A method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic

Abstract

The invention discloses a method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic. The method comprises the following steps: extracting black garlic by ultrasonic-assisted extraction to obtain an extract; subjecting the obtained extract to solid phase extraction and then PITC derivatization to obtain a sample solution; and subjecting the obtained sample solution to UPLC-MS detection to obtain the contents of selenomethionine and selenomethylselenocysteine ​​in the black garlic through calculation.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic by combining UPLC-MS (ultra-performance liquid chromatography-single quadrupole mass spectrometry) with pre-column derivatization. Background Art

[0002] Black garlic, also known as black garlic or fermented black garlic, is a deep-processed garlic product made from fresh raw garlic fermented in a high temperature and high pressure environment. After the garlic is fermented, the cloves turn dark brown, the pungent smell and spicy feeling disappear, and the taste is soft, glutinous and sweet. A large number of studies have shown that compared with garlic, fermented black garlic has enhanced biological activity and increased content of various nutrients. It has physiological functions such as anti-oxidation, anti-tumor, liver protection, regulating blood sugar and blood lipid levels, anti-allergy, and anti-inflammatory.

[0003] Selenium is one of the essential trace elements for the human body. It participates in many physiological activities of the human body and is very important for human health. Insufficient selenium intake will reduce the human body's immunity and cause various diseases, such as Keshan disease, big bone disease, endemic myocarditis, etc. Excessive selenium will cause human poisoning. Therefore, it is crucial for the human body to supplement an appropriate amount of selenium in time. Selenium is divided into inorganic selenium and organic selenium. Organic selenium is more suitable for human absorption and utilization due to its low toxicity and high biological activity. Obtaining selenium from plants is currently recognized as the main way for the human body to absorb selenium. Organic selenium in plants mainly exists in the form of selenomethionine and selenomethylselenocysteine. Black garlic has become one of the popular selenium supplement foods because of its selenium content. Therefore, the ability to quickly and accurately detect the content of selenomethionine and selenomethylselenocysteine ​​in black garlic can provide an important reference for the quality and safety evaluation of black garlic.

[0004] At present, the detection methods of selenomethionine and selenomethylselenocysteine ​​mainly include gas chromatography-mass spectrometry, liquid chromatography-atomic fluorescence spectrometry, and high performance liquid chromatography-inductively coupled plasma mass spectrometry. At present, there is no method for PITC derivatization detection of selenomethionine and selenomethylselenocysteine ​​in black garlic. Summary of the invention

[0005] The invention provides a method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic. The invention proposes to establish a high performance liquid chromatography-single quadrupole mass spectrometry method based on pre-column PITC derivatization by optimizing extraction, extraction and derivatization condition parameters, so as to simultaneously and rapidly detect the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic, and analyze the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic from 5 different origins, so as to provide a reference basis for the quality of black garlic.

[0006] A method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic, comprising:

[0007] (1) peeling the black garlic, evenly crushing it into a paste, and then vacuum drying it; taking the dried black garlic and adding a methanol aqueous solution in proportion, mixing it thoroughly, and then ultrasonicating it at 20 to 50° C. for 10 to 40 minutes; and taking the supernatant after centrifugation;

[0008] (2) subjecting the obtained supernatant to solid phase extraction and eluting with methanol-water solution, collecting all the eluates; concentrating the obtained eluate by rotary evaporation and dissolving it again in methanol-water solution, mixing it evenly and filtering it to obtain the filtrate (extract);

[0009] (3) Adding the internal standard solution, triethylamine acetonitrile solution and PITC acetonitrile solution to the obtained filtrate (extract), mixing thoroughly and performing a derivatization reaction at 20-60° C. for 1-3 h; after the reaction is completed, taking the supernatant into an automatic sampling bottle for testing;

[0010] (4) The obtained sample solution is subjected to UPLC-MS detection, the absorption peak area is recorded and substituted into the standard curve, and the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic are calculated.

[0011] The method of the invention comprises the following steps: extracting black garlic by ultrasonic-assisted extraction to obtain an extract; subjecting the obtained extract to solid phase extraction and then PITC derivatization to obtain a sample solution; and subjecting the obtained sample solution to UPLC-MS detection to calculate and obtain the contents of selenomethionine and selenomethylselenocysteine ​​in the black garlic.

