Method for determining content of glycyrrhetinic acid in Baijiu by adopting ultra-high performance liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for determining the content of glycyrrhetinic acid in Baijiu by adopting an ultra-high performance liquid chromatography-mass spectrometry method, and belongs to the technical field of food inspection, the method comprises the following steps: preparing glycyrrhetinic acid standard working solutions with different concentrations, analyzing the glycyrrhetinic acid standard working solutions with different concentrations by adopting an UPLC-MS / MS method, and determining the content of glycyrrhetinic acid in the Baijiu by adopting an ultra-high performance liquid chromatography-mass spectrometry method. Making a standard curve according to the peak area response value and the concentration, pretreating a to-be-detected sample, and measuring the pretreated sample on a machine; according to the method, the existence condition of glycyrrhetinic acid in white spirit can be accurately distinguished according to specific qualitative ions of glycyrrhetinic acid, false positive detection caused by the separation degree of common high performance liquid chromatography can be effectively avoided, the method is high in detection capacity, the detection limit is as low as 0.3 microgram / L, and the quantification limit is as low as 1 microgram / L; the pretreatment process is simple and convenient, the sample treatment time is about 5 minutes, no organic or toxic reagent participates in, and the timeliness and the safety are very high.

Description

Technical Field

[0001] The present invention relates to the technical field of food inspection, and in particular to a method for determining the content of glycyrrhetinic acid in liquor by ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Glycyrrhetinic acid, mainly present in licorice plants, is one of the main sources of licorice sweetness. In liquor production, glycyrrhetinic acid, as a sweetener (with a sweetness 250 times that of sucrose), may be used in liquor blending, especially in low-end liquor, to improve the taste. However, according to the "Standard for the Use of Food Additives GB 2760-2014", it belongs to non-fermentation source substances and is prohibited from being artificially added. If illegally added to liquor, it may cover up the roughness of low-quality liquor bodies. However, since it is a non-fermentation product, it is necessary to detect and confirm compliance.

[0003] Therefore, the problem of detecting the content of glycyrrhetinic acid in liquor involves many aspects such as detection technology, regulatory restrictions, and health impacts.

[0004] Currently, for the detection of the content of glycyrrhetinic acid in liquor, there are mainly: The paper "Determination of Glycyrrhizic Acid and Glycyrrhetinic Acid in Liquor by SFOD-LPME-HPLC" published by Huang Yuan et al. discloses a method for detecting glycyrrhetinic acid in liquor by combining single-drop microextraction in floating organic droplets (SFOD-LPME) with HPLC. Its detection limit is 0.004 mg / L, the quantification limit is 0.01 mg / L, and the recovery rate of the spiked samples is between 89.2% and 105.0%. This method has at least the following disadvantages: First, it requires pre-treatments such as de-alcoholization and multiple extractions. On the one hand, the pre-treatment is relatively complicated and the detection efficiency is low. On the other hand, glycyrrhetinic acid may be lost during multiple extraction treatments, which may result in false negatives. Then, there is still room for further improvement in terms of detection sensitivity, accuracy, and reliability.

[0005] The paper "Simultaneous Determination of Multiple Artificial Sweeteners in Alcoholic Beverages by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry" published by Zhou Bin et al. discloses a method for simultaneously determining artificial sweeteners such as acesulfame potassium, sodium saccharin, sodium cyclamate, sucralose, aspartame, alitame, and neotame by ultra-high performance liquid chromatography-tandem mass spectrometry. However, the detection of glycyrrhetinic acid is not disclosed in this method.

