A method for simultaneously detecting multiple sugars and sugar alcohols

Abstract

The present application discloses a method for simultaneously detecting multiple sugars and sugar alcohols, comprising the following steps: 1) preparing a sample solution to be detected; 2) using ultra-high performance liquid chromatography-mass spectrometry to qualitatively or quantitatively detect sugars and sugar alcohols in the sample solution to be detected; wherein, the chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry are: chromatographic column: Waters Acquity BEH Amide 2.1×100mm; column temperature: 10°C; flow rate: 0.2-0.5 mL / min; injection volume: 0.1-1 μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile, and gradient elution is performed. The present application can simultaneously detect ten sugars and sugar alcohols, and can also simultaneously detect isomers of multiple sugars, with very high sensitivity.

Description

Technical Field

[0001] This application relates to the technical field of analytical detection, and particularly relates to a method for simultaneously detecting multiple sugars and sugar alcohols. Background Art

[0002] Sugar is a sweet substance for consumption, mainly extracted from substances such as rice, wheat, sugarcane, and beet, and can be divided into monosaccharides, disaccharides, and polysaccharides. Common monosaccharides include glucose, fructose, galactose, etc., and disaccharides include maltose, lactose, sucrose, etc. Sugar alcohol is a polyol that can be prepared from the corresponding sugar, has certain properties of sugar but lower calories. Common sugar alcohols include sorbitol, erythritol, maltitol, xylitol, etc. Sugars and sugar alcohols have a wide range of applications in the food industry, such as being used as sweeteners, humectants, preservatives, etc. Currently, the common methods for detecting sugars mainly include acid hydrolysis-Rhine-Eynon method, Rhine-Eynon method, ion chromatography, and high-performance liquid chromatography. The common method for detecting sugar alcohols is high-performance liquid chromatography. The current mainstream quantitative detection methods for sugars and sugar alcohols are ion chromatography and high-performance liquid chromatography respectively, and there are mainly three problems as follows: Chromatography only relies on retention time for qualitative analysis, and has relatively weak anti-interference ability, and is prone to false positives when encountering co-eluting impurities; it is unable to simultaneously detect sugars and sugar alcohols, and the same sample needs to be detected twice, resulting in low detection efficiency; the method sensitivity is not high. Moreover, currently there is no method that can simultaneously detect nine common sugars and sugar alcohols.

[0003] Therefore, there is an urgent need to develop a highly sensitive method for simultaneously detecting multiple sugars and sugar alcohols. Summary of the Invention

[0004] The purpose of this application is to provide a method for simultaneously detecting sugars and sugar alcohols. This method can simultaneously detect 6 sugars and 4 sugar alcohols, and can also simultaneously detect isomers of multiple sugars, and has very high sensitivity.

[0005] This application provides a method for simultaneously detecting sugars and sugar alcohols, including the following steps:

[0006] 1) Prepare a sample solution to be detected;

[0007] 2) Use ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to qualitatively or quantitatively detect sugars and sugar alcohols in the sample solution to be detected;

[0008] Among them, the chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry method are: chromatographic column: Waters Acquity BEH Amide 2.1×100 mm; column temperature: 10°C; flow rate: 0.2 - 0.5 mL / min; injection volume: 0.1 - 1 μL; mobile phase: phase A is 0.1% formic acid in water, phase B is acetonitrile, and gradient elution is used.

[0009] Optionally, the sugar is one or more of fructose, glucose, sucrose, maltose, lactose, and galactose.

[0010] Optionally, the sugar alcohol is one or more of xylitol, sorbitol, maltitol, and erythritol.

[0011] Optionally, before step 1), the sample solution is pretreated with a treating agent, where the treating agent consists of a zinc acetate solution and a potassium ferrocyanide solution or the treating agent is n-hexane.

[0012] Optionally, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry method are as follows: ion source: ESI; scanning mode: negative mode; scanning pattern: MRM; drying gas temperature: 300 °C; drying gas flow rate: 9 L / min; sheath gas temperature: 350 °C; sheath gas flow rate: 12 L / min.

[0013] Optionally, the parent ion-daughter ion pairs for mass spectrometry analysis in the quantitative detection are: erythritol, m / z 121.1→89.1; fructose, m / z 179.1→89.1; galactose, m / z 179.1→89.1; glucose, m / z 179.1→89.1; lactose, m / z 387.2→341.2; maltitol, m / z 343.1→179.1; maltose, m / z 387.2→341.2; sorbitol, m / z181.1→89.1; sucrose, m / z 387.2→341.2; xylitol, m / z 151.1→89.1.