[0012] Optionally, in step (1), the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 0 to 80%; the mass volume ratio of black garlic to methanol aqueous solution is 1 g:10 to 25 mL.

[0013] Optionally, in step (2), the methanol aqueous solution is a methanol aqueous solution with a volume percentage concentration of 10 to 80%.

[0014] Optionally, in step (1), the methanol aqueous solution is a methanol aqueous solution with a volume percentage concentration of 20%; the mass volume ratio of black garlic to methanol aqueous solution is 1g:10mL; after sufficient mixing, ultrasonic treatment is carried out at 40°C for 30min; in step (2), the methanol aqueous solution is a methanol aqueous solution with a volume percentage concentration of 40%.

[0015] Optionally, in step (3), the volume ratio of the filtrate, the triethylamine acetonitrile solution and the PITC acetonitrile solution is 5:2.5:1; wherein the concentration of triethylamine in the triethylamine acetonitrile solution is 1 mol / L; and the concentration of PITC in the PITC acetonitrile solution is 0.1 mol / L.

[0016] Optionally, in step (3), the internal standard solution is methionine-D3.

[0017] Optionally, the concentration of the internal standard solution is 1 mg / mL; the volume ratio of the added amount of the internal standard solution to the filtrate is 0.5:5.

[0018] Optionally, the derivatization reaction temperature is 20° C., and the derivatization reaction time is 1 h.

[0019] Optionally, in step (4), the detection conditions are:

[0020] Chromatographic conditions: ACOQUITY BEH C18 column; flow rate: 0.3 mL min -1 ; Column temperature: 45°C; Injection volume: 5 μL; Detection wavelength: 338 nm; Mobile phase: acetonitrile A-0.01% formic acid aqueous solution B, gradient elution;

[0021] Mass spectrometry conditions: electrospray ion source with negative ion detection; electrospray capillary voltage was set to 3.0 kV, nitrogen was used as drying gas for solvent evaporation, evaporation temperature was 172 °C, ion transfer tube temperature was 300 °C, and selective ion monitoring mode.

[0022] Optionally, the concentration of selenomethionine in the filtrate (extract) is 0.019-1.25 μg·mL -1 , selenomethylselenocysteine ​​concentration is 0.078-5.00 μg·mL -1 , the linear relationship is good under this condition.

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] (1) The present invention selects an ultrasonic-assisted water extraction method with low cost, simple operation and short time consumption to extract selenomethionine and selenomethylselenocysteine ​​from black garlic, and optimizes the extraction method from four aspects: extraction solvent, solid-liquid ratio, extraction time and extraction temperature. The results show that the content of selenomethionine and selenomethylselenocysteine ​​is the highest when the extraction solvent is 20% methanol, the solid-liquid ratio is 1:10, the extraction time is 30 minutes and the extraction temperature is 40°C.

[0025] (2) The content of selenomethionine and selenomethylselenocysteine ​​in black garlic is relatively low. The present invention selects a pre-column derivatization amino acid method to improve the sensitivity and specificity of selenomethionine and selenomethylselenocysteine ​​detection in black garlic samples; compared with the post-column derivatization method (which requires a dedicated amino acid analyzer), it has low cost and high sensitivity, and is convenient for laboratory application.

[0026] (3) The present invention selects PITC as a derivatization reagent to perform a derivatization reaction to detect selenomethionine and selenomethylselenocysteine ​​in black garlic. The PITC derivatization method has good selectivity and can react with primary amino acids and secondary amino acids at the same time. The derivatization product has good stability, and the reaction conditions are mild and the cost is low.

[0027] (4) During the research process of the present invention, it was found that the matrix effect of the LC-MS method was relatively large and had a strong ion suppression effect. Therefore, the present invention selected isotope methionine as the internal standard.

