily side dishes, such as sugar garlic and vinegar garlic. Sulfur-containing compounds, as the main flavor substances and bioactive substances of garlic, give garlic a unique flavor and unique efficacy. Since fresh garlic has a strong pungent smell and a distinct garlic odor, the development of garlic products is limited. In terms of primary processing and utilization, the main consumption forms of garlic in my country's market are fresh garlic, dried garlic, pickled garlic, garlic slices, garlic powder, garlic cloves, garlic paste, garlic juice, etc. Enterprises combine food processing and production technology to produce ready-to-eat foods such as garlic sausage and garlic bread. As people's ideological awareness and consumption level of pursuing food quality and health continue to strengthen, the consumer group of high-end garlic by-products and processed products continues to increase. However, due to the differences in the content of garlic flavor precursors and the influence of processing conditions, the flavor substances and flavor characteristics produced by degradation are different, which often causes instability in the quality and flavor of related products, bringing difficulties to food companies in product quality control. Therefore, establishing an efficient detection method for garlic flavor precursors is of great significance for the quality evaluation and selection of garlic products and the quality control of garlic-flavored foods.

[0004] Unbroken garlic does not have a spicy flavor because the flavor precursors in garlic are non-volatile S-alkyl (en)ylcysteine ​​S-oxides (ACSOs), which mainly include: S-methylcysteine ​​sulfoxide (commonly known as methyl alliin, Methiin), S-propylcysteine ​​sulfoxide (commonly known as propyl alliin, Propiin), S-1-propenylcysteine ​​sulfoxide (commonly known as isoalliin) and S-allylcysteine ​​sulfoxide (commonly known as alliin, Alliin), among which alliin is the most important flavor precursor known to date. When garlic is crushed, the alliinase in the vacuole contacts the alliin in the cell matrix, and volatile thiosulfinate products are instantly produced, among which diallyl thiosulfinate (i.e., allicin) has the highest content, usually accounting for 70% to 80% of all thiosulfinates. The unique pungent and spicy flavor components of garlic are mainly composed of a series of fat-soluble organic sulfides, and the composition and content of these substances are important quality indicators in garlic products. Important flavor precursors in garlic also contain water-soluble sulfur compounds, the main component of which is S-allyl-L-cysteine ​​(S-allyl-L-cysteine, SAC), which is produced by the decomposition of γ-glutamyl-S-allyl-L-cysteine ​​under the action of γ-glutamyl transpeptidase (γ-glutamylendo peptidase). The physical and chemical properties of S-allyl-L-cysteine ​​are: colorless and odorless crystals, which can be stably dissolved in neutral or slightly acidic solids and aqueous solutions.

[0005] In recent years, the detection technology of garlic flavor precursors is divided into two categories: indirect determination and direct determination. The indirect determination method is to indirectly obtain the content of flavor precursor compounds by detecting the downstream metabolites of alliin. Among the indirect determination methods, the pyruvate method is the most widely used. The indirect determination method is relatively simple, but it cannot obtain the specific composition of flavor precursor compounds. Moreover, some flavor precursor compounds will not be converted into allicin under the action of alliinase, so the determination of pyruvate cannot obtain complete flavor precursor compound information. In addition, other commonly used indirect determination methods, such as sulfur determination method, thin layer scanning method, phenylhydrazone method, and ninhydrin method, generally have the problems of poor specificity and inaccuracy. The direct determination method directly determines the garlic flavor precursors, which can better evaluate the quality of garlic; but the chemical enzyme inactivation method has a more complicated operation process, and because the enzymatic hydrolysis reaction of alliin occurs instantly after the substrate contacts the enzyme, the enzyme inactivation effect is not easy to control, resulting in poor repeatability of the test results; on the other hand, garlic flavor precursors have similar structures, and chromatographic separation is more difficult. Summary of the invention

[0006] In view of the above-mentioned prior art and existing problems, the present invention provides a simple, practical, accurate and reliable liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors, which can meet the needs of simultaneous determination of major S-alkyl (enyl)-cysteine ​​sulfoxides and S-allyl-L-cysteine ​​in garlic, and provide an effective technical means for the quality evaluation of garlic.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors, and to simultaneously detect alliin, isoalliin, methyl alliin, propyl alliin and S-allyl-L-cysteine ​​by liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0008] (1) The sample is treated and crushed by microwave, and then extracted by immersion and ultrasonic method to obtain a sample for machine testing;

[0009] (2) On-machine detection: Chromatographic conditions: chromatographic column is C18-AQ; mobile phase is formic acid solution with a volume concentration of 0.1%, flow rate is 0.4 mL / min, elution time is 35 min; injection volume is 10 μL; mass spectrometry conditions: ESI source operates in positive ionization mode, capillary voltage is 4000 V; desolvation nitrogen temperature is 300°C, flow rate is 6 L / min; nebulizer gas pressure is 15 psi; multiple reaction monitoring mode.