[0006] If the sample is directly detected by HPLC after dilution, although this method can complete the detection of glycyrrhetinic acid under the condition of simplifying the pretreatment process, due to the existence of hundreds of trace components in distilled liquor, after the sample passes through the chromatographic column, glycyrrhetinic acid cannot be well separated from other components, thus there is a great possibility of false positives; in addition, its detection sensitivity is also relatively low, and even if glycyrrhetinic acid is separated from other components, there is still a possibility of false negatives. Summary of the Invention

[0007] The object of the present invention is to provide a method for determining the content of glycyrrhetinic acid in liquor by ultra-high performance liquid chromatography-tandem mass spectrometry to solve the above problems.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for determining the content of glycyrrhetinic acid in liquor by ultra-high performance liquid chromatography-tandem mass spectrometry, comprising the following steps: (1) Prepare standard working solutions of glycyrrhetinic acid with different concentrations; (2) Analyze the standard working solutions of glycyrrhetinic acid with different concentrations by UPLC-MS / MS method, and then make a standard curve with the peak area response value and concentration; (3) Pretreat the sample to be tested; (4) Measure the pretreated sample on the machine.

[0009] The principle of the determination method of the present invention is as follows: After glycyrrhetinic acid in liquor is separated by ultra-high performance liquid chromatography, it enters the mass spectrometry detector for detection. The presence or absence of the substance is determined by the qualitative ions of glycyrrhetinic acid detected, and the content of the substance is determined by the quantitative characteristic ions of glycyrrhetinic acid.

[0010] As a preferred technical solution, in step (1), the preparation method of the standard working solution of glycyrrhetinic acid is as follows: 1) Prepare a standard stock solution of glycyrrhetinic acid with a concentration of 1.0 mg / ml; 2) Measure 1 ml of the standard stock solution of glycyrrhetinic acid into a 100 ml volumetric flask, and make up to 100 ml with pure methanol to obtain intermediate solution A with a concentration of 10 mg / L; 3) Accurately measure 1 ml of intermediate solution A into a 100 ml volumetric flask, and make up to 100 ml with pure water to obtain intermediate solution B with a concentration of 100 μg / L; 4) Measure 1 ml, 2 ml, 4 ml, 8 ml, and 10 ml of intermediate solution B into 100 ml volumetric flasks in sequence, and make up with pure water to obtain a series of standard working solutions with concentrations of 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, and 10 μg / L.

[0011] As a preferred technical solution, in step (2), the chromatographic conditions in the UPLC-MS / MS method are as follows: Mobile phase A: aqueous formic acid solution with a volume fraction of 0.04%; Mobile phase B: methanol-acetonitrile solution with a volume ratio of 1:1; Gradient elution.

[0012] As a further preferred technical solution, the gradient elution program is shown in Table 1: Table 1 Gradient elution program Among them, the ratio of mobile phase A to mobile phase B is in volume percentage.

[0013] As a preferred technical solution, in step (2), the mass spectrometry conditions in the UPLC-MS / MS method are as follows: Ion mode: electrospray ionization source; Ion source body: ESI electrospray ionization source; Scanning mode: positive ion mode; (ESI+, 1.5 KV), Detection mode: multiple reaction monitoring mode (MRM) Cone orifice voltage: -60 v Desolvation gas temperature: 400 °C Desolvation gas flow rate: 700 L / h Curtain gas flow rate: 50 L / h Glycyrrhetinic acid parent ion: 471.35, quantitative daughter ion: 149, collision energy 30 eV; qualitative daughter ion 107, collision energy 35 eV.

[0014] As a preferred technical solution, in step (3), the method for pre-treating the sample is to remove ethanol by nitrogen blowing and then make up the volume with mobile phase B.

[0015] As a further preferred technical solution, the nitrogen blowing treatment temperature is 55-60 °C. At this temperature, the main organic solvent ethanol in the wine body can be volatilized by nitrogen blowing. When the temperature is lower than this range, the nitrogen blowing treatment is difficult. Although too high a temperature can accelerate the volatilization of ethanol, it will inevitably bring about a solvent entrainment effect, resulting in the loss of glycyrrhetinic acid. Considering the influence of temperature on the stability of glycyrrhetinic acid, 55-60 °C is selected. The nitrogen blowing purge gas dosage is controlled at 2-3 psi to adapt to the vortex of the sample in the tube. Too high a pressure will cause droplet splashing and the loss of glycyrrhetinic acid, while too low a pressure will not cause the volatilization of ethanol.