[0028] (5) There is currently no method for simultaneously detecting selenomethionine and selenomethylselenocysteine ​​in black garlic by PITC derivatization. The present invention establishes an analytical method for determining selenomethionine and selenomethylselenocysteine ​​in black garlic based on ultra-high performance liquid chromatography-tandem single quadrupole mass spectrometry. The concentrations of selenomethionine and selenomethylselenocysteine ​​are 0.019-1.25 μg mL -1 , 0.078-5.00μg·mL -1 The linear relationship is good, and the correlation coefficient R 2 At 0.9995-0.9996, the detection limits were 4.844mL and 0.605ng / mL respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of PITC derivatization of selenomethionine (A) and PITC derivatization of selenomethylselenocysteine ​​(B);

[0030] Figure 2 The results of the effects of different extraction conditions on the extraction efficiency of selenomethionine in Example 2 are shown in Figure 2 (A is a comparison of the results of different extraction solvents; B is a comparison of the results of different solid-liquid ratios; C is a comparison of the results of different extraction times; and D is a comparison of the results of different extraction temperatures);

[0031] Figure 3 This is a graph showing the effect of different elution reagents on the extraction efficiency of selenomethionine in Example 3;

[0032] Figure 4 The results of the effects of different derivatization conditions on the derivatization products in Example 4 are shown in Figure 4 (A is a comparison of the results of different derivatization times; B is a comparison of the results of different derivatization temperatures);

[0033] Figure 5 The chromatograms are of selenomethionine (A), selenomethylselenocysteine ​​(B) and internal standard: methionine-D3d3 (C) in Example 5 (wherein, the left figure is the chromatogram of the blank control; the middle figure is the chromatogram of the black garlic sample; the right figure is the chromatogram of the standard). DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0036] Materials and Reagents

[0037] 5 portions of black garlic (commercially available, sample information see Table 1).

[0038] Selenomethionine standard was purchased from Sigma-Aldrich; selenomethylselenocysteine ​​standard was purchased from Maclean; methionine-D3d3 was purchased from Sigma-Aldrich; o-phthalaldehyde was purchased from Tokyo Chemical Industry Co., Ltd.; mercaptopropionic acid was purchased from Beijing J&K Technology Co., Ltd.; triethylamine was purchased from Beijing J&K Technology Co., Ltd.; acetonitrile (analytical grade) and methanol (analytical grade) were both purchased from Beijing J&K Technology Co., Ltd.; formic acid was purchased from Tokyo Chemical Industry Co., Ltd.; ultrapure water; commercially available black garlic.

[0039] Table 1 Sample information

[0040]

[0041] Instruments and Equipment

[0042] TGL-16B high-speed centrifuge (Shanghai Anting Scientific Instrument Factory); XH-B vortex mixer (Jiangsu Tianling Instrument Co., Ltd.); DW-86L338J medical cryopreservation box (Qingdao Haier Biomedical Co., Ltd.); KM-410C ultrasonic cleaner (KM-410C ultrasonic cleaner); CV100-DNA vacuum centrifugal concentrator (Beijing Ji Aim Technology Co., Ltd.); Varian 600M nuclear magnetic resonance instrument (Varian, USA); XH-B vortex mixer (Jiangsu Tianling Instrument Co., Ltd.); single quadrupole liquid chromatography-mass spectrometry (Thermo Fisher Scientific, USA); ACOQUITY BEH C18 chromatographic column (Thermo Fisher Scientific, USA); ME104E analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); SPE-24D solid phase extractor (Shanghai Xiyang Instrument Co., Ltd.); C18 solid phase extraction cartridge (Thermo Fisher Scientific, USA); oil-free vacuum pump (Shanghai Xiyang Instrument Co., Ltd.); DZF-6050 vacuum drying oven (Gongyi Yuhua Instrument Co., Ltd.); 2XZ-2 rotary vane vacuum pump (Linhai Tanshi Vacuum Equipment Co., Ltd.)

[0043] Solution preparation

[0044] Accurately weigh 10 mg of selenomethionine and 10 mg of selenomethylselenocysteine, dissolve them in 80% (v / v) methanol and make up to volume in a 10 mL volumetric flask, shake well to obtain a 1 mg / mL standard stock solution, and store at 4°C: when used, use 80% (v / v) methanol to prepare a series of standard working solutions of different concentrations, prepare them before use; Accurately weigh 10 mg of methionine-d3, dissolve them in 80% (v / v) methanol and make up to volume in a 10 mL volumetric flask, shake well to obtain a 1 mg / mL standard stock solution, and store at 4°C.

