Method for simultaneously determining sugar and sugar alcohol sweet substances in white spirit

Abstract

The invention belongs to the technical field of substance detection, and particularly relates to a method for simultaneously determining sugar and sugar alcohol sweet substances in white spirit. In order to rapidly, accurately, effectively and simultaneously determine a variety of sugar and sugar alcohol sweet substances in Baijiu, a high performance liquid chromatography-tandem mass spectrometry method is adopted, and determination conditions of chromatography and mass spectrometry are limited; according to the method, the aim of simultaneously determining 14 sweet substances, namely cane sugar, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol and inositol, in the Baijiu is fulfilled. The method provided by the invention has good accuracy and precision, and experimental results show that the standard addition recovery rate of the determined white spirit blank sample at different levels is 86.4-107.6%, the precision is less than 10%, and the detection requirements can be completely met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of substance detection, and particularly relates to a method for simultaneously determining sugar and sugar alcohol sweet substances in liquor. BACKGROUND

[0002] The traditional sweetness of liquor mainly comes from natural sugar and sugar alcohol substances (such as 2,3-butanediol, glycerol, etc.) produced in the fermentation process, which endow the liquor body with a sweet and mellow taste. However, in actual production, some enterprises may illegally add artificial sweeteners (such as sodium saccharin, cyclamate, etc.) or non-fermentation-derived sugar alcohol substances (such as xylitol, maltitol, etc.) to improve the taste or reduce costs, which violates the standard regulation that solid-state liquor cannot add non-fermentation-derived flavoring substances. Therefore, accurately and efficiently determining the content of sugar and sugar alcohol sweet substances in liquor is of great significance for understanding liquor quality, controlling production process, and protecting consumer health.

[0003] For the detection of sugar and sugar alcohol sweet substances in liquor, currently, there are mainly ion chromatography, high performance liquid chromatography-fluorescence detection method (HPLC-FLD), high performance liquid chromatography-differential refractometry (HPLC-RID), etc. However, these methods have the defects of complex operation, poor universality, and inability to simultaneously detect multiple sugar and sugar alcohol sweet substances in liquor. In order to overcome the shortcomings of the existing detection methods, meet the detection needs of the liquor industry for multiple sugar and sugar alcohol sweet substances, and improve the detection efficiency and accuracy, it is urgent to establish a method for rapidly, accurately and effectively simultaneously determining multiple sugar and sugar alcohol sweet substances in liquor. SUMMARY

[0004] In order to rapidly, accurately and effectively simultaneously determine multiple sugar and sugar alcohol sweet substances in liquor, the application provides a new method for simultaneously determining sugar and sugar alcohol sweet substances in liquor.

[0005] To achieve the above application purposes, the technical solutions adopted by the application are as follows:

[0006] In a first aspect, the application provides a method for simultaneously determining sugar and sugar alcohol sweet substances in liquor, which adopts high performance liquid chromatography-tandem mass spectrometry, and the liquid chromatography conditions include:

[0007] Chromatographic column: Agilent Proshell 120-OH5 column;

[0008] Mobile phase: A is 5 mmol / L ammonium acetate solution containing 0.001% ammonium fluoride, and B is 5 mmol / L ammonium acetate 92% acetonitrile aqueous solution;

[0009] Gradient elution program 0-10.0 min, 95% B; 10.0-15.0 min, 95% B-75% B; 15.0-20.0 min, 75% B-60% B; 20.0-22.0 min, 60% B; 22.0-22.2 min, 60% B-95% B; 22.2-24.5 min, 95% B;

[0010] Column temperature: 40℃;

[0011] Flow rate: 0.25 mL / min;

[0012] Injection volume: 5 μL;

[0013] The mass spectrometry condition parameters are as follows:

[0014] Compound name Parent ion m / z Daughter ion m / z De-clustering voltage V Fragmentation voltage V Sucrose 341.1 118.9*，89.0，71.2 380 15，20，25 Ribose 149.0 88.9*，58.9 380 3，15 Xylose 149.1 89.0*，71.0，59.1 380 3，5，13 Isomaltotriose 503.0 442.9*，382.9，179.3 380 5，15，18 Palatinose 341.1 221.0*，179.0，89.2 380 5，5，18 Maltitol 343.2 179.0*，119.0，89.1 380 12，15，18 Sorbitol 181.0 89.1*，71.1，59.1 380 12，20，20 Xylitol 151.1 89.1*，71.2，59.2 380 10，15，23 Erythritol 121.1 89.1*，71.1 380 5，15 Lactitol 342.9 179.1*，88.9，59.1 380 10，18，40 Palatinose 343.1 179.0*，88.9，59.0 380 12，20，35 Mannitol 180.9 101.0*，71.1，59.2 380 12，20，18 Arabitol 150.9 101.0*，71.2，59.1 380 10，18，20 Inositol 178.9 87.1*，71.1，41.2 380 18，25，38 .