[0016] Compared with the prior art, the advantages of the present invention are as follows: The present invention can accurately identify the presence of glycyrrhetinic acid in liquor according to its specific qualitative ions, effectively avoiding false positive detections caused by the separation degree of ordinary high performance liquid chromatography (HPLC). Moreover, this method has strong detection ability, with a detection limit as low as 0.3 μg / L and a quantification limit as low as 1 μg / L. The pretreatment process is simple, the sample treatment takes about 5 minutes, and no organic or toxic reagents are involved, with high timeliness and safety. Description of the Drawings

[0017] Figure 1 It is a chromatogram of detecting glycyrrhetinic acid by using mobile phase A and mobile phase B of the present invention; Figure 2 It is a chromatogram obtained by the present invention through multiple repeated experiments on standard products of the same concentration; Figure 3 It is the standard curve graph of the present invention; Figure 4 It is a result graph of detecting a certain strong aroma liquor by using the method of the present invention; Figure 5 It is a result graph of detecting a certain strong aroma liquor by using the traditional HPLC method; Figure 6 It is an HPLC graph when the mobile phase is an aqueous solution of formic acid with a volume fraction of 0.04% + methanol; Figure 7 It is an HPLC graph when the mobile phase is an aqueous solution of formic acid with a volume fraction of 0.04% + acetonitrile; Figure 8 It is a spectrogram obtained by using different spray voltages; Figure 9 It is a spectrogram obtained by using different cone hole voltages; Figure 10 It is a result graph of detecting a certain strong aroma liquor by using the traditional HPLC method in Example 7; Figure 11 It is a result graph of detecting a certain strong aroma liquor by using the method of the present invention. Detailed Embodiments

[0018] The present invention will be further described below in conjunction with embodiments. Embodiment

[0019] A method for determining the content of glycyrrhetinic acid in liquor by using ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps: (1) Prepare standard working solutions of glycyrrhetinic acid with different concentrations; (2) Analyze the standard working solutions of glycyrrhetinic acid with different concentrations by using UPLC-MS / MS method, and then make a standard curve with the peak area response value and the concentration; (3) Pretreat the sample to be tested; (4) Measure the pre - treated samples on the machine.

[0020] Specifically: 1.1 Reagents Water (H2O): The third - grade water for laboratory specified in GB / T 6682; Acetonitrile: UPLC grade; Methanol: UPLC grade; Formic acid: UPLC grade; Glycyrrhetinic acid standard: Purity ≥ 99wt.%.

[0021] 1.2 Solution preparation Mobile phase A: Aqueous formic acid solution with a volume fraction of 0.04%; Mobile phase B: Methanol - acetonitrile solution with a volume ratio of 1:1; Glycyrrhetinic acid standard stock solution (1.0 mg / ml): Accurately weigh 0.1 g of glycyrrhetinic acid (accurate to 0.0001 g) into a 100 - mL volumetric flask, dilute to the mark with methanol, store in the refrigerator, and the validity period is one month; Preparation of glycyrrhetinic acid standard working solution: Pipette 1 ml of the stock solution into a 100 - ml volumetric flask, dilute to 100 ml with pure methanol to obtain an intermediate solution A of 10 mg / L. Accurately pipette 1 ml of intermediate solution A into a 100 - ml volumetric flask, dilute to 100 ml with pure water to obtain an intermediate solution B of 100 μg / L; Pipette 1 ml, 2 ml, 4 ml, 8 ml, and 10 ml of intermediate solution B into 100 - ml volumetric flasks in sequence, and dilute with pure water to obtain a series of standard working solutions with concentrations of 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, and 10 μg / L. This working solution should be prepared and used immediately.

[0022] 1.3 Instruments and equipment Ultra - performance liquid chromatography - mass spectrometry: UPLC - MS / MS; Ultrasonic oscillator; Vacuum filtration device.

