CN108169385B - Method for simultaneously and rapidly detecting six saccharides in health wine by connecting QDa in series through ultra-high performance liquid chromatography - Google Patents
Method for simultaneously and rapidly detecting six saccharides in health wine by connecting QDa in series through ultra-high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for simultaneously and rapidly detecting six saccharides in health-care wine by connecting QDa in series through ultra-high performance liquid chromatography, wherein a wine sample to be detected is subjected to pretreatment and then is detected by adopting an ultra-high performance liquid chromatograph equipped with a QDa detector to obtain a chromatogram of the sample to be detected, qualitative analysis is carried out according to selected ions, and then the wine sample of the health-care wine to be detected is subjected to quantitative analysis according to a standard curve of the six saccharides. The method is simple, rapid, accurate and reliable, can be used for simultaneously detecting the content of 6 saccharides (glucose, fructose, sucrose, lactose, maltose and inositol) in the health-care wine, and provides scientific basis for accurate judgment and rapid detection of the saccharides in the health-care wine.
Description
Technical Field
The invention relates to a method for simultaneously and rapidly detecting six saccharides in health-care wine by connecting QDa in series through ultra-high performance liquid chromatography, belonging to the technical field of detection and analysis.
Background
The sugar and the sugar alcohols are carbohydrates, are natural ingredients in food and health care wine, and have important nutritional values. Some sugars are added to the processed food in order to enhance the aroma or to simulate fresh food. In recent years, with the increasing prevalence of obesity and diabetes in the population of developed countries, the need for further monitoring of carbohydrate intake has increased. Therefore, in order to meet the increasingly stringent regulatory requirements, accurate sugar content information needs to be provided on food labels. The analysis of sugars and sugar alcohols is very challenging as reported in the existing literature, because their compound structures lack chromophores and the various molecules are very similar, many of which are isomers of each other.
The prior art uses a liquid chromatograph (equipped with an RI or ELS detector). RI detection requires careful control of the mobile phase to avoid any change in the analysis and therefore requires isocratic elution. With RI detection, it is difficult to change mobile phase components from one analysis to the next because when different mobile phase components are used, the RI detector can take hours to reach equilibrium, and even when a new batch of the same mobile phase is used, the RI can still detect small changes resulting in baseline changes. In view of the fact that ELS detection is relatively more reliable in the presence of changes in the mobile phase composition, but ELS often fails to achieve the sensitivity and selectivity required for the detection of carbohydrates in complex food matrices, it is imperative to establish a method for the simultaneous rapid detection of carbohydrates. The invention uses the ultra-high performance liquid chromatography to connect QDa detectors in series, completely avoids the problems, greatly reduces the difficulty of saccharide analysis, and provides scientific basis for accurate judgment and rapid detection of saccharide substances in the health wine.
Disclosure of Invention
The invention aims to provide a method for simultaneously and rapidly detecting six saccharides in health care wine by connecting QDa in series by using ultra-high performance liquid chromatography to avoid the defects in the prior art. The six saccharides are glucose, fructose, sucrose, lactose, maltose and inositol. The method can provide scientific basis for accurate judgment and rapid detection of the saccharides in the health-care wine.
The invention relates to a method for simultaneously and rapidly detecting six saccharides in health-care wine by connecting QDa in series by using ultra-high performance liquid chromatography, which comprises the following steps:
step 1: pretreatment
Measuring 1.0-5.0 mL of a to-be-detected health-care wine sample into a 10mL plastic centrifuge tube, adding 8-10 mL of acetonitrile solution with the volume concentration of 75%, swirling for 1-5 min, centrifuging for 5-10 min at 1000 rpm, and filtering by a 0.22-micrometer filter membrane needle filter to obtain a to-be-detected sample;
step 2: drawing of standard curve
Preparing 1000mg/L stock solutions of five kinds of edible sugar (fructose, glucose, sucrose, maltose and lactose) and inositol by using water respectively, and then diluting by using acetonitrile aqueous solution (50:50, v/v) to prepare 50mg/L mixed stock solution (the concentration of each component in the mixed stock solution is 50 mg/L); diluting the obtained mixed stock solution with acetonitrile aqueous solution (50:50, v/v) to obtain mixed standard working solutions with different concentrations, detecting by using an ultra-high performance liquid chromatograph equipped with an QDa detector, and performing linear regression on the corresponding mass concentration of the substance to be detected by using the peak area of the substance to be detected to obtain linear regression equations of the six saccharide substances, wherein the curve corresponding to each linear regression equation is the standard curve of the corresponding saccharide substance;
in the step 2, the concentration of the mixed standard working solution is 5-1000 ng/mL, and at least 5 point values with different concentrations are prepared.