[0045] Example 1

[0046] (1) Extraction and solid phase extraction of selenomethionine and selenomethylselenocysteine ​​from black garlic

[0047] Extraction: Select a certain amount of black garlic sample (this embodiment is tested with S1 in Table 1), peel it, evenly crush it into a mud, and store it at 4°C for later use. After drying it in a vacuum drying oven for 24 hours, accurately weigh 0.20g of the black garlic sample, accurately add 2.003.00mL of 20% (v / v) methanol, vortex vigorously for 10min, ultrasonicate for 10min at 40°C, centrifuge it at 10000rp / m for 15min, take 2mL of the supernatant respectively and add it to a 5ml centrifuge tube for purification.

[0048] Solid phase extraction: Activate the SPE column with 2 mL of methanol and 2 mL of water in turn, then transfer 2 mL of the extract from the previous step to the column, elute the sample with 1 mL of 40 (v / v)% methanol aqueous solution at a rate of 1 ml / min, and collect all the eluate. The eluate is concentrated to near dryness using a rotary evaporator, dissolved with 200 μL of 20% methanol aqueous solution, vortexed and mixed, and then filtered through a 0.45 μm filter membrane. The filtrate is placed in a centrifuge tube for derivatization. (2) PITC derivatization of selenomethionine and selenomethylselenocysteine

[0049] Take 200 μL of the standard solution or the black garlic extract after solid phase extraction purification, add 20 μL of the internal standard solution (DL-methionine-D3), 100 μL of triethylamine acetonitrile solution (where the concentration of triethylamine is 1 mol / L) and 40 μL of PITC acetonitrile solution (where the concentration of PITC is 0.1 mol / L) respectively, vortex vigorously to mix, and then stand at room temperature for 1 hour for derivatization reaction ( Figure 1 ). After the reaction is completed, add 800 μL of n-hexane to extract the excess PITC. Vortex mix and let stand for 10 minutes. After stratification, take 50 μL of the lower layer of PITC derivative solution and add 450 μL of 20% (v / v) acetonitrile solution containing 0.1% formic acid. Mix well and centrifuge at 13000 rpm / min for 10 minutes. Take 200 μL of the supernatant and put it in an automatic injection bottle for testing.

[0050] (3) UPLC-MS detection

[0051] Test conditions

[0052] Chromatographic conditions: ACOQUITY BEH C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm); flow rate: 0.3 mL min-1; column temperature: 45 °C; injection volume: 5 μL; detection wavelength: 338 nm; mobile phase: acetonitrile (A)-0.01% formic acid aqueous solution (B), gradient elution, elution program see Table 2.

[0053] Mass spectrometry conditions: The electrospray ion source used negative ion detection; the electrospray capillary voltage was set to 3.0 kV, nitrogen was used as the drying gas for solvent evaporation, the evaporation temperature was 172 °C, the ion transfer tube temperature was 300 °C, and the selective ion monitoring mode (SIM) was used. The quantitative analysis parameters of each test substance after derivatization are shown in Table 3.

[0054] Table 2 Liquid chromatography gradient elution program

[0055]

[0056] Table 3 Mass spectrometry parameters of materials after derivatization

[0057]

[0058] (4) Linear relationship and limit of quantification

[0059] The selenomethionine standard solution was diluted step by step into a series of standard working solutions of 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, and 0.019 μg / ml, and the selenomethylselenocysteine ​​standard solution was diluted step by step into a series of standard working solutions of 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, and 0.078 μg / ml. After preparation according to the above method, the samples were injected and tested, and the standard curve was made with the standard concentration as the abscissa and the absorption peak area as the ordinate, and the linear equation, linear coefficient and linear range were calculated.

[0060] The detection limit (LOD) and quantification limit (LOQ) were determined using a 3-fold signal-to-noise ratio (S / N=3) and a 10-fold signal-to-noise ratio (S / N=10). The results of the linear relationship, detection limit, and quantification limit are shown in Table 4.

[0061] As shown in Table 4, selenomethionine and selenomethylselenocysteine ​​showed a good linear relationship in the linear range of 0.019-5.00ug / ml, with a correlation coefficient R 2 ≥0.9995, the detection limits were 4.844ng / ml and 0.605ng / ml respectively, and the quantification limits were 14.532ng / ml and 1.815ng / ml respectively.