[0010] Furthermore, the parameters of the analytes in the multiple reaction monitoring mode scan are as follows:

[0011] Alliin: retention time is 12.42min, precursor ion is 178.6m / z, fragment ion 41m / z is the qualitative ion, collision energy is 20eV; fragment ion 88m / z is the quantitative ion, collision energy is 5eV;

[0012] Isolic acid: retention time is 14.50min, precursor ion is 178.6m / z, fragment ion 41m / z is the qualitative ion, collision energy is 20eV; fragment ion 88m / z is the quantitative ion, collision energy is 5eV;

[0013] Methyl alliin: retention time is 7.43min, precursor ion is 152m / z, fragment ion 88m / z is the qualitative ion, collision energy is 15eV; fragment ion 152m / z is the quantitative ion, collision energy is 0eV;

[0014] Propyl alliin: retention time is 16.76min, precursor ion is 180.7m / z, fragment ion 43m / z is the qualitative ion, collision energy is 15eV; fragment ion 88m / z is the quantitative ion, collision energy is 10eV;

[0015] S-allyl-L-cysteine: retention time is 28.65min, precursor ion is 163.6m / z, fragment ion 73m / z is the qualitative ion, collision energy is 15eV; fragment ion 145m / z is the quantitative ion, collision energy is 5eV.

[0016] Furthermore, the power of microwave treatment was 900 W and the time was 1 min; the power of ultrasound treatment was 500 W and the time was 15 min.

[0017] Furthermore, the solvent used in the immersion and ultrasonic methods is phosphoric acid with a volume concentration of 0.01%.

[0018] Furthermore, the internal standard method was adopted, and the internal standard was L-norleucine, and its concentration was 200 ng / mL.

[0019] Furthermore, the parameters of L-norleucine in the multiple reaction monitoring mode were as follows: retention time was 26.67 min, precursor ion was 132.2 m / z, fragment ion 132.2 m / z was the qualitative ion, and collision energy was 0 eV; fragment ion 86.2 m / z was the quantitative ion, and collision energy was 10 eV.

[0020] Furthermore, the specifications of the chromatographic column C18-AQ are 5 μm 100A250 mm×4.6 mm.

[0021] Furthermore, the sample is garlic, a garlic product or onion.

[0022] Furthermore, the sample is fresh garlic, dried garlic, pickled garlic or onion.

[0023] The beneficial effects of the present invention are as follows: the present invention starts from two aspects of problems, uses microwave enzyme inactivation before garlic is crushed, effectively prevents the occurrence of enzymatic hydrolysis reaction, ensures that flavor precursors are not destroyed, and optimizes the chromatographic separation method and combines the high selectivity of tandem mass spectrometry, selects a suitable chromatographic column and mobile phase, optimizes the elution mode, optimizes the mass spectrometry parameters, and gives full play to the advantages of microwave enzyme inactivation method with good repeatability and high efficiency and liquid chromatography tandem mass spectrometry with high sensitivity and high selectivity, and finally establishes a simple and practical new method for simultaneously detecting five substances, namely alliin, isoalliin, methyl alliin, propyl alliin and S-allyl-L-cysteine ​​in garlic. The method has simple sample processing, small amount of organic reagent, high accuracy and good reproducibility, and the methodological evaluation results all meet the requirements of national standards, can meet the needs of synchronous determination of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in garlic, and provides effective technical means for quality evaluation of garlic. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a representative MRM chromatogram of the main S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine; wherein A is alliin, B is isoalliin, C is propyl alliin, D is L-norleucine (internal standard), E is S-allyl-L-cysteine, and F is an unknown interfering peak;

