Method for simultaneously detecting various sugars and sugar alcohols

Abstract

The invention discloses a method for simultaneously detecting various sugars and sugar alcohols. The method comprises the following steps: 1) preparing a sample solution to be detected; (2) carrying out qualitative or quantitative detection on sugar and sugar alcohol in the sample solution to be detected by adopting an ultra-high performance liquid chromatography-mass spectrometry method; wherein the chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry method are as follows: the chromatographic column is Waters Acquity BEH Amide 2.1 * 100 mm, the column temperature is 20-30 DEG C, and the column temperature is 20-30 DEG C; the column temperature is 10 DEG C; the flow velocity is 0.2 to 0.5 mL / min; the sample size is 0.1 to 1 [mu] L; mobile phases are as follows: a phase A is 0.1% formic acid water, a phase B is acetonitrile, and gradient elution is performed. The method can be used for simultaneously detecting ten sugars and sugar alcohols, can also be used for simultaneously detecting isomers of various sugars, and has very high sensitivity.

Description

Technical Field

[0001] The present application relates to the technical field of analysis and detection, and in particular to a method for simultaneously detecting multiple sugars and sugar alcohols. Background Art

[0002] Sugar is a sweet substance for consumption, mainly extracted from rice, wheat, sugar cane, beets, etc. It 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 made from the corresponding sugar. It has some properties of sugar but lower calories. Common sugar alcohols include sorbitol, erythritol, maltitol, xylitol, etc. Sugar and sugar alcohol are widely used in the food industry, such as sweeteners, moisturizers, preservatives, etc. At present, the commonly used methods for detecting sugars are acid hydrolysis-Rhine-Enon method, Rhine-Enon method, ion chromatography, and high-performance liquid chromatography. The commonly used 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. The main problems are the following three points: Chromatography only relies on retention time for qualitative analysis, and its anti-interference ability is relatively weak. It is easy to cause false positives when encountering co-eluting impurities; it is impossible to detect sugars and sugar alcohols at the same time, and the same sample needs to be tested twice, resulting in low detection efficiency; the method sensitivity is not high. Moreover, there is currently no method that can detect the nine commonly used sugars and sugar alcohols at the same time.

[0003] Therefore, there is an urgent need to develop a highly sensitive method that can simultaneously detect multiple sugars and sugar alcohols. Summary of the invention

[0004] The purpose of the present application is to provide a method for simultaneously detecting sugars and sugar alcohols, which can simultaneously detect 6 kinds of sugars and 4 kinds of sugar alcohols, and can also simultaneously detect isomers of multiple sugars with very high sensitivity.

[0005] The present application provides a method for simultaneously detecting sugars and sugar alcohols, comprising the following steps: 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; Among them, the chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry method are: chromatographic column: WatersAcquity 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, and gradient elution.

[0006] Optionally, the sugar is one or more of fructose, glucose, sucrose, maltose, lactose and galactose.

[0007] Optionally, the sugar alcohol is one or more of xylitol, sorbitol, maltitol and erythritol.

[0008] Optionally, the sample solution is pre-treated with a treatment agent before step 1), wherein the treatment agent consists of a zinc acetate solution and a potassium ferrocyanide solution or the treatment agent is n-hexane.

[0009] Optionally, 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.

[0010] Optionally, 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 / z181.1→89.1; sucrose, m / z 387.2→341.2; xylitol, m / z 151.1→89.1.

[0011] Optionally, 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.

[0012] Optionally, the gradient elution procedure is as follows: Time Mobile Phase A 0~0.5min 10%A; 0.5~21.6 min 10%A~30%A; 21.6~25.6 min 30%A; 25.6~25.7 min 10%A.

[0013] Optionally, the quantitative detection is an external standard quantitative detection.

[0014] Optionally, the external standard method quantitative detection includes preparing standard solutions of sugars and sugar alcohols respectively, taking the standard solutions to prepare standard samples of a series of concentrations respectively, determining 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, and establishing standard curves for sugars and sugar alcohols respectively, and calculating the sugar and sugar alcohol contents in the sample solutions based on these standard curves.