[0062] Table 4 Linear equations, linear coefficients, linear ranges, detection limits and quantification limits of selenomethionine and selenomethylselenocysteine

[0063]

[0064] (5) Precision, matrix effect and spike recovery

[0065] The products of the standard products of 0.313μg / mL, 0.625μg / mL, and 1.250μg / mL were tested 5 times on the same day, and the results showed that the average RSD was less than 5%, less than 8% within 24 hours. The 36 and 48 hours exceeded 10%, indicating that the samples were best tested within 24 hours of derivatization. 20μl of the internal standard solution of equal concentration was added to 200μl of black garlic extract and 80% solvent, respectively. After derivatization according to the above method, the product peak area IS-PITC was measured, and the matrix effect was calculated 6 times in parallel.

[0066] The results showed that the matrix effect value of this method was 56.07+0.11%, indicating that the matrix effect of this method was large and had a strong ion suppression effect. It was necessary to use an isotope internal standard for quantitative detection. Three mixed standard solutions of 0.625μg / mL, 1.250μg / mL, and 2.50μg / mL were added to the extract, and the samples were pretreated and measured together. Each sample concentration was measured in parallel 3 times. The results showed that the recovery rates of selenomethionine and selenomethylselenocysteine ​​were between 71.4% and 91.4%. This method showed that although isotope methionine was used as an internal standard, the spike recovery rate was low at low and high concentrations due to the large ion suppression effect. The spike recovery rate was 91.4% at a concentration of 1.25ug / mL, indicating that the method can meet the qualitative and quantitative detection of selenomethionine and selenomethylselenocysteine ​​in black garlic, and it is best to detect it within the concentration range of 0.8-2.0ug / mL.

[0067] Example 2 Optimization of extraction

[0068] In the condition optimization example, black garlic was tested with S1 in Table 1.

[0069] Referring to the test process of Example 1, the effects of different extraction solvents on the extraction efficiency were first compared. The solid-liquid ratio was 1g:15mL, the extraction method was ultrasonic, the extraction time was 20min, and the extraction temperature was 30°C. The changes in the contents of selenomethionine and selenomethylselenocysteine ​​in the extract were studied when the extraction solvents were pure water, 20% methanol (v / v), 40% methanol (v / v), and 80% methanol (v / v) (other extraction steps were the same as in Example 1). The results are shown in Figure 2 As shown in A. Figure 2 As shown in Figure A, different extractants have no significant effect on the extraction of selenomethionine. When the extraction solvent is 20% (v / v) methanol, the selenomethionine content in the extract after solid phase extraction (corresponding to Figure 2 Therefore, 20% (v / v) methanol was selected as the extraction solvent for selenomethionine and selenomethylselenocysteine.

[0070] At the same time, the changes in the contents of selenomethionine and selenomethylselenocysteine ​​in the black garlic samples were compared when the material-liquid ratio (mass volume ratio (g:mL) of black garlic to 20% (v / v) methanol in the extraction step) was 1g:10mL, 1g:15mL, 1g:20mL, and 1g:25mL (other extraction steps were the same as those in Example 1). The results are as follows: Figure 2 As shown in B. Figure 2 As shown in Figure B, with the increase of the solid-liquid ratio, the content of selenomethionine in the extract after solid phase extraction (corresponding to Figure 2The vertical coordinate of B in the figure gradually decreases, so the appropriate liquid ratio of selenomethionine and selenomethylselenocysteine ​​is 1g:10mL.

[0071] Then, the changes in the contents of selenomethionine and selenomethylselenocysteine ​​in the black garlic samples were compared when the extraction time was 10 min, 20 min, 30 min, and 40 min (other extraction steps were the same as in Example 1). The results are as follows: Figure 2 As shown in C. Figure 2 As shown in Figure 5, with the increase of extraction time, the content of selenomethionine in the extract after solid phase extraction (corresponding to Figure 2 The ordinate of C in the figure shows a gradually increasing trend. When the extraction time is 30 min, the content of selenomethionine in the extract after solid phase extraction is the highest. Therefore, 30 min is selected as the extraction time for selenomethionine and selenomethylselenocysteine.