[0015] Further, the mass spectrometry condition comprises:

[0016] Ionization mode: electrospray ionization, negative ion mode (ESI-);

[0017] Mass spectrometry scanning mode: multiple reaction ion pair monitoring (MRM);

[0018] Capillary voltage: -2800 V;

[0019] Ion source temperature: 280℃;

[0020] Dry gas flow rate: 11 L / min;

[0021] Atomization gas pressure: 45 psi;

[0022] Sheath gas temperature: 350℃;

[0023] Sheath gas flow rate: 11 L / min;

[0024] Nozzle voltage: 500 V.

[0025] Further, the detection method comprises the following steps:

[0026] Fourteen sweet substances are prepared into series of standard samples with different concentrations, and the sample to be detected is extracted to obtain a test sample; then the test sample and the standard sample are detected by using high performance liquid chromatography-tandem mass spectrometry, the concentrations of the fourteen sweet substances in the test sample are obtained, and the content of each sweet substance in the test sample is calculated.

[0027] Further, the detection method specifically comprises the following steps:

[0028] (1) Preparation of mixed standard solution: 14 sweeteners were weighed and prepared into a mixed standard stock solution, which was ready for use;

[0029] (2) Preparation of series standard solution: the mixed standard solution was prepared into a series standard solution;

[0030] (3) Preparation of sample: after the liquor was diluted with water, the sweeteners were extracted completely by oscillation and ultrasonic treatment, and the supernatant obtained by centrifugal separation was the liquor sample;

[0031] (4) Qualitative detection of sample: the sample and the series standard were determined according to the conditions of high performance liquid chromatography-tandem mass spectrometry, the retention time of the sweeteners in the sample and the standard was recorded, the relative abundance of the qualitative ion pair was taken as the percentage relative to the strongest ion abundance, and the relative ion abundance of the corresponding components in the sample and the standard with the same concentration was recorded; when the chromatographic peak in the sample was detected within the range of ±2.5% of the chromatographic peak retention time of the 14 sweeteners in the standard, and the relative ion abundance deviation was within the specified range, it was determined that the sample contained the corresponding sweetener; otherwise, the sample did not contain the corresponding sweetener;

[0032] (5) Quantitative determination of sample: when the sample contained the corresponding sweetener, quantitative detection was performed;

[0033] According to the conditions of high performance liquid chromatography-tandem mass spectrometry, the series standard was determined, the quantitative ion chromatographic peak area of the 14 sweeteners in the standard was obtained, the mass concentration of the sweeteners in the standard was taken as the abscissa, and the peak area of the quantitative ion chromatographic peak was taken as the ordinate, and the standard curve of the 14 sweeteners was drawn;

[0034] According to the conditions of liquid chromatography-mass spectrometry, the sample was determined, the quantitative chromatographic peak area of the 14 sweeteners in the sample was obtained, and the concentration of the 14 sweeteners in the sample was obtained according to the standard curve drawn; the content of the sweeteners in the sample was calculated according to the following formula:

[0035]

[0036] X, the content of the sweetener in the sample, unit: mg / kg;

[0037] C, the concentration of the sweetener in the sample obtained by detection, unit: ng / mL;

[0038] V, the volume of the sample treated, unit: mL;

[0039] m, the sample amount of liquor, unit: g.

[0040] Preferably, the 14 sweet substances are sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol and inositol.

[0041] Preferably, in steps (1) and (2), a blank matrix is used to prepare a standard sample for quantitative detection; the blank matrix refers to liquor without the 14 sweet substances.

[0042] Preferably, in step (3), the amount of water added should meet the requirements of the detection limit; the detection limit of sucrose, ribose, xylose, isomaltotriose, isomaltulose and erythritol is 0.020 mg / kg, and the quantitative limit is 0.040 mg / kg; the detection limit of maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabitol and inositol is 0.010 mg / kg, and the quantitative limit is 0.020 mg / kg.

[0043] Preferably, in step (4), the maximum allowable deviation of relative ion abundance is not more than the following specified range: relative ion abundance > 50%, maximum allowable deviation ± 20%; relative ion abundance 20-50%, maximum allowable deviation ± 25%; relative ion abundance 10-20%, maximum allowable deviation ± 30%; relative ion abundance ≤ 10%, maximum allowable deviation ± 50%.