[0015] In summary, the present application includes at least one of the following beneficial technical effects: 1. The method of the present application has high sensitivity and can be applied not only to the simultaneous detection of various sugars and sugar alcohols in various foods, filling the gap in the prior art, but also to the detection of environmental samples and biological samples; 2. The method of the present application adds confirmation of the ratio of qualitative ions on the basis of qualitative determination by chromatographic retention time, thus improving the accuracy of qualitative determination and greatly reducing the occurrence of false positives; 3. The method of the present application can detect nine commonly used sugars and sugar alcohols simultaneously without having to perform the test twice, and can also detect galactose simultaneously; 4. The method of the present application can detect multiple sugar isomers and can provide isomer ratio information when isomer testing is required. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the MRM spectrum of each sugar alcohol; Figure 2 is the MRM spectrum of each sugar; Figure 3 is the MRM spectrum of each sugar; Figure 4 The MRM spectra of the quantitative ion pair and the qualitative ion pair of α-lactose (left) and β-lactose (right) are superimposed; Figure 5 The MRM spectra of the quantitative ion pair and the qualitative ion pair of α-maltose (left) and β-maltose (right) are superimposed; Figure 6 is the spectrum under comparative method 1; Figure 7 is the spectrum under comparative method 2; Figure 8 This is the spectrum in Example 1. DETAILED DESCRIPTION

[0017] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present application, the technical solution of the present invention is described in detail below in conjunction with the following specific embodiments and the accompanying drawings of the specification, but it should not be understood as limiting the scope of implementation of the present invention. Example 1 Detection method

[0018] 1.1 Detection steps Take 1g of sample in a 50mL centrifuge tube, add 20mL of pure water and vortex mix. If the dissolution is slow, the water temperature can be appropriately increased. For insoluble samples, homogenization extraction can be used, and insoluble substances can be removed by centrifugation after extraction. For samples with high protein content or turbidity after extraction, add 5mL of 1 mol / L zinc acetate solution (mixed with 21.9g zinc acetate and 3mL acetic acid, and diluted to 100mL with pure water) and 5mL of 0.25 mol / L potassium ferrocyanide solution (10.6g potassium ferrocyanide diluted to 100mL with pure water) to remove protein; for samples with high oil content, add 20mL of n-hexane to remove oil.

[0019] Transfer the extract to a 100 mL volumetric flask and dilute to the mark with acetonitrile + water = 50:50. Pipette the above solution, dilute it by a certain multiple, pass it through the membrane and perform UPLC-MS / MS detection.

[0020] 1.2 Instrument conditions Chromatographic conditions: Chromatographic column: Waters ACQUITY BEH Amide 1.7um, 2.1x100 mm Mobile phase: Phase A is 0.1% formic acid water, phase B is acetonitrile Flow rate: 0.300mL / min Column temperature: 10°C Injection volume: 1 μL The specific gradient elution conditions are shown in Table 1: Table 1

[0021] Mass spectrometry conditions: Ion source: ESI Scanning mode: Negative mode Scan mode: MRM Drying gas temperature: 300℃ Drying gas flow rate: 9L / min Sheath gas temperature: 350℃ Sheath gas flow rate: 12L / min The specific quantitative and qualitative parent ion-daughter ion pairs are shown in Table 2: Table 2

[0022] 1.3 Qualitative analysis Positive judgment criteria: The relative error of the retention time of the target sugar or sugar alcohol in the sample compared with the retention time of the corresponding standard chromatographic peak should be within ±2.5%, and the qualitative ion abundance ratio should meet the requirements of Table 3.

[0023] Table 3 Relative abundance% Permissible deviation% >50 ±20 >20~50 ±25 >10~20 ±30 ≤10 ±50 1.4 Quantitative analysis Select at least five concentration points to draw a standard curve based on response and concentration, and use the external standard method for quantification:

[0024] Where: X—the content of sugar or sugar alcohol in the sample, in grams per 100 grams (g / 100g); c—the concentration of the sample solution obtained from the standard curve, in micrograms per milliliter (μg / mL); f—dilution factor; V—sample solution fixed volume, in milliliters (mL); m—sample mass, in grams (g); 10000—Unit conversion factor. Example 2 Methodology Validation

[0025] 2.1 Linear range The linear range of the method of the present application is shown in Table 4. For sugars with isomers, the concentration is calculated by summing the peak areas.