[0072] Finally, this example compares the changes in the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic samples when the extraction temperatures are 10°C, 20°C, 30°C, and 40°C, respectively (other extraction steps are the same as in Example 1). Figure 2 As shown in D. Figure 2 As shown in D, with the increase of extraction temperature, the content of selenomethionine in the extract after solid phase extraction (corresponding to Figure 2 The vertical coordinate of D in the figure gradually increases. When the extraction temperature is 40℃, the content of selenomethionine in the extract after solid phase extraction is the highest. Therefore, 40℃ is selected as the extraction temperature of selenomethionine and selenomethylselenocysteine.

[0073] Figure 2 The overall results showed that the contents of selenomethionine and selenomethylselenocysteine ​​in the extract after solid phase extraction were the highest when the extraction solvent was 20% methanol, the solid-liquid ratio was 1g:10mL (the mass volume ratio of black garlic to 20% methanol in the extraction step (g:mL)), the extraction time was 30min, and the extraction temperature was 40°C.

[0074] Example 3 Optimization of solid phase extraction

[0075] In the condition optimization example, black garlic was tested with S1 in Table 1.

[0076] In the solid phase extraction process, elution is one of the key factors. This example compares the effects of different extraction solvents on the extraction efficiency. Elution solvents of 10% methanol (v / v), 20% methanol (v / v), 40% methanol (v / v), 60% methanol (v / v), and 80% methanol (v / v) were selected to compare the extraction effects (other extraction conditions refer to the extraction steps of Example 1). In the process of studying solid phase extraction, the extraction step refers to the optimal extraction conditions determined in Example 2: extraction solvent 20% methanol, solid-liquid ratio selected 1g:10mL (mass volume ratio of black garlic to 20% methanol in the extraction step (g:mL)), extraction time 30min, extraction temperature 40°C.

[0077] The results are as follows Figure 3 As shown in Figure 2, with the increase of methanol volume fraction, the content of selenomethionine in the extract after solid phase extraction (corresponding to Figure 3 The selenomethionine content in the extract after solid phase extraction was the lowest when the elution solvent was 80% methanol. Therefore, the 40% methanol solution was selected as the elution solution in the experiment.

[0078] Example 4

[0079] The derivatization method was optimized by referring to the PITC derivatization method in Example 1, and black garlic selected S1 in Table 1. This example investigated the effects of different derivatization times (1 h, 1.5 h, 2 h, 2.5 h, 3 h) (otherwise the same as the PITC derivatization process in Example 1) and different derivatization temperatures (20° C., 40° C., 60° C.) (otherwise the same as the PITC derivatization process in Example 1) on the derivatized products to determine the optimal derivatization time and temperature.

[0080] Other operations are the same as in Example 1.

[0081] The results are as follows Figure 4 As shown. Figure 4 From Figure A, we can see that the derivatization time has little effect on the derivatization results, and the derivatization product is more stable when the derivatization time is 1h. Figure 4 As shown in Figure B, the yield of the derivatization product decreases with the increase of the derivatization temperature, and the derivatization product is relatively stable when the derivatization temperature is 20°C. Therefore, the derivatization product of selenocysteine ​​and selenomethylselenocysteine ​​is most stable when the reaction is carried out at 20°C for 1h, which is the optimal derivatization condition.

[0082] Example 5

[0083] Content determination results of black garlic samples from different origins.

[0084] Five samples of black garlic from five origins numbered S1-S5 (Table 1) were prepared, each with 0.2 g. The black garlic from each origin was extracted according to the optimal extraction conditions in Example 2. The black garlic extract was purified according to the optimal extraction method in Example 3. The extracted extract was derivatized according to the optimal derivatization method in Example 4. Finally, the sample was injected according to the chromatographic and mass spectrometric conditions in Example 1. The HPLC chromatograms of selenomethionine, selenomethylselenocysteine ​​and internal standard: methionine-d3 in the blank control, typical black garlic samples and standard samples were as shown in the figure. Figure 5 The average contents of selenomethionine and selenomethylselenocysteine ​​in black garlic samples from various origins are shown in Table 5.