[0023] 1.4 Instrument reference conditions Chromatographic column: ACQUITY UPLC BEH C18 1.7μm 2.1×50mm; Flow rate: 0.3 mL / min; Injection volume: 4 μL; Column temperature: 35°C; Gradient elution program is shown in Table 1: Table 1 Gradient elution program Among them, the ratio of mobile phase A to mobile phase B is in volume percentage; Mass spectrometry end conditions and ion pair conditions: Ion mode: Electrospray ionization source; Ion source body: ESI electrospray ionization source; Scanning mode: positive ion mode (ESI+, spray voltage 1.5 KV); Detection mode: multiple reaction monitoring mode (MRM); Cone voltage: -60 v; Desolvation gas temperature: 400 °C; Desolvation gas flow rate: 700 L / h; Curtain gas flow rate: 50 L / h; The parent ion of glycyrrhetinic acid: 471.35, the quantitative daughter ion: 149, collision energy 30 eV; the qualitative daughter ion 107, collision energy 35 eV. 1.5 Preparation of standard curve Analyze the above series of standard sample working solutions in sequence, and then prepare a standard curve with the peak area response value and concentration.

[0024] 1.6 Determination of samples First, perform sample pretreatment: Take 2 ml of distilled white liquor in a graduated tube, set the temperature of the nitrogen blower to 60 °C, the purge gas pressure to 3 psi, evaporate the ethanol from the sample by nitrogen blowing, and then dilute it to 2 ml with mobile phase B, filter it through a membrane and analyze it on the machine.

[0025] Example 2 Optimization of determination conditions During the condition optimization process, an ACQUITY UPLC BEH C18 1.7um 2.1×50mm chromatographic column was selected, and aqueous formic acid solution (0.04% by volume) + methanol, aqueous formic acid solution (0.04% by volume) + acetonitrile, aqueous formic acid solution (0.04% by volume) + methanol-acetonitrile solution with a volume ratio of 1:1 were respectively investigated as the mobile phase. The inventor found that when using aqueous formic acid solution (0.04% by volume) + methanol, the peak of glycyrrhetinic acid had serious tailing and a bad peak shape, as Figure 6 shown; when using aqueous formic acid solution (0.04% by volume) + acetonitrile, the tailing phenomenon of the glycyrrhetinic acid peak was significantly reduced, but the elution rate of glycyrrhetinic acid with pure acetonitrile was too fast, as Figure 7 shown. When synchronously determining other sweet substances, there may be a problem of poor peak separation. Therefore, considering the advantages of methanol and acetonitrile comprehensively, an aqueous formic acid solution (0.04% by volume) + methanol-acetonitrile solution with a volume ratio of 1:1 was selected as the gradient elution mobile phase, and the gradient elution program was as shown before, thus obtaining an ideal peak shape and elution time of glycyrrhetinic acid, as Figure 1 shown.

[0026] During the condition optimization process, buffer salts were not used mainly because the steps in preparing the buffer salt mobile phase were rather cumbersome. From weighing to dissolution, volume fixation, filtration, etc., it took half an hour, which was time-consuming. Moreover, buffer salts were prone to salting-out effects during use, causing certain blockages. And after a long time, salts would precipitate in the ion source cavity, making the cleaning process rather troublesome.

[0027] During the optimization of the spray voltage, the inventors respectively investigated the effects of 0.5, 1.0, 1.5, 2.0, and 2.5 KV on the detection sensitivity of glycyrrhetinic acid. Using a 4 μg / L standard product for testing, the obtained Figure 8 spectra were as follows; By comparing Figure 8 the five spectra in

[0028] it was found that when 1.5 KV was used as the spray voltage, a response result with a peak height of 438592 could be obtained at a retention time of 9.77 minutes (for glycyrrhetinic acid), which was the maximum value in the experimental sequence. Therefore, the spray voltage was set at 1.5 KV. Figure 9 spectra were as follows; From Figure 9 the results shown, when the cone voltage changed from -40 V to -60 V, the response of glycyrrhetinic acid increased. However, when it exceeded -60 V, the response hardly increased anymore. Therefore, only setting the cone voltage at -60 V could meet the requirements, and there was no need to pursue too high a voltage.

[0029] Example 3 Reproducibility Experiment Using the 10 μg / L standard working solution prepared in Example 1 for four repeated experiments, the results were as Figure 2 follows; As can be seen from Figure 2 among the above four standard product injections, the areas of the target peaks obtained were 40752.04, 40806.83, 41630.82, and 41872.91 in sequence. The standard deviation was 0.138%, and the retention times after the four injections were all 9.76 min, with the peak emergence position being constant, proving good reproducibility.