[0026] Table 4

[0027] 2.2 Accuracy and precision Three levels and six parallel spike tests were performed on blank beverage samples. The spike concentrations and test results of all sugars and sugar alcohols are shown in Table 5: Table 5 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 2.3 Sensitivity The quantification limits of all sugars and sugar alcohols are shown in Table 6. The signal-to-noise ratio at these quantification limits meets the requirement of >10.

[0028] Table 6 project Limit of quantification (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 The comparison results of the method of the present application and the existing method for detecting commonly used sugars and sugar alcohols are shown in Table 7: The present method has higher sensitivity, the sensitivity of the five sugars is increased by 100-5000 times compared with the existing method, and the sensitivity of the four sugar alcohols is increased by 24-1400 times compared with the existing method, which can be applied to more application scenarios.

[0029] Table 7 Example 3 Sample test results

[0030] The quantitative test results of the samples are as follows Figure 1-Figure 3 As shown, from Figure 1-Figure 3 It can be seen that all sugars and sugar alcohols have good peak shapes, and the same sugars or sugar alcohols collected by mass spectrometry are effectively separated, which can achieve accurate quantification.

[0031] The qualitative test results of the samples are as follows Figure 4 and Figure 5 As shown, from Figure 4 and Figure 5 It can be seen that the added qualitative ion pairs can further confirm whether it is the target peak, the anti-interference ability is significantly improved, and the occurrence of false positives is reduced. Example 4 Comparison of different methods

[0032] Comparative method 1: 0.1% ammonia water and acetonitrile were used as mobile phases, the column temperature was set to 35°C, and the rest was consistent with Example 1; Comparative method 2: 0.1% formic acid water and acetonitrile were used as mobile phases, the column temperature was set to 35° C., and the rest was consistent with Example 1.

[0033] The lactose detection results in Comparative Method 1, Comparative Method 2 and Example 1 are as follows Figure 6-Figure 8 shown.

[0034] pass Figure 6 and Figure 7 It can be seen from the comparison that the peak shape of comparison method 2 is significantly improved compared with the commonly used comparison method 1 (alkaline mobile phase), the peak width of lactose is reduced from 0.4min to 0.2min, and the peak width of fructose and glucose is also reduced from 0.5min to 0.2min (not shown). 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 alkaline or neutral conditions, and the sensitivity is fully improved. 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 worse than 0.1% formic acid water.

[0035] from Figure 7 and Figure 8It can be seen that Example 1 uses a lower column temperature, and the isomers of lactose α-lactose and β-lactose can be observed in the spectrum. Figure 1-3 It can also be seen that other sugars with isomers can also be well separated. Therefore, when isomer testing is required, the ratio information of isomers can be provided, which has great practicality.

[0036] Of course, the above description is only a specific embodiment of the present application and is not intended to limit the scope of implementation of the present invention. All equivalent changes or modifications made according to the features and principles described in the patent application scope of the present invention should be included in the patent application scope 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; Among them, the chromatographic separation conditions of the ultra-high performance liquid chromatography-mass spectrometry method are: chromatographic column: Waters Acquity BEH Amide 2.1×100mm; column temperature: 10°C; flow rate: 0.2-0.5mL / min; injection volume: 0.1-1μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile, gradient elution.

2. A method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The sugar is one or more of fructose, glucose, sucrose, maltose, lactose and galactose.

3. A method for simultaneously detecting sugars and sugar alcohols according to claim 1, characterized in that: The sugar alcohol is one or more of xylitol, sorbitol, maltitol and erythritol.

4. The 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.

5. 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.

6. The method for simultaneous detection of 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.

7. 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.

8. 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.

9. 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.

10. A method for simultaneously detecting sugars and sugar alcohols according to claim 9, 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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