[0014] Optionally, the parent ion-daughter ion pairs for mass spectrometry analysis in the qualitative detection are: erythritol, m / z 121.1→100.9, 121.1→71.2; fructose, m / z 179.1→59.1, 179.1→71.2; galactose, m / z 179.1→71.1, 179.1→59.1; glucose, m / z 179.1→59.1, 179.1→119; lactose, m / z 387.2→179.1, 387.2→161.1; maltitol, m / z 343.1→89.1, 343.1→59.1; maltose, m / z 387.2→221.1, 387.2→161.1; sorbitol, m / z 181.1→101.1, 181.1→71.2; sucrose, m / z 387.2→179.1, 387.2→89.1; xylitol, m / z 151.1→71.2, 151.1→101.1.

[0015] Optionally, the gradient elution program is as follows:

[0016] Time Mobile phase A

[0017] 0 - 0.5 min, 10%A;

[0018] 0.5 - 21.6 min, 10%A - 30%A;

[0019] 21.6 - 25.6 min, 30%A;

[0020] 25.6 - 25.7 min, 10%A.

[0021] Optionally, the quantitative detection is external standard method quantitative detection.

[0022] Optionally, the external standard method quantitative detection includes separately preparing standard solutions of sugars and sugar alcohols, preparing a series of concentration standard samples from the standard solutions, measuring the peak areas of the standard samples by ultra - performance liquid chromatography - tandem mass spectrometry, performing linear regression of the peak areas against the concentrations, separately establishing standard curves for sugars and sugar alcohols, and calculating the contents of sugars and sugar alcohols in the sample solution therefrom.

[0023] In summary, the present application includes at least one of the following beneficial technical effects:

[0024] 1. The method of the present application has high sensitivity, can not only be applicable to the simultaneous detection of multiple sugars and sugar alcohols in various foods, filling the gap in the prior art, but also be applicable to the detection of environmental samples and biological samples, etc.;

[0025] 2. Based on the qualitative analysis of chromatographic retention time, the method of the present application adds the confirmation of the qualitative ion ratio, improving the accuracy of qualitative analysis and greatly reducing the generation of false positives;

[0026] 3. The method of the present application can simultaneously detect nine common sugars and sugar alcohols without the need for two separate operations, and can also simultaneously detect galactose;

[0027] 4. The method of the present application can detect multiple sugar isomers and can provide the ratio information of isomers when the isomers need to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the MRM spectrogram of each sugar alcohol;

[0029] Figure 2 It is the MRM spectrogram of each sugar;

[0030] Figure 3 It is the MRM spectrogram of each sugar;

[0031] Figure 4 It is the MRM spectrogram of the superposition of the quantitative ion pairs and qualitative ion pairs of α - lactose (left) and β - lactose (right);

[0032] Figure 5 MRM spectrogram of the superposition of the quantitative ion pairs and qualitative ion pairs of α-maltose (left) and β-maltose (right);

[0033] Figure 6 Spectrogram under Comparative Method 1;

[0034] Figure 7 Spectrogram under Comparative Method 2;

[0035] Figure 8 Spectrogram under Example 1. Detailed implementation mode

[0036] For a clearer understanding of the technical features, objectives, and beneficial effects of this application, the technical solutions of the present invention will be described in detail below in combination with the following specific examples and the accompanying drawings of the specification. However, it should not be construed as a limitation on the scope of implementation of the present invention.

[0037] Example 1 Detection method

[0038] 1.1 Detection steps

[0039] Take 1 g of the sample in a 50 mL centrifuge tube, add 20 mL of pure water and vortex to mix evenly. If the dissolution is slow, the water temperature can be appropriately increased. For insoluble samples, homogenization extraction can be used, and after extraction, centrifuge to remove insoluble substances. For samples with a high protein content or samples that are turbid after extraction, add 5 mL of 1 mol / L zinc acetate solution (obtained by mixing 21.9 g of zinc acetate with 3 mL of acetic acid and diluting to 100 mL with pure water) and 5 mL of 0.25 mol / L potassium ferrocyanide solution (obtained by diluting 10.6 g of potassium ferrocyanide to 100 mL with pure water) to remove proteins; for samples with a high oil content, add 20 mL of n-hexane to remove oils.

[0040] Transfer the extract to a 100 mL volumetric flask and make up to the scale line with acetonitrile + water = 50:50. Pipette the above solution, dilute it by a certain multiple, filter through a membrane and then detect by UPLC-MS / MS.