[0085] Table 5. Sample content determination results

[0086]

[0087] As shown in Table 5, selenomethionine and selenomethylselenocysteine ​​were detected in the black garlic samples from the five origins. Among them, the content of selenomethionine in black garlic produced in Qinghai was the highest, and the content of selenomethionine in black garlic produced in Yunnan was the lowest; the content of selenomethylselenocysteine ​​in black garlic produced in Jiangsu was the highest, followed by black garlic produced in Qinghai, and the content of selenomethylselenocysteine ​​in black garlic produced in Yunnan was the lowest. This shows that the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic produced in Qinghai are both high.

[0088] In summary:

[0089] (1) The present invention uses an ultrasonic-assisted water extraction method with low cost, simple operation and short time consumption to extract selenomethionine and selenomethylselenocysteine ​​from black garlic, and optimizes the extraction method from four aspects: extraction solvent, solid-liquid ratio, extraction time and extraction temperature. The results show that the content of selenomethionine and selenomethylselenocysteine ​​is the highest when the extraction solvent is 20% methanol, the solid-liquid ratio is 1:10, the extraction time is 30 minutes and the extraction temperature is 40°C.

[0090] (2) The content of selenomethionine and selenomethylselenocysteine ​​in black garlic is relatively low. The present invention adopts the pre-column derivatization amino acid method to improve the sensitivity and specificity of selenomethionine and selenomethylselenocysteine ​​detection in black garlic samples. According to the different derivatization times, the derivatization method can be divided into two types: pre-column derivatization and post-column derivatization. Compared with pre-column derivatization, the post-column derivatization method requires a dedicated amino acid analyzer, which is more expensive, less sensitive, and not convenient for laboratory application.

[0091] (3) The experiment of the present invention selects PITC as a derivatization reagent, and performs a derivatization reaction to detect selenomethionine and selenomethylselenocysteine ​​in black garlic. At present, the most commonly used methods for pre-column derivatization of amino acids are mainly (PITC) method and o-phthalaldehyde (OPA) method. The advantages of the OPA method are rapid reaction, but strong selectivity. It can only be used for the derivatization of primary amino acids, and secondary amino acids cannot be detected at the same time. In addition, the stability of the derivatized products is poor and the cost is high. The PITC derivatization method has good selectivity, can react with primary amino acids and secondary amino acids at the same time, the derivatized products are stable, and the reaction conditions are mild and the cost is low.

[0092] (4) There is currently no method for detecting selenomethionine and selenomethylselenocysteine ​​in black garlic by PITC derivatization. The present invention establishes an analytical method for determining selenomethionine and selenomethylselenocysteine ​​in black garlic based on ultra high performance liquid chromatography-tandem single quadrupole mass spectrometry, and optimizes sample pretreatment conditions and derivatization conditions. The experiment selected a relatively fast ultrasonic water extraction method for sample extraction, and after removing macromolecular interference with a C18 solid phase extraction column, PITC derivatization was performed, and then the sample was loaded for analysis. The concentrations of selenomethionine and selenomethylselenocysteine ​​were 0.019-1.25 and 0.078-5.00 μg·mL, respectively. -1 The linear relationship is good, and the relationship R 2 At 0.9995-0.9996, the detection limits were 4.844mL and 0.605ng / mL respectively.

[0093] (5) The experimental results of the present invention show that the matrix effect of the LC-MS method is relatively large and has a strong ion suppression effect. Therefore, the isotope methionine-D3 is selected as the internal standard in this experiment. The methodological results show that the established LC-MS method can be used for the quantitative detection of selenomethionine and selenomethylselenocysteine ​​in black garlic.

[0094] (6) The experimental results of the present invention show that the content of selenomethionine in the black garlic samples from the five origins is relatively low, and the content varies greatly. The content of selenomethionine in the black garlic produced in Yunnan is much lower than that in the other four origins, and the content of selenomethionine in the black garlic produced in Qinghai is the highest. The content of selenomethylselenocysteine ​​in the black garlic from each origin is also quite different. The content of selenomethylselenocysteine ​​in the black garlic produced in Yunnan is the lowest, the content of selenomethionine in the black garlic produced in Jiangsu is much higher than that in the other four origins, and the black garlic produced in Qinghai is second. The content of selenomethylselenocysteine ​​in the black garlic samples from each origin is much higher than that of selenomethionine, which can reach 5-20 times. In short, there are large differences in the content of selenomethylselenocysteine ​​and selenomethionine in the black garlic samples from the five origins, which may be related to the growth and cultivation conditions such as climate and soil. Among them, the content of selenomethionine and selenomethylselenocysteine ​​in the black garlic produced in Qinghai is relatively high, which may be a better selenium supplement food.