[0044] Preferably, in step (5), the response value of the measured substance in the sample should be within the linear range of the instrument; if the concentration of the measured substance in the sample exceeds the range of the standard curve, the sample should be diluted and then measured.

[0045] Beneficial effects:

[0046] 1. The present application uses high performance liquid chromatography-tandem mass spectrometry to achieve the simultaneous determination of 14 sweet substances, i.e., sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol and inositol in liquor by limiting chromatographic and mass spectrometric determination conditions, and the method of the present application fills the technical gap in the field of liquor for the simultaneous determination of the 14 sweet substances.

[0047] 2. The present application simultaneously optimizes the extraction treatment and separation conditions, and the qualitative and quantitative methods, thereby providing technical support for cracking down on the behavior of using sweet substances beyond the scope in liquor.

[0048] 3. Using the method of the present application, one set of instrument, one reagent system and one operation can simultaneously obtain the content of 14 sweet substances, which can reduce the testing workload, save analysis reagents and effectively reduce the analysis cost.

[0049] 4. The method has good accuracy and precision, and the experimental results show that the recovery rate of the determined white spirit blank sample at different levels is 86.4% to 107.6%, and the precision is less than 10%, which can completely meet the detection requirements. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is the total ion chromatogram of the standard sample in the blank matrix in Example 1, and the concentrations of sucrose, ribose, xylose, isomaltotriose, isomaltulose, erythritol are all 100 ng / mL, and the concentrations of maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabitol, inositol are all 50 ng / mL.

[0051] Figure 2 is the total ion chromatogram of the sample prepared by adding standard to the blank white spirit in Example 1: the concentrations of sucrose, ribose, xylose, isomaltotriose, isomaltulose, erythritol are all 100 ng / mL, and the concentrations of maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabitol, inositol are all 50 ng / mL.

[0052] Figure 3 is the sucrose content distribution diagram obtained by detecting the white spirit in Example 1.

[0053] Figure 4 is the ribose content distribution diagram obtained by detecting the white spirit in Example 1.

[0054] Figure 5 is the xylose content distribution diagram obtained by detecting the white spirit in Example 1.

[0055] Figure 6 is the isomaltotriose content distribution diagram obtained by detecting the white spirit in Example 1.

[0056] Figure 7 is the isomaltulose content distribution diagram obtained by detecting the white spirit in Example 1.

[0057] Figure 8 is the maltitol content distribution diagram obtained by detecting the white spirit in Example 1.

[0058] Figure 9 is the sorbitol content distribution diagram obtained by detecting the white spirit in Example 1.

[0059] Figure 10 is the xylitol content distribution diagram obtained by detecting the white spirit in Example 1.

[0060] Figure 11 is the erythritol content distribution diagram obtained by detecting the white spirit in Example 1.

[0061] Figure 12 is a lactitol content distribution chart obtained from the detection of the white liquor in Example 1.

[0062] Figure 13 is an isomaltitol content distribution chart obtained from the detection of the white liquor in Example 1.

[0063] Figure 14 is a mannitol content distribution chart obtained from the detection of the white liquor in Example 1.

[0064] Figure 15 is an arabitol content distribution chart obtained from the detection of the white liquor in Example 1.

[0065] Figure 16 is an inositol content distribution chart obtained from the detection of the white liquor in Example 1.

[0066] Figure 17 is a total content distribution chart of sweet substances (7 kinds) obtained from the detection of the white liquor of Enterprise 1 in Example 1.

[0067] Figure 18 is a sorbitol content distribution chart obtained from the detection of the white liquor of Enterprise 1 in Example 1.

[0068] Figure 19 is a sweet substance (sorbitol) content distribution chart obtained from the detection of the white liquor of Enterprise 2 in Example 1.

[0069] Figure 20 is a sweet substance (sorbitol) content distribution chart obtained from the detection of the white liquor of Enterprise 3 in Example 1.

[0070] Figure 21 is a sweet substance (sorbitol) content distribution chart obtained from the detection of the white liquor of Enterprise 4 in Example 1.

[0071] Figure 22 is a total content distribution chart of sweet substances (5 kinds) obtained from the detection of the white liquor of Enterprise 5 in Example 1.

[0072] Figure 23 is a sweet substance (sorbitol) content distribution chart obtained from the detection of the white liquor of Enterprise 5 in Example 1.

[0073] Figure 24 is a sweet substance (sorbitol) content distribution chart obtained from the detection of the white liquor of Enterprise 6 in Example 1. DETAILED DESCRIPTION

[0074] The following will list specific embodiments to explain the scheme of the present application. Those skilled in the art will understand that the following examples are only for illustration of the present application, and should not be regarded as limiting the scope of the present application. The specific techniques or conditions not noted in the examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not noted by the manufacturer are all conventional products that can be obtained by purchase.