[0025] Figure 2 The separation effect of garlic flavor precursors under different liquid chromatography conditions; A is alliin, B is isoalliin, C is propyl alliin, D is L-norleucine (internal standard), E is S-allyl-L-cysteine, and F is an unknown interference peak;

[0026] Wherein, a is a Waters XSELECTM HSS T3 (3.5 μm 2.1×150 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-4 min (5% B), 4-5 min (5-80% B), 5-12 min (80% B);

[0027] b is a Waters XSELECTM HSS T3 (3.5 μm 2.1×150 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-4 min (0% B), 4-5 min (0-80% B), 5-12 min (80% B);

[0028] c is a Caprisil C18-AQ (5 μm 100A250 mm×4.6 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-15 min (0% B), 15-18 min (50%-90% B), 18-26 min (90% B);

[0029] d is a Caprisil C18-AQ (5 μm 100A250 mm×4.6 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-18 min (0% B), 18-21 min (0-80% B), 21-26 min (80% B);

[0030] e is a Caprisil C18-AQ (5 μm 100A250 mm×4.6 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-18 min (0% B), 18-21 min (0-90% B), 21-26 min (90% B);

[0031] f is a Caprisil C18-AQ (5 μm 100A250 mm×4.6 mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, B is pure acetonitrile, 0-18 min (0% B), 18-26 min (0-90% B);

[0032] g is a Caprisil C18-AQ (5μm 100A250mm×4.6mm) chromatographic column, the mobile phase is 0.1% (v / v) formic acid solution, and the elution time is isocratic elution for 35min. DETAILED DESCRIPTION

[0033] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0034] Materials and reagents: 50 mL plastic centrifuge tube (BIOLGIX, China); 0.22 μm organic nylon filter membrane (Beijing Compuxing Technology Co., Ltd.); garlic, onion, and pickled garlic (online shopping platforms and markets); formic acid and phosphoric acid (Beijing Chemical Plant); acetonitrile and methanol (chromatographic grade); alliin standard, isoalliin standard, propyl alliin standard, S-allyl-L-cysteine ​​standard, and L-norleucine standard (Shanghai Anpu Technology Co., Ltd.).

[0035] Instruments and equipment: Agilent Technologies 1200 Series liquid chromatography-tandem mass spectrometer, Caprisil C18-AQ chromatographic column (5μm 100A250mm×4.6mm), Waters XSELECTM HSS T3 (3.5μm 2.1×150mm) chromatographic column; Centrifuge5810R centrifuge (Eppendorf, Germany); IKAMS 3basic vortex mixer (IKA, Germany); multi-function crusher (Midea Group Co., Ltd.); variable frequency microwave oven (Midea Group Co., Ltd.); BSA 224S-CW electronic analytical balance (accuracy 0.0001, Beijing Sartorius Scientific Instrument Co., Ltd.); Milli-Q water purifier (Shanghai Murray Biotechnology Co., Ltd.).

[0036] Example 1: Preparation of standard solution

[0037] (1) Alliin standard stock solution (0.5 mg / mL): Accurately weigh 5.0 mg of alliin standard, dissolve it in 2 mL of water, and then make up to 10 mL with pure methanol. Store at -18°C away from light. The shelf life is 1 month.

[0038] (2) Isollylic acid standard stock solution (0.5 mg / mL): Accurately weigh 5.0 mg of isollylic acid standard, dissolve it in pure methanol and make up to 10 mL. Store at -18°C in the dark. The shelf life is 1 month.

[0039] (3) Propyl alliin standard stock solution (1 mg / mL): Accurately weigh 10.0 mg of propyl alliin standard, dissolve it in pure methanol and make up to 10 mL. Store at -18°C away from light. The shelf life is 1 month.

[0040] (4) S-allyl-L-cysteine ​​standard stock solution (1 mg / mL): Accurately weigh 10.0 mg of S-allyl-L-cysteine ​​standard, dissolve it in pure methanol and make up to 10 mL. Store at -18°C in the dark. The shelf life is 1 month.

[0041] (5) L-norleucine internal standard stock solution (1 mg / mL): Accurately weigh 10.0 mg of L-norleucine standard, dissolve it in pure methanol and make up to 10 mL. Store at -18°C in the dark. The shelf life is 1 month.