[0095] In summary, there are few reports on the quantitative detection of organic selenium in garlic. The present invention establishes a UHPLC-MS method based on PITC pre-column derivatization to simultaneously determine selenomethionine and selenomethylselenocysteine ​​in black garlic, and examines the extraction solvent, liquid-to-solid ratio, extraction time, extraction temperature and other factors to extract the two target compounds in garlic. At the same time, the derivatization conditions are optimized. The results show that the established UHPLC-MS detection method is fast and reliable, providing a reference for the quality detection of black garlic. The content of selenomethylselenocysteine ​​in black garlic from various places is 5-20 times that of selenomethionine. The differences in selenomethionine and selenomethylselenocysteine ​​in black garlic from different origins are obvious, which may be related to the growth and cultivation conditions such as climate and soil.

[0096] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for simultaneously detecting the contents of selenomethionine and selenomethylselenocysteine ​​in black garlic, characterized in that: include: (1) peeling the black garlic, crushing it into a paste, and then vacuum drying it; taking the dried black garlic and adding methanol aqueous solution in proportion, mixing it thoroughly, and then ultrasonicating it at 20-50° C. for 10-40 minutes; and taking the supernatant after centrifugation; (2) subjecting the obtained supernatant to solid phase extraction through a C18 solid phase extraction column and eluting with a methanol aqueous solution, and collecting all the eluates; concentrating the obtained eluate by rotary evaporation, dissolving it again in a methanol aqueous solution, mixing it evenly, and filtering to obtain the filtrate; The methanol aqueous solution is a methanol aqueous solution with a volume percentage concentration of 40%; (3) adding an internal standard solution, a triethylamine acetonitrile solution and a PITC acetonitrile solution to the obtained filtrate, mixing thoroughly and performing a derivatization reaction at 20 to 60° C. for 1 to 3 hours; after the reaction is completed, taking the supernatant into an automatic injection bottle for testing; (4) subjecting the obtained sample solution to be tested to UPLC-MS detection, recording the absorption peak area and substituting it into the standard curve, and calculating the content of selenomethionine and selenomethylselenocysteine ​​in black garlic; The test conditions are: Chromatographic conditions: BEH C18 column; flow rate: 0.3 mL min -1 ; Column temperature: 45°C; injection volume: 5 μL; detection wavelength: 338 nm; mobile phase: acetonitrile A-0.01% formic acid aqueous solution B, gradient elution; Mass spectrometry conditions: electrospray ion source with negative ion detection; electrospray capillary voltage was set to 3.0 kV, nitrogen was used as drying gas for solvent evaporation, evaporation temperature was 172 °C, ion transfer tube temperature was 300 °C, and selective ion monitoring mode.

2. The method according to claim 1, characterized in that In step (1), the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 0 to 80%; the mass volume ratio of black garlic to methanol aqueous solution is 1g:10 to 25mL.

3. The method according to claim 1, characterized in that In step (1), the methanol aqueous solution is a methanol aqueous solution with a volume percentage concentration of 20%; the mass volume ratio of black garlic to methanol aqueous solution is 1g:10mL; after being fully mixed, ultrasonic treatment is carried out at 40°C for 30min.

4. The method according to claim 1, characterized in that: In step (3), the volume ratio of the filtrate, the triethylamine acetonitrile solution and the PITC acetonitrile solution is 5:2.5:1; wherein the concentration of triethylamine in the triethylamine acetonitrile solution is 1 mol / L; and the concentration of PITC in the PITC acetonitrile solution is 0.1 mol / L.

5. The method according to claim 1, characterized in that In step (3), the internal standard solution is methionine-D3.

6. The method according to claim 1, characterized in that The temperature of the derivatization reaction was 20°C, and the time of the derivatization reaction was 1 h.

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