[0075] In the following examples, 5 sugars: sucrose, ribose, xylose, isomaltotriose, isomaltulose, 8 sugar alcohols: maltitol, sorbitol, xylitol, erythritol, lactitol, isomalt, mannitol, arabinitol, and 1 cyclic alcohol: inositol standard are commercially available; other chemical reagents are chromatographically pure, and are commercially available.

[0076] Example 1

[0077] In this example, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is used to simultaneously determine sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabinitol, and inositol in Baijiu (liquor).

[0078] I. Determination of blank Baijiu

[0079] The blank Baijiu does not contain the above-mentioned 14 sugars or sugar alcohols, i.e., does not contain sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabinitol, and inositol, and is used for verification of the method described in the present application. In this example, the blank Baijiu is commercially available and is detected by liquid chromatography-tandem mass spectrometry to confirm that it does not contain the above-mentioned 14 substances.

[0080] 1. Preparation of standards

[0081] (1) Preparation of mixed standard solution

[0082] Take 200 μL of sucrose, ribose, xylose, isomaltotriose, isomaltulose, erythritol standard solution with concentration of 10.0 mg / mL, 100 μL of maltitol, sorbitol, xylitol, lactitol, isomalt, mannitol, arabitol, inositol standard solution into the same 10 mL volumetric flask, dilute to the mark with water, shake well, and then use as mixed standard solution A, the concentration of 5 kinds of sugar and erythritol is 200 μg / mL, and the concentration of 8 kinds of sugar alcohol is 100 μg / mL; accurately take 100 μL of mixed standard solution A into a 10 mL volumetric flask, dilute to the mark with water, shake well, and then use as mixed standard solution B, the concentration of 5 kinds of sugar and erythritol is 2.0 μg / mL, and the concentration of 8 kinds of sugar alcohol is 1.0 μg / mL, and then store in a brown stock solution bottle at 4 ℃ for standby.

[0083] (2) Preparation of series of standard samples

[0084] Take 0.002 mL, 0.005 mL of mixed standard solution A and 0.01 mL, 0.02 mL, 0.05 mL, 0.1 mL of mixed standard solution B into a 1 mL volumetric flask, dilute to the mark with a blank matrix, shake well, and then obtain six series of standard sample solutions containing 14 kinds of sweet substances, the concentration of sucrose, ribose, xylose, isomaltotriose, isomaltulose, and erythritol is 20.0 μg / L, 40.0 μg / L, 100.0 μg / L, 200.0 μg / L, 400.0 μg / L, and 1000.0 μg / L; the concentration of maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabitol, and inositol is 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 500.0 μg / L.

[0085] 2. Preparation of samples

[0086] (1) Extraction and treatment of the measured liquor

[0087] Take 2.5 g of mixed liquor into a 5 mL volumetric flask, dilute to the mark with water, mix well, transfer to a centrifugal tube, and then extract and treat the sweet substances by means of 33 / 40 KHz dual-frequency ultrasonic wave for 5 min, and then centrifuge at 12000 r / min for 5 min, and then the supernatant is the liquor sample.

[0088] (2) Preparation of blank matrix solution

[0089] Take the liquor which is confirmed by liquid chromatography-tandem mass spectrometry to be free of 14 kinds of sweet substances, and then treat it by the same method as in "(1) Extraction and treatment of the measured liquor", and then obtain the blank matrix solution.

[0090] 3. Instrument conditions

[0091] (1) Liquid phase condition

[0092] Column: Agilent Proshell 120-OH5 column (2.1 mm x 150 mm, 2.7 μm); mobile phase: A was 5 mmol / L ammonium acetate solution (containing 0.001% ammonium fluoride), B was 5 mmol / L ammonium acetate solution in 92% acetonitrile, gradient elution program 0-10.0 min, 95% B; 10.0-15.0 min, 95% B-75% B; 15.0-20.0 min, 75% B-60% B; 20.0-22.0 min, 60% B; 22.0-22.2 min, 60% B-95% B; 22.2-24.5 min, 95% B; column temperature: 40 °C; flow rate: 0.25 mL / min; injection volume: 5 μL.

[0093] (2) Mass spectrometry condition

[0094] Ionization mode: electrospray ionization, negative ion mode (ESI - ); mass spectrometry scanning mode: multiple reaction ion pair monitoring (MRM); capillary voltage: -2800 V; ion source temperature: 280 °C; drying gas flow rate: 11 L / min; atomizing gas pressure: 45 psi; sheath gas temperature: 350 °C; sheath gas flow rate: 11 L / min; nozzle voltage: 500 V; the optimized mass spectrometry parameters are shown in Table 1.