[0041] 1.2 Instrument conditions

[0042] Chromatographic conditions:

[0043] Chromatographic column: Waters ACQUITY BEH Amide 1.7um, 2.1x100 mm

[0044] Mobile phase: Phase A is 0.1% formic acid in water, and Phase B is acetonitrile

[0045] Flow rate: 0.300 mL / min

[0046] Column temperature: 10 °C

[0047] Sample injection volume: 1 μL

[0048] The specific gradient elution conditions are shown in Table 1:

[0049] Table 1

[0050]

[0051] Mass spectrometry conditions:

[0052] Ion source: ESI

[0053] Scanning mode: negative mode

[0054] Scanning pattern: MRM

[0055] Drying gas temperature: 300 °C

[0056] Drying gas flow rate: 9 L / min

[0057] Sheath gas temperature: 350 °C

[0058] Sheath gas flow rate: 12 L / min

[0059] The specific quantitative and qualitative parent ion-daughter ion pairs are shown in Table 2:

[0060] Table 2

[0061]

[0062] 1.3 Qualitative analysis

[0063] Positive judgment criteria: When comparing the retention time of the target sugar or sugar alcohol in the sample with the retention time of the corresponding standard chromatographic peak, the relative error should be within ±2.5%, and the qualitative ion abundance ratio should meet the requirements of Table 3.

[0064] Table 3

[0065] Relative abundance % Allowable deviation % >50 ±20 >20~50 ±25 >10~20 ±30 ≤10 ±50

[0066] 1.4 Quantitative analysis

[0067] Select at least five concentration points, plot a standard curve with response and concentration, and use the external standard method for quantification:

[0068]

[0069] In the formula:

[0070] X—the content of sugar or sugar alcohol in the sample, in grams per 100 grams (g / 100g);

[0071] c—the concentration of the sample solution obtained from the standard curve, in micrograms per milliliter (μg / mL);

[0072] f—dilution factor;

[0073] V—the volume of the sample solution for constant volume, in milliliters (mL);

[0074] m—the mass of the sample, in grams (g);

[0075] 10000—unit conversion factor.

[0076] Example 2 Methodology Validation

[0077] 2.1 Linear Range

[0078] The linear range of the method of this application is shown in Table 4. For sugars with isomers, the concentration is calculated by adding the peak areas.

[0079] Table 4

[0080]

[0081] 2.2 Accuracy and Precision

[0082] A three-level six-parallel spiking test was carried out on blank beverage samples. The spiking concentrations and detection results of all sugars and sugar alcohols are shown in Table 5:

[0083] Table 5

[0084] Spiked concentration (g / 100g) Recovery rate range (%) Relative standard deviation (%) 0.01 79-113 9.83 0.05 85-105 6.52 0.25 91-102 4.11

[0085] 2.3 Sensitivity

[0086] The quantitative limits of all sugars and sugar alcohols are shown in Table 6. The signal-to-noise ratio at this quantitative limit meets the requirement of >10.

[0087] Table 6

[0088] Item Quantification limit (μg / mL) Signal-to-noise ratio Erythritol 0.5 19.5 Fructose 0.05 20.1 Galactose 0.05 23.5 Glucose 0.05 31.0 Lactose 0.01 85.3 Maltitol 0.005 67.3 Maltose 0.05 24.3 Sorbitol 0.05 42.1 Sucrose 0.001 100.3 Xylitol 0.05 37.1

[0089] The comparison results of detecting common sugars and sugar alcohols by the method of this application and existing methods are shown in Table 7: This method has higher sensitivity. The sensitivity of five sugars has increased by 100 - 5000 times compared with existing methods, and the sensitivity of four sugar alcohols has increased by 24 - 1400 times compared with existing methods, and it can be applied to more application scenarios.

[0090] Table 7

[0091]

[0092] Example 3 Sample Detection Results

[0093] The quantitative detection results of the samples are asFigure 1 - Figure 3 As shown, from Figure 1 - Figure 3 it can be seen that all the sugar and sugar alcohol peaks have good shapes, and the sugars or sugar alcohols with the same mass spectrometry acquisition ion pairs are effectively separated, enabling accurate quantification.

[0094] The results of the qualitative detection of the sample are as Figure 4 and Figure 5 shown. From Figure 4 and Figure 5 it can be seen that the increased qualitative ion pairs can further confirm whether it is the target peak, significantly improving the anti-interference ability and reducing the generation of false positives.

[0095] Example 4 Comparison using different methods

[0096] Comparative method 1: Using 0.1% ammonia water and acetonitrile as the mobile phase, the column temperature is set at 35°C, and the rest is the same as in Example 1;

[0097] Comparative method 2: Using 0.1% formic acid water and acetonitrile as the mobile phase, the column temperature is set at 35°C, and the rest is the same as in Example 1.

[0098] The lactose detection results in Comparative method 1, Comparative method 2, and Example 1 are as Figure 6 - Figure 8 shown.