[0042] (6) Mixed standard intermediate solution (10 μg / mL): Accurately pipette 200.00 μL each of alliin and isoalliin standard stock solutions (0.5 mg / mL) and 100.00 μL each of propyl alliin and S-allyl-L-cysteine ​​standard stock solutions (1 mg / mL), dilute with 0.01% (v / v) phosphoric acid solution and make up to 10 mL. Store at -18°C in the dark. The shelf life is 1 month.

[0043] (7) Internal standard working solution (10 μg / mL): Accurately pipette 100 μL of L-norleucine internal standard stock solution, dilute it with 0.01% (v / v) phosphoric acid solution and make up to 10 mL.

[0044] (8) Series of standard solutions

[0045] Accurately pipette 20 μL, 50 μL, 100 μL, 200 μL, 500 μL, 1000 μL and 1250 μL of the mixed standard intermediate solution into a series of 10 mL volumetric flasks, add 200 μL of internal standard working solution to each of them, and use 0.01% (v / v) phosphoric acid solution to prepare a series of standard solutions with standard concentrations of 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL and 1250 ng / mL and an internal standard concentration of 200 ng / mL, which are used on a liquid chromatography-mass spectrometry instrument.

[0046] Example 2: Sample pretreatment

[0047] (1) Take 50 g of the edible part of the garlic sample, inactivate the enzyme in a microwave oven at 900 W for 1 min, cool to room temperature, use water to make up for the mass lost during the microwave inactivation process, then add water at a ratio of 1 g:1 mL of sample mass to water, use a crusher to make garlic mince, place in a sealed container, and store in a freezer at -18°C.

[0048] (2) Weigh 2 g of the sample treated in step (1) into a conical flask, add 0.01% (v / v) phosphoric acid solution, manually shake for 1 min, then ultrasonically extract at a power of 500 W for 15 min, let stand until it returns to room temperature, transfer to a 100 mL volumetric flask, add 2000 μL of internal standard working solution, and make up to volume with 0.01% (v / v) phosphoric acid solution. Take 10 mL of the solution in a centrifuge tube, centrifuge at 8000 r / min for 15 min, and pass the supernatant through a 0.22 μm organic nylon filter membrane for testing.

[0049] Example 3: LC-MS / MS detection

[0050] (1) Chromatographic conditions: The chromatographic column was Caprisil C18-AQ (5 μm 100A 250 mm×4.6 mm); the mobile phase was 0.1% (v / v) formic acid solution, the flow rate was 0.4 mL / min, and the elution time was 35 min; the injection volume was 10 μL.

[0051] (2) Mass spectrometry conditions: ESI source operated in positive ionization mode, capillary voltage 4000 V; desolvation nitrogen temperature 300 °C, flow rate 6 L / min; nebulizer gas pressure: 15 psi; multiple reaction monitoring mode (MRM) scanning, detailed parameters are shown in Table 1.

[0052] Table 1 MRM parameters for the contents of S-alkyl(enyl)-cysteine ​​sulfoxide, S-allyl-L-cysteine ​​and internal standard in garlic

[0053]

[0054] Note: * is the quantitative ion

[0055] (3) Representative MRM chromatograms

[0056] Representative MRM chromatograms of major flavor precursors (alliin, isoalliin, propyl alliin, and S-allyl-L-cysteine) and internal standard (L-norleucine) are shown in Figure 1 .

[0057] Example 4: Qualitative and quantitative methods

[0058] MassHunterB.07.01 software was used for qualitative and quantitative analysis. Qualitative analysis mainly depends on the retention time of liquid chromatography and mass spectrometry data. The retention time requires that the relative deviation between the retention time of the analyzed substance and the standard sample under the same experimental conditions is allowed to be within ±2.5%. For mass spectrometry data, data processing, such as spectrum accumulation, background subtraction or spectrum subtraction, is used to effectively compare the two, and the deviation of the relative ion abundance and the ion abundance of the standard substance does not exceed the specified range in Table 2, which can determine the presence of the target compound in the sample. Quantitative analysis uses L-norleucine as an internal standard, and the internal standard method is used for quantification.