[0095] Table 1. Main mass spectrometry condition parameters of compounds

[0096] Compound name Parent ion m / z Daughter ion m / z De-clustering voltage V Fragmentation voltage V Sucrose 341.1 118.9*，89.0，71.2 380 15，20，25 Ribose 149.0 88.9*，58.9 380 3，15 Xylose 149.1 89.0*，71.0，59.1 380 3，5，13 Isomaltotriose 503.0 442.9*，382.9，179.3 380 5，15，18 Palatinose 341.1 221.0*，179.0，89.2 380 5，5，18 Maltitol 343.2 179.0*，119.0，89.1 380 12，15，18 Sorbitol 181.0 89.1*，71.1，59.1 380 12，20，20 Xylitol 151.1 89.1*，71.2，59.2 380 10，15，23 Erythritol 121.1 89.1*，71.1 380 5，15 Lactitol 342.9 179.1*，88.9，59.1 380 10，18，40 Palatinose 343.1 179.0*，88.9，59.0 380 12，20，35 Mannitol 180.9 101.0*，71.1，59.2 380 12，20，18 Arabitol 150.9 101.0*，71.2，59.1 380 10，18，20 Inositol 178.9 87.1*，71.1，41.2 380 18，25，38

[0097] Note: * indicates quantitative ions, the parameters may be different for different mass spectrometry instruments, and the mass spectrometry parameters should be optimized to the best before determination

[0098] 4. Qualitative determination

[0099] According to the high performance liquid chromatography-tandem mass spectrometry condition, the sample and series of standard samples are determined, the chromatographic retention time of the sweet substances in the sample and the standard sample is recorded, the relative abundance of the qualitative ion pair is taken as the percentage relative to the strongest ion abundance, and the relative ion abundance of the corresponding components in the sample and the standard sample with the same concentration is recorded; when the chromatographic peak of the sample detected in the sample and the chromatographic peak of sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol, inositol in the standard sample are within the range of ±2.5% in the variation range of the chromatographic peak retention time, and the relative ion abundance is allowed to deviate from the range specified in Table 2, it is determined that the sample contains the corresponding sweet substance. Otherwise, the sample does not contain the corresponding sweet substance.

[0100] Table 2 Qualitative allowable deviation range of relative abundance of ions in test sample

[0101] Relative ion abundance (%) ＞50 20～50 10～20 ≤10 Maximum allowed deviation (%) ±20 ±25 ±30 ±50

[0102] Since the measured liquor in the example is blank liquor, the test sample does not contain 14 sweet substances.

[0103] 5. Quantitative determination

[0104] (1) Preparation of standard curve

[0105] According to the high performance liquid chromatography-tandem mass spectrometry conditions, a series of 14 sweet substance standards were determined, and the quantitative ion chromatographic peak areas of sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol, and inositol in each standard were obtained. The mass concentration of the sweet substance in the standard was taken as the abscissa, and the peak area of the quantitative ion chromatographic peak was taken as the ordinate. The standard curves of sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabitol, and inositol were drawn. The linear equation and the correlation coefficient (r) obtained from the standard curve are shown in Table 3.

[0106] Table 3 Linear equation and linear range of 14 compounds in blank matrix

[0107] Name Concentration range ng / mL Linear equation r Sucrose 20.0～1000 y = 171.523444x - 64.128211 0.9996 Ribose 20.0～1000 y = 12.852198x + 0.525713 0.9998 Xylose 20.0～1000 y = 16.351309x + 18.506835 0.9993 Isomaltotriose 20.0～1000 y = 1.995961x + 3.527789 1.0000 Palatinose 20.0～1000 y = 5.039273x - 3.629120 0.9998 Erythritol 20.0～1000 y = 20.395343x + 19.397040 0.9999 Maltitol 10.0～500 y = 36.235379x - 46.499939 1.0000 Sorbitol 10.0～500 y = 90.993646x - 235.850537 1.0000 Xylitol 10.0～500 y = 302.076470x - 394.259569 0.9998 Lactitol 10.0～500 y = 14.448383x + 16.048133 0.9996 Palatinose 10.0～500 y = 24.627153x - 21.189264 0.9999 Mannitol 10.0～500 y = 86.659885x - 107.815573 0.9997 Arabitol 10.0～500 y = 216.399949x - 347.441214 0.9999 Inositol 10.0～500 y = 653.027142x + 156.738096 0.9998

[0108] (2) Determination of test sample solution

[0109] According to the high performance liquid chromatography-tandem mass spectrometry conditions, the test sample was determined, and the chromatographic peak area of the 14 sweet substances in the test sample was 0. The determination concentration of the 14 sweet substances in the liquor of this example was 0 ng / mL.