[0099] By Figure 6 and Figure 7 comparison, it can be known that the peak shape of Comparative method 2 is significantly improved compared to the commonly used Comparative method 1 (basic mobile phase). The lactose peak width is reduced from 0.4 min to 0.2 min, and the peak widths of fructose and glucose are also reduced from 0.5 min to 0.2 min (not shown in the figure). The applicant unexpectedly found that the parent ion changed from [M - 1] to [M + HCOO - ], which is more suitable for detection than [M - 1] under basic or neutral conditions, fully improving the sensitivity. The applicant also conducted many other tests, changing the main components of mobile phase A (triethylamine, ammonium formate, ammonium formate + formic acid, acetic acid, ammonium acetate, ammonium acetate + acetic acid), and adjusting the proportion of formic acid, but the tests all failed, and the separation effect was significantly inferior to 0.1% formic acid water.

[0100] From Figure 7 and Figure 8 it can be seen that Example 1 adopted a lower column temperature, and the lactose isomers α-lactose and β-lactose can be observed in the chromatogram. From Figure 1 - 3 it is also known that other sugars with isomers can also be well separated. Therefore, when it is necessary to test isomers, the proportion information of the isomers can be provided, which has great practicality.

[0101] Certainly, the above are only specific embodiments of the present application and do not limit the scope of implementation of the present invention. Any equivalent changes or modifications made according to the features and principles described in the scope of the patent application of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A method for simultaneously detecting sugars and sugar alcohols, characterized in that: The following steps are involved: 1) Prepare the sample solution to be tested; 2) Using ultra-high performance liquid chromatography-mass spectrometry to qualitatively or quantitatively detect sugars and sugar alcohols in the sample solution to be tested; The chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry method are as follows: chromatographic column: Waters Acquity BEH Amide 2.1×100 mm; column temperature: 10°C; flow rate: 0.2-0.5 mL / min; injection volume: 0.1-1 μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile, gradient elution; The sugar is one or more of fructose, glucose, sucrose, maltose, lactose and galactose; The sugar alcohol is one or more of xylitol, sorbitol, maltitol and erythritol.

2. A method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: Prior to step 1), the sample solution is pre-treated with a treatment agent, wherein the treatment agent is composed of a zinc acetate solution and a potassium ferrocyanide solution or the treatment agent is n-hexane.

3. The method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry method are: ion source: ESI; scanning mode: negative mode; scanning mode: MRM; drying gas temperature: 300°C; drying gas flow rate: 9 L / min; sheath gas temperature: 350°C; sheath gas flow rate: 12 L / min.

4. The method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The parent ion-daughter ion pairs of mass spectrometry analysis in the quantitative detection are: erythritol, m / z 121.1→89.1; fructose, m / z 179.1→89.1; galactose, m / z 179.1→89.1; glucose, m / z 179.1→89.1; lactose, m / z 387.2→341.2; maltitol, m / z 343.1→179.1; maltose, m / z 387.2→341.2; sorbitol, m / z 181.1→89.1; sucrose, m / z 387.2→341.2; xylitol, m / z 151.1→89.

1.

5. The method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The parent ion-daughter ion pairs of mass spectrometry analysis in the qualitative detection are: erythritol, m / z 121.1→100.9, 121.1→71.2; fructose, m / z 179.1→59.1, 179.1→71.2; galactose, m / z 179.1→71.1, 179.1→59.1; glucose, m / z 179.1→59.1, 179.1→119; lactose, m / z 387.2→179.1, 387.2→161.1; maltitol, m / z 343.1→89.1, 343.1→59.1; maltose, m / z 387.2→221.1, 387.2→161.1; sorbitol, m / z 181.1→101.1, 181.1→71.2; sucrose, m / z 387.2→179.1, 387.2→89.1; xylitol, m / z 151.1→71.2, 151.1→101.

1.

6. The method for simultaneous detection of sugars and sugar alcohols according to claim 1, characterized in that: The gradient elution procedure is as follows: Time Mobile Phase A 0~0.5min 10%A; 0.5~21.6min 10%A~30%A; 21.6~25.6min 30%A; 25.6~25.7min 10%A.

7. The method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The quantitative detection is an external standard method quantitative detection.

8. The method for simultaneously detecting sugars and sugar alcohols according to claim 7, characterized in that: The external standard method quantitative detection includes preparing standard solutions of sugar and sugar alcohol respectively, taking the standard solutions to prepare standard samples of a series of concentrations respectively, measuring the peak area of ​​the standard samples by ultra-high performance liquid chromatography-tandem mass spectrometry, performing linear regression of the peak area against the concentration, and establishing standard curves of sugar and sugar alcohol respectively, and calculating the sugar and sugar alcohol contents in the sample solution based on the standard curves.

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