[0059] Table 2 Maximum allowable deviation of relative ion abundance in qualitative analysis

[0060]

[0061] Example 5: Linearity, Limit of Detection and Limit of Quantitation

[0062] According to the possible contents of S-alkyl(enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in garlic, a series of standard solutions with a concentration range of 20 to 1250 ng / mL were analyzed and tested.2 >0.996 shows a good linear relationship. The samples (selected samples with low or undetected content of the analyte) were spiked and measured, and the LOD (Limit of Detection) and LOQ (Limit of Quantitation) were calculated according to 3 times the signal-to-noise ratio and 10 times the signal-to-noise ratio. The LOD and LOQ of this method were 0.027-5.551 μg / kg and 0.091-18.502 μg / kg, respectively. The detection limit and quantification limit of alliin measured by this method were lower than the LOD of alliin in NY / T 3871-2021 Determination of alliin in garlic-high performance liquid chromatography: 0.012 g / kg and LOQ: 0.040 g / kg. The LOD and LOQ of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in garlic are shown in Table 3.

[0063] Table 3 LOD and LOQ of S-alkyl(enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in garlic

[0064]

[0065] Note 1: x is the ratio of the mass concentration of the substance to be measured to the mass concentration of the corresponding internal standard, and y is the ratio of the peak area of ​​the quantitative ion of the substance to be measured to the peak area of ​​the quantitative ion of the corresponding internal standard.

[0066] Note 2: Methyl alliin is not easily available as a standard sample, so it is not evaluated in this paper. However, referring to analogs such as alliin, isoalliin and propyl alliin, the LOD and LOQ should be of the same order of magnitude, and the linearity should also be similar.

[0067] Example 6: Accuracy and Precision

[0068] Considering the contents of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in most garlic, the spike concentrations were determined to be 5, 20, and 40 μg / kg, and the spike recovery and RSD of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​were 94.89%-118.32% and 3.38%-14.85%, respectively, which were better than the requirements of GB 5009.295-2023 National Food Safety Standard Chemical Analysis Method Validation General Requirements. As shown in Table 4.

[0069] Table 4 Accuracy and precision of determination of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​in garlic (n=6)

[0070]

[0071] Note: As the standard sample of methyl alliin is not easy to purchase, it is not evaluated in this invention.

[0072] Example 7: Detection of actual samples

[0073] In this experiment, a total of 18 common types of garlic on the market, including fresh garlic, dried garlic, single-headed garlic, triple-clove garlic, horse-tooth garlic, Cangshan garlic, Shandong Jinxiang garlic, and shallot, as well as 1 kind of pickled garlic and 2 common types of onions on the market were collected for research. The test results are shown in Table 5.

[0074] Table 5 Detection results of S-alkyl (enyl)-cysteine ​​sulfoxide and S-allyl-L-cysteine ​​content in different varieties of garlic and its products

[0075]

[0076]

[0077] Note: Since the standard sample of methyl alliin is not easy to purchase, the qualitative detection of methyl alliin is given by referring to the ion pair information in the literature (Rektorisova M., Hrbek V., Jiru M., et al. Variability in S-Alk(en)yl-L-Cysteine ​​Sulfoxides in Garlic within a Seven-Month Period Determined by a Liquid Chromatography–Tandem Mass Spectrometry Method. Plant Foods for Human Nutrition, 2020, doi: 10.1007 / s11130-020-00817-z), and the quantitative detection is carried out with alliin as a reference.

[0078] The comparison of the total content of detected substances in different varieties of garlic and their products is shown in Table 5. Except for small horse teeth garlic and garlic heads, alliin accounts for more than 90% of the total substances in garlic. Among them, the dried garlic A purchased from the market has the highest alliin content, accounting for 99.42% of the total content of the detected substances. The content of propyl alliin in garlic heads is the highest, reaching 82.52%.

[0079] The alliin content of dried garlic is generally higher than that of fresh garlic, which is related to the different degrees of pungent odor it produces, and is highly related to the different spicy tastes and processing and eating methods it possesses. Among the garlic varieties containing alliin, the allium garlic and the small horse tooth garlic have the lowest content, at 82.88μg / g and 26.94μg / g, respectively. Their content is even 1 / 100 of the dried garlic with the highest alliin content, which is why these two types of garlic have a distinct flavor difference from other garlics.

[0080] The content of isoalliin in fresh garlic is generally higher than that in dried garlic. The content of isoalliin in the flavor precursor of onion is relatively high, and isoalliin is an important flavor precursor of onion. It will convert the substances degraded by isoalliin into tear-inducing factors that make people cry. Other allium plants do not contain this enzyme.