[0030] Example 4 Establishment of the Standard Curve The series of standard working solutions of 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, and 10 μg / L prepared in Example 1 were analyzed in sequence. After obtaining the analysis results, a standard curve graph was made, as Figure 3 follows, From Figure 3It can be seen that the linear correlation equation is: y = 334.567x + 61.7441, and the correlation coefficient r 2 is 0.995796, with a linear ideal state, meeting the requirements of actual use.

[0031] Under the same conditions, the concentrations of glycyrrhetinic acid in different wine bodies were tested, and the signal-to-noise ratios S / N≥3 and S / N≥10 were used as the criteria for qualitative and quantitative determination. The detection limit of this method was found to be 0.3 μg / L, and the quantification limit was 1 μg / L; It is proved that compared with the detection limit of 0.004 mg / L and the quantification limit of 0.01 mg / L of the prior art such as "Determination of glycyrrhizic acid and glycyrrhetinic acid in Chinese liquor by SFOD-LPME-HPLC method", the detection limit and quantification limit of the method of the present invention are lower and more sensitive.

[0032] Example 5 Addition recovery and precision Take strong-flavor Chinese liquors 1# and 2# respectively, add standard substances and form glycyrrhetinic acid-added samples with three concentrations of 2 μg / L, 6 μg / L and 10 μg / L respectively, and conduct 3 parallel determinations at each level. The results are shown in Table 2 below: Table 2 Detection results of different samples

[0033] The results show that the parallelism and addition recovery results of glycyrrhetinic acid at the three concentration points (low, medium, and high) are relatively ideal. The addition recovery rate is between 96% and 100.5%, meeting the range requirement of the addition recovery rate between 95% and 105%, meeting the test requirements; and the relative standard deviation is less than 2%, with good overall precision. Example

[0034] Qualitative detection comparison between UPLC-MS / MS of the present invention and traditional HPLC By using the UPLC-MS / MS method of the present invention (using the detection conditions of Example 1 above) and the traditional HPLC method to detect the same sample, such as a certain strong-flavor raw liquor, the comparison charts are obtained as follows Figure 4 and Figure 5 shown, Chromatographic conditions of the traditional HPLC method: Chromatographic column: Sepax HP-C18 column, 5um, 250mm×4.69mm; Mobile phase: 80% methanol + 20% (volume fraction), isocratic elution; Flow rate: 1 mL / min; Injection volume: 10 µL; Column temperature: 30°C; Detector: UV detector; Detector wavelength: 250 nm.

[0035] Component determination description: When using the UPLC-MS / MS detection method of the present invention to determine the presence or absence of a certain substance, it is necessary to simultaneously meet the requirements that the specific quantitative ions, qualitative ions, and retention time of the substance are in line with the requirements. None of the three can be missing. Therefore, the determination conditions of this method are numerous and strict, which can narrow down the determination situation within a very narrow range, that is, the probability of false positives is extremely low. When using HPLC detection, usually under given conditions, the only determination criterion used is the retention time, and the determination conditions are too single, resulting in a greater probability of false positive detection.

[0036] Such as Figure 4 and Figure 5 , in the UPLC-MS / MS detection, at the retention time of 9.76 min, neither the quantitative ion 149 nor the qualitative ion 107 shows a peak shape, that is, the characteristic ions are missing. Therefore, based on the specificity of the specific ions of the substance, it can be accurately determined that glycyrrhetinic acid does not exist in this sample; compared with ( Figure 2 HPLC test results), in this figure, an obvious glycyrrhetinic acid peak appears at the retention time of 23.667 min. However, based on this determination method, only the retention time is used for qualitative analysis, and the limiting conditions are too few to determine the presence or absence of this substance. Therefore, compared with Figure 4 , it can be known that Figure 5 the detection result is a false positive, that is, glycyrrhetinic acid does not exist in this sample.