[0110] 6. Calculation of sweet substance content

[0111] The content of the sweet substance was calculated using the following formula:

[0112]

[0113] X, the content of the sweet substance in the test sample, in mg / kg;

[0114] C, the concentration of the sweet substance in the test sample obtained by detection, in ng / mL;

[0115] V, the volume of the sample solution, in mL;

[0116] m, the sample amount of liquor, in g.

[0117] In the detection of blank baijiu, the sample was diluted to 5 mL, and the baijiu sample was 2.5 g. However, the concentrations of the 14 sweet substances in the sample were 0 ng / mL, and the calculated contents of sucrose, ribose, xylose, isomaltotriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulose, mannitol, arabinose, and inositol in the detected baijiu were all 0 mg / kg.

[0118] 7. Method validation

[0119] (1) Matrix effect

[0120] Since the mass spectrometric response is easily disturbed by the sample matrix, the relative response value method was used to investigate the matrix effect (ME) in baijiu during the experiment. The matrix effect was evaluated by calculating the slope ratio of the matrix matching standard curve and the pure solvent standard curve. The pure solvent standard curve represents a series of standards composed of mixtures of 14 sweet substances with different concentrations prepared by ethanol water (ethanol: water = 25:75, by volume), which were detected according to the high-performance liquid chromatography-tandem mass spectrometry conditions (the linear equation and correlation coefficient are shown in Table 4). The matrix matching standard curve represents a series of standards composed of mixtures of 14 sweet substances with different concentrations prepared by the blank matrix solution obtained by extracting and processing the blank baijiu according to the above method, which were detected according to the high-performance liquid chromatography-tandem mass spectrometry conditions. Generally, when the ME value is between 80% and 120%, it indicates that the matrix effect is within an acceptable range, and the matrix effect can be ignored in actual detection. Otherwise, the influence of the matrix effect on actual detection should be considered. The ME of the 14 sweet substances in baijiu is shown in Table 4. The results show that erythritol has a strong matrix enhancement effect, xylose and ribose have a weak matrix inhibition effect, and the matrix effects of the remaining 11 sweet substances can be ignored. In actual detection, blank matrix should be used to prepare standards for quantitative detection.

[0121] Table 4 Linear relationship of 14 sweet substances in solvent and matrix effect in blank baijiu

[0122]

[0123]

[0124] (2) Linear range and correlation coefficient

[0125] According to the response of 14 sweet substances on high performance liquid chromatography-tandem mass spectrometer, the 14 sweet substance series standards were prepared with blank matrix solution, under the optimized chromatographic conditions, the peak area (y) was plotted against the corresponding mass concentration (x), the standard curve was drawn, and the linear regression equation and the correlation coefficient of each measured sweet substance were calculated. Table 3 shows that the linear relationship is good when the concentration of sucrose, ribose, xylose, isomaltotriose, isomaltulose, erythritol is in the range of 20.0-1000.0 ng / mL, the concentration of maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabinol, inositol is in the range of 10.0-500.0 ng / mL, and the correlation coefficient r is all greater than 0.999.

[0126] (3) Recovery and precision

[0127] According to the determination steps 1-6 of the blank white wine, the blank white wine sample without the measured sweet substance was added with 14 sweet substance standards for standard addition recovery test, 6 samples were prepared at different addition levels, the accuracy and precision of the method were investigated, and the results are shown in Table 5. The average recovery rate of each addition level of the 14 sweet substances is between 86.4% and 107.6%, the precision is less than 10%, the accuracy and precision of the method meet the requirements of quantitative analysis. In addition, 3 samples at the limit of quantification level were taken from the blank white wine sample, and were determined at 0d, 2d, 4d, and 7d, respectively, and the recovery rate was used to express. As shown in Table 6, the 14 sweet substances at the limit of quantification level are still relatively stable after the white wine sample is placed for 7 days, and the content change is less than 10%, and the inter-day precision is between 1.6% and 8.3%.