[0081] Compared with the other four characteristic components, the content of propyl alliin in scallion garlic is the highest, accounting for 82.52% of the total detected substances. The content of precursor substances is: propyl alliin>methyl alliin>alliin>S-allyl-L-cysteine. According to literature research, scallion does not have the garlic odor and pungent garlic odor and spicy taste of ordinary garlic. It is juicy, crisp, tender, and spicy. It is often pickled with sugar for consumption.

[0082] The content of S-allyl-L-cysteine ​​in small horsetooth garlic is higher than that in other varieties of garlic. The content of S-allyl-L-cysteine ​​in sugar garlic is the highest, which is consistent with the conclusion in the literature. In addition, the amount of flavor substance precursors in sugar garlic is very small. These flavor substance precursors may be generated by the addition of seasonings or decomposition during the processing process.

[0083] Comparative Example 1: Optimization of separation conditions

[0084] Waters XSELECTM HSS T3 (3.5 μm) was used, which has a good retention effect on polar substances.

[0085] 2.1×150mm) chromatographic column, mobile phase A is 0.1% (v / v) formic acid solution, mobile phase B is pure acetonitrile, flow rate is 0.2mL / min, gradient elution, and separation conditions are optimized. Figure 2 a It can be seen that alliin, isoalliin and propyl alliin cannot be separated when eluted with 5% organic phase from 0 to 4 minutes. After 4 minutes, as the proportion of organic phase increases, the internal standard L-norleucine and S-allyl-L-cysteine ​​flow out of the chromatographic column with an ideal retention time, but the peaks overlap. Changing the proportion of mobile phase, the results are as follows Figure 2 As shown in Figure b, when 100% mobile phase A is used within 0-4 min, the separation effect of alliin, isoalliin and propyl alliin is significantly improved, but it is still not ideal. Since alliin and isoalliin are isomers, they cannot be distinguished by mass spectrometry and must be separated in liquid chromatography. Therefore, the Waters XSELECTM HSS T3 (3.5μm 2.1×150mm) chromatographic column cannot meet the requirements.

[0086] A Caprisil C18-AQ (5 μm 100A 250 mm × 4.6 mm) column was used, mobile phase A was 0.1% (v / v) formic acid solution, mobile phase B was pure acetonitrile, the flow rate was 0.4 mL / min, the ratio of mobile phase A and B was adjusted, and the separation conditions were further optimized. The results are shown in Figure 2. Figure 2 c~f. Using 100% mobile phase A for elution within 0~18min can fully separate alliin, isoalliin and propyl alliin. After 18min, when the proportion of organic phase is increased, L-norleucine and S-allyl-L-cysteine ​​cannot be separated and the peak shape becomes worse. By changing the gradient elution to isocratic elution and using 0.1% (v / v) formic acid solution as the mobile phase, alliin, isoalliin, methyl alliin, propyl alliin and S-allyl-L-cysteine ​​are satisfactorily separated, see Figure 2 g.

[0087] Therefore, a Caprisil C18-AQ (5 μm 100A 250 mm×4.6 mm) chromatographic column was used, the mobile phase was 0.1% (v / v) formic acid solution, the flow rate was 0.4 mL / min, and the elution time was 35 min of isocratic elution.

[0088] Comparative Example 2: Comparison with the standard method

[0089] The statistical comparison of the test results of garlic samples by this method and NY / T 3871-2021 "High Performance Liquid Chromatography for the Determination of Alliin in Garlic" is shown in Table 6. At the α=0.05 level, there is no significant difference in the test results of alliin in garlic by the two methods by t-test. Compared with NY / T 3871-2021, this method can simultaneously identify and quantify the five flavor precursors in garlic, achieving a more accurate overall evaluation of the characteristic components of flavor substances in garlic.

[0090] Table 6 Comparison of this method with the standard method

[0091]

[0092] In summary, the present invention selects a suitable chromatographic column, mobile phase, optimizes and elution mode, and optimizes mass spectrometry parameters, and finally establishes a new method for simultaneously detecting four S-alkyl (enyl)-cysteine ​​sulfoxides and S-allyl-L-cysteine ​​in garlic by liquid chromatography-tandem mass spectrometry. The method has simple sample processing, small amount of organic reagents, high accuracy, good reproducibility, and methodological evaluation results meet the requirements of national standards, can meet the needs of simultaneous determination of S-alkyl (enyl)-cysteine ​​sulfoxides and S-allyl-L-cysteine ​​in garlic, and provides an effective technical means for the quality evaluation of garlic.