In the step 2, the detection conditions of the ultra-high performance liquid chromatograph are set as follows:
a chromatographic column: a BEH Amide saccharide substance analytical column of 2.1X 150mm,1.7 μm; a vortex instrument and a pipette;
the column temperature is 30-50 ℃;
the temperature of the sample chamber is 10-20 ℃;
mobile phase: phase A is 75% acetonitrile water solution (A), phase B is 10mmol NH4HCO3Solution (containing 0.1% NH)4OH solution);
the flow rate is 0.15-0.30 mL/min;
the elution mode is isocratic elution, and the elution volume ratio of the mobile phase A to the mobile phase B is as follows: 15: 85;
the sample injection amount is 0.5-2 mu L;
the detection time is 15-20 min;
QDa the setting conditions of the detector are:
the system comprises the following steps: ACQUITY QDa Mass Spectroscopy Detector, Performance mode;
ionization mode: ESI-;
capillary voltage: 0.8V;
taper hole voltage: 4.0V;
temperature of the probe: 600 ℃;
acquisition rate: 1 HZ;
full scanning: 50-450 m/z;
the SIR mass numbers are shown in table 1 below:
TABLE 1
| Name of Compound | SIR(m/z) | Mode of ion formation |
| Fructose | 215.1 | [M+CL-]- |
| Glucose | 215.1 | [M+CL-]- |
| Inositol | 179.2 | [M-H+]- |
| Lactose | 377.2 | [M+CL-]- |
| Maltose | 377.2 | [M+CL-]- |
| Sucrose | 341.3 | [M-H+]- |
Description of the drawings: multiple mass number monitoring was performed for each carbohydrate. In order to improve the response of substances in mass spectrometry and reduce the detection limit and the quantitative limit of the substances, the invention uses a novel ion forming mode, and fructose, glucose, lactose and maltose use a chlorination mode to form a chlorine adduct.
And step 3: detection of health wine sample to be detected
Taking 1.0-2.0 mL of the sample to be detected obtained in the step 1, detecting under the same detection conditions in the step 2 to obtain a chromatogram of the sample to be detected, performing qualitative analysis according to the selected ions, and then performing quantitative analysis on the health wine sample to be detected according to the standard curves of the six saccharides.
The following sensitivity tests were performed on the method of the invention: the sensitivity test comprises the sensitivity of an instrument and the sensitivity of a method, the sensitivity of the instrument is expressed by the detection limit of the instrument, and the minimum concentration of the mixed standard solution with the signal-to-noise ratio of more than or equal to 3 is taken as the detection limit of the instrument; the sensitivity of the method is expressed by the quantitative limit of the method, and the minimum concentration of the mixed standard solution with the signal-to-noise ratio of more than or equal to 9 is taken as the quantitative limit of the method. The resulting correlation data are shown in Table 2.
The following accuracy and reproducibility experiments were performed on the method of the present invention: selecting the same health wine sample as a blank sample after pretreatment, dividing the blank sample into 3 parts, respectively adding mixed standard working solutions to perform a standard addition recovery experiment, and calculating the recovery rate; selecting 1 health wine sample, treating 6 samples according to the same pretreatment method, respectively carrying out experiments, and judging the reproducibility of the analysis method by calculating the range of Relative Standard Deviation (RSD). The accuracy of the process is expressed in terms of recovery, see table 3, and the reproducibility of the process is expressed in terms of Relative Standard Deviation (RSD), see table 4. It can be seen that the recovery rate is 80-120%, and RSD is less than 10%.
TABLE 3 spiking recovery test for six saccharides
TABLE 4 reproducibility test of six sugar substances
The invention uses QDa mass spectrum detector to detect, adopts full scan mode (SIR), the sensitivity meets the requirement of low limit detection of sample, and the detector is convenient to use, plug-and-play, does not need tuning, and can rapidly detect saccharide and inositol in white spirit. The detection limit and the quantitative limit of the six kinds of saccharides can completely meet the low content determination requirement of the wine sample, and the recovery rate and the reproducibility of each compound can meet the quantitative test requirement.
The invention has the beneficial effects that:
1. the invention establishes a method for simultaneously and rapidly detecting six saccharides in the health-care wine by connecting an ultra-High Performance Liquid Chromatography (HPLC) with QDa detectors in series, can accurately perform qualitative and quantitative detection on the six saccharides in the white wine, and provides scientific basis for accurate judgment and rapid detection of the saccharides in the white wine.
2. The ultra-high performance liquid chromatography tandem QDa