[0037] Example 7 False negative verification The content of glycyrrhetinic acid in a certain super-grade strong-flavor liquor was simultaneously detected by using the traditional HPLC method (the chromatographic conditions are the same as those in Example 6 above) and the UPLC-MS / MS method (the detection conditions of Example 1 above).

[0038] The results showed that: the traditional HPLC method did not detect the presence of glycyrrhetinic acid component at the retention time of 23.667 min (see the red line marked part in Figure 10 ); while when using the UPLC-MS / MS method to detect this sample, glycyrrhetinic acid component was detected in this sample (see Figure 11 at 9.76 min), and the quantitative sub-ion 149 and qualitative sub-ion 107 of glycyrrhetinic acid existed simultaneously. Therefore, it is considered that glycyrrhetinic acid exists in this super-grade strong-flavor liquor, and the content of glycyrrhetinic acid is quantitatively determined to be: 1.2 μg / L.

[0039] The above comparative experiments prove that the method of the present invention can reduce the false negative results during determination, thus significantly improving the accuracy of the test.

[0040] In summary, using UPLC-MS / MS to detect glycyrrhetinic acid has obvious qualitative advantages. At the same time, the detection limit of ordinary HPLC remains at the mg / L level, while the detection limit of UPLC-MS / MS has reached the μg / L level, with stronger detection ability. Therefore, using UPLC-MS / MS to detect glycyrrhetinic acid has more obvious advantages.

[0041] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the content of glycyrrhetinic acid in Chinese liquor by ultra - high performance liquid chromatography - mass spectrometry, characterized in that, It includes the following steps: (1) Prepare standard working solutions of glycyrrhetinic acid with different concentrations; (2) Analyze the standard working solutions of glycyrrhetinic acid with different concentrations by UPLC-MS / MS method, and then make a standard curve with the peak area response value and concentration; (3) Pretreat the sample to be tested; (4) Measure the pretreated sample on the machine.

2. The method according to claim 1, characterized in that, In step (1), the preparation method of the standard working solution of glycyrrhetinic acid is as follows: 1) Prepare a standard stock solution of glycyrrhetinic acid with a concentration of 1.0 mg / ml; 2) Take 1 ml of the standard stock solution of glycyrrhetinic acid and place it in a 100-ml volumetric flask, and make up the volume to 100 ml with pure methanol to obtain intermediate solution A with a concentration of 10 mg / L; 3) Accurately take 1 ml of intermediate solution A and place it in a 100-ml volumetric flask, and make up the volume to 100 ml with pure water to obtain intermediate solution B with a concentration of 100 μg / L; 4) Take 1 ml, 2 ml, 4 ml, 8 ml, and 10 ml of intermediate solution B in sequence and place them in 100-ml volumetric flasks. After making up the volume with pure water, a series of standard working solutions with concentrations of 1 μg / L, 2 μg / L, 4 μg / L, 8 μg / L, and 10 μg / L are obtained.

3. The method according to claim 1, wherein In step (2), the chromatographic conditions in the UPLC-MS / MS method are as follows: Mobile phase A: Aqueous formic acid solution with a volume fraction of 0.04%; Mobile phase B: Methanol-acetonitrile solution with a volume ratio of 1:1; Gradient elution.

4. The method according to claim 3, characterized in that, The gradient elution program is as follows: ； Among them, the ratio of mobile phase A to mobile phase B is in volume percentage.

5. The method according to claim 1, characterized in that In step (2), the mass spectrometry conditions in the UPLC-MS / MS method are as follows: Ion mode: Electrospray ionization source; Ion source body: ESI electrospray ionization source; Scanning mode: Positive ion mode; Detection mode: Multiple reaction monitoring mode; Parent ion of glycyrrhetinic acid: 471.35, quantitative daughter ion: 149, collision energy 30 eV; qualitative daughter ion 107, collision energy 35 eV.

6. The method according to claim 3, wherein In step (3), the method for pretreating the sample is to remove ethanol by nitrogen blowing and then make up the volume with mobile phase B.

7. The method according to claim 6, wherein The temperature for nitrogen blowing treatment is 55-60 °C, and the purge gas pressure is 2-3 psi.

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