[0093] The application method is to test different varieties of garlic and garlic products. It is found that except for small horse garlic and scallion, alliin accounts for more than 90% of the total substances in garlic. The alliin content in dried garlic is generally higher than that in fresh garlic, and its total amount of flavor precursors is the highest, which is closely related to its ability to present the unique flavor required as a condiment. The isoalliin content in fresh garlic is generally higher than that in dried garlic. Triple clove garlic and fresh garlic have higher isoalliin content. The composition of flavor precursors in scallion is different from that of other varieties of garlic, and the most important substance is propyl alliin. This is why scallion is juicy, spicy, tender, sweet and crispy. The flavor precursors detected in sugar garlic are very trace, and these flavor precursors may be generated by the addition of seasonings or reaction decomposition during processing.

[0094] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors, characterized in that: The method comprises the following steps: simultaneously detecting alliin, isoalliin, methylalliin, propylalliin and S-allyl-L-cysteine ​​by liquid chromatography-tandem mass spectrometry. (1) The sample is treated and crushed by microwave, and then extracted by immersion and ultrasonic method to obtain a sample for machine testing; (2) On-machine detection: Chromatographic conditions: chromatographic column is C18-AQ; mobile phase is formic acid solution with a volume concentration of 0.1%, flow rate is 0.4 mL / min, elution time is 35 min; injection volume is 10 μL; mass spectrometry conditions: ESI source operates in positive ionization mode, capillary voltage is 4000 V; desolvation nitrogen temperature is 300°C, flow rate is 6 L / min; nebulizer gas pressure is 15 psi; multiple reaction monitoring mode is adopted.

2. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The parameters of the analytes in the multiple reaction monitoring mode are as follows: Alliin: retention time is 12.42min, precursor ion is 178.6m / z, fragment ion 41m / z is the qualitative ion, collision energy is 20eV; fragment ion 88m / z is the quantitative ion, collision energy is 5eV; Isolic acid: retention time is 14.50min, precursor ion is 178.6m / z, fragment ion 41m / z is the qualitative ion, collision energy is 20eV; fragment ion 88m / z is the quantitative ion, collision energy is 5eV; Methyl alliin: retention time is 7.43min, precursor ion is 152m / z, fragment ion 88m / z is the qualitative ion, collision energy is 15eV; fragment ion 152m / z is the quantitative ion, collision energy is 0eV; Propyl alliin: retention time is 16.76min, precursor ion is 180.7m / z, fragment ion 43m / z is the qualifier ion, collision energy is 15eV; The fragment ion 88 m / z was used as the quantitative ion, and the collision energy was 10 eV; S-allyl-L-cysteine: retention time is 28.65min, precursor ion is 163.6m / z, fragment ion 73m / z is the qualifier ion, collision energy is 15eV; The fragment ion 145 m / z was used as the quantitative ion, and the collision energy was 5 eV.

3. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The power of microwave treatment was 900 W and the time was 1 min; the power of ultrasound treatment was 500 W and the time was 15 min.

4. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The solvent used in the immersion and ultrasonic method is phosphoric acid with a volume concentration of 0.01%.

5. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The internal standard method was adopted, and the internal standard was L-norleucine with a concentration of 200 ng / mL.

6. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 5, characterized in that: The parameters of L-norleucine in the multiple reaction monitoring mode are as follows: retention time is 26.67 min, precursor ion is 132.2 m / z, fragment ion 132.2 m / z is a qualitative ion, collision energy is 0 eV; fragment ion 86.2 m / z is a quantitative ion, collision energy is 10 eV.

7. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The specifications of the chromatographic column C18-AQ are 5μm 100A 250mm×4.6mm.

8. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 1, characterized in that: The sample is garlic, garlic products or onion.

9. The liquid chromatography-tandem mass spectrometry detection method for garlic flavor precursors according to claim 8, characterized in that: The sample is fresh garlic, dried garlic, pickled garlic or onion.

Images