[0128] Table 5 Recovery and precision results of 14 sweet substances (n=6)

[0129]

[0130]

[0131] Table 6 Inter-day precision results of 14 sweet substances (n=5)

[0132]

[0133] (4) Limit of detection and limit of quantification

[0134] The limits of detection (LOD) were determined by adding spikes to blank spirits and diluting them stepwise. Spiked samples were obtained using the same extraction method as the blank spirits, and detection was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The LOD and LOQ were determined based on the spiked levels corresponding to the signal-to-noise ratios (S / N) of the characteristic chromatographic peaks for each sweet substance (S / N = 3 and S / N = 10). The LOD for sucrose, ribose, xylose, isomaltriosanol, isomaltulose, and erythritol was ultimately determined to be 0.020 mg / kg, and the LOQ was 0.040 mg / kg. The LOD for maltitol, sorbitol, xylitol, lactitol, isomaltulose, mannitol, arabinitol, and inositol was determined to be 0.010 mg / kg, and the LOQ was 0.020 mg / kg.

[0135] II. Actual Baijiu Testing

[0136] The actual baijiu testing employed the same method as the blank baijiu testing, measuring 640 batches of pre-packaged baijiu purchased. This included 300 batches of strong-aroma baijiu, 142 batches of sauce-aroma baijiu, 78 batches of light-aroma baijiu, and 120 batches of other aroma types such as Lao Bai Gan, Feng Xiang, and mixed aroma. At least one sample of 2.5g was taken from each batch. All 14 sweet substances were detected, with sorbitol showing the highest detection rate, followed by mannitol and erythritol. Specific detection details of sweet substances in the actual baijiu are shown in Tables 7 and 8. Figure 3 to Figure 16 The test results show that the detection rate of sweet substances in commercially available baijiu is similar across different aroma types, but the total content of sweet substances varies considerably. The types and amounts of sweet substances detected in different aroma types of baijiu products also differ significantly. This may be related to the differences in the types and quality of yeast, raw grains, and production processes used in different baijiu products.

[0137] Table 7. Detection and content range of sweet substances in Baijiu (Chinese liquor)

[0138]

[0139] Table 8. Detection of sweet substances in different types of Baijiu

[0140]

[0141] III. Comparison of sweet substances detected in baijiu from different companies

[0142] Using the same method as for the blank baijiu samples, 156 batches of pre-packaged baijiu products from 6 distilleries (confirmed not to have added sweeteners during production) were tested. At least one sample of 2.5g was taken from each batch. Sweeteners were detected in all 156 batches of baijiu samples. The results are shown in Table 9. Figure 17 to Figure 24From Table 9, it can be seen that 7 sweet substances were detected in the liquor products of 6 enterprises, except for xylose detected in the product of one enterprise, the rest were sugar alcohol sweet substances, sorbitol was detected in the liquor products of 6 enterprises, and multiple sweet substances were detected in the products of two enterprises. From the detection results of Table 9, it can be seen that the same enterprise has basically the same type of sweet substances detected, but the content has certain differences; different enterprises have certain differences in the number, type and content of sweet substances detected. Figure 17 to Figure 24 It can be seen that the same enterprise has basically the same type of sweet substances detected, but the content has certain differences; different enterprises have certain differences in the number, type and content of sweet substances detected.

[0143] Table 9 Detection of sweet substances in liquor of different enterprises

[0144]

[0145] Four, comparison of sweet substances detected in different series of liquor of the same enterprise

[0146] The same method as the determination of the blank liquor sample was used to determine 69 batches of pre-packaged liquor products (different series) from one liquor enterprise (Enterprise 1), and at least one sample was taken from each batch of liquor during determination, and the sample weight was 2.5 g. Sweet substances were detected in 69 batches of liquor samples, and the detection results are shown in Table 10. It can be seen that the same type of compounds was detected in the different series of products of the enterprise, but the content had certain differences, and the content of sorbitol in the 5 series of liquor products was basically consistent.

[0147] Table 10 Comparison of sweet substances detected in different series of liquor of the same enterprise

[0148]

Claims

1. A method for simultaneously determining sugars and sugar alcohols (sweet substances) in baijiu (Chinese liquor), characterized in that, High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used, and the HPLC conditions included: Column: Agilent Proshell 120-OH5 column; Mobile phase: A is a 5 mmol / L ammonium acetate solution containing 0.001% ammonium fluoride, and B is a 5 mmol / L 92% acetonitrile aqueous solution of ammonium acetate; Gradient elution program: 0–10.0 min, 95% B; 10.0–15.0 min, 95% B–75% B; 15.0–20.0 min, 75% B–60% B; 20.0–22.0 min, 60% B; 22.0–22.2 min, 60% B–95% B; 22.2–24.5 min, 95% B; Column temperature: 40℃; Flow rate: 0.25 mL / min; Injection volume: 5 μL; The mass spectrometry conditions are as follows: 。 2. The method according to claim 1, characterized in that, The mass spectrometry conditions include: Ionization modes: electrospray ionization, negative ion mode; Mass spectrometry scanning mode: Multiple reaction pair monitoring; Capillary voltage: -2800V; Ion source temperature: 280℃; Drying gas flow rate: 11 L / min; Nebulizer gas pressure: 45 psi; Sheath gas temperature: 350℃; Sheath gas flow rate: 11 L / min; Nozzle voltage: 500V.

3. The method according to claim 1 or 2, characterized in that, The detection method includes the following steps: Fourteen sweet substances were prepared into a series of standard samples with different concentrations. The liquor to be tested was extracted to obtain the sample. Then, high performance liquid chromatography-tandem mass spectrometry was used to detect the sample and the standard samples to obtain the concentration of the 14 sweet substances in the sample. The content of each sweet substance in the sample was then calculated.

4. The method according to claim 3, characterized in that, The detection method specifically includes the following steps: (1) Preparation of mixed standard solution: Weigh 14 kinds of sweet substances to prepare mixed standard stock solution for later use; the 14 kinds of sweet substances are sucrose, ribose, xylose, isomalttriose, isomaltulose, maltitol, sorbitol, xylitol, erythritol, lactitol, isomaltulitol, mannitol, arabinitol and inositol; (2) Preparation of a series of standard solutions: Take the mixed standard solution and prepare a series of standard solutions; (3) Sample preparation: After diluting the liquor with water, mix it by shaking, then extract the sweet substances to be tested completely by ultrasound, and then extract the supernatant by centrifugation to obtain the liquor sample. (4) Qualitative detection of samples: The samples and a series of standards were determined according to the high performance liquid chromatography-tandem mass spectrometry conditions. The chromatographic retention time of sweet substances in the samples and standards was recorded. The percentage relative to the abundance of the strongest ion was used as the relative abundance of the qualitative ion pair. The relative ion abundance of the corresponding components in the samples and standards with equivalent concentrations was recorded. When a chromatographic peak with a retention time variation of ±2.5% with 14 sweet substances in a certain standard was detected in the sample, and the allowable deviation of the relative ion abundance did not exceed the specified range, it was determined that the sample contained the corresponding sweet substance. Otherwise, the sample did not contain the corresponding sweet substance. (5) Quantitative determination of samples: After determining that the sample contains the corresponding sweet substances, quantitative detection is performed; According to the high performance liquid chromatography-tandem mass spectrometry conditions, a series of standard samples were measured to obtain the quantitative ion chromatographic peak areas of 14 sweet substances in each standard sample. The standard curves of the 14 sweet substances were plotted with the mass concentration of sweet substances in the standard sample as the abscissa and the peak area of ​​the quantitative ion chromatographic peak as the ordinate. The sample was analyzed using liquid chromatography-mass spectrometry (LC-MS) to obtain the quantitative chromatographic peak areas of 14 sweet substances in the sample. The concentrations of these 14 sweet substances were then determined based on the plotted standard curve. The content of the sweet substances in the sample was calculated using the following formula: X represents the content of sweet substances in the sample, expressed in mg / kg. C, the concentration of sweet substances in the sample obtained by detection, in ng / mL; V, the volume to be brought to a fixed volume during sample treatment, in mL; m represents the sample size of the liquor, expressed in grams.

5. The method according to claim 4, characterized in that, In steps (1) and (2), a blank matrix is ​​used to prepare a standard sample for quantitative detection; the blank matrix refers to a type of liquor that does not contain 14 kinds of sweet substances.

6. The method according to claim 4, characterized in that, In step (3), the amount of water added should meet the requirements of the detection limit. The detection limit for sucrose, ribose, xylose, isomalttriose, isomaltulose, and erythritol is 0.020 mg / kg, and the quantitation limit is 0.040 mg / kg. The detection limit for maltitol, sorbitol, xylitol, lactitol, isomaltulitol, mannitol, arabinitol, and inositol is 0.010 mg / kg, and the quantitation limit is 0.020 mg / kg.

7. The method according to claim 4, characterized in that, In step (4), the allowable deviation of relative ion abundance shall not exceed the following ranges: relative ion abundance > 50%, maximum allowable deviation ±20%; relative ion abundance 20-50%, maximum allowable deviation ±25%; relative ion abundance 10-20%, maximum allowable deviation ±30%; relative ion abundance ≤ 10%, maximum allowable deviation ±50%.

8. The method according to claim 4, characterized in that, In step (5), the response values ​​of the analyte in the sample should all be within the linear range of the instrument. If the concentration of the analyte in the sample exceeds the range of the standard curve, the sample should be diluted before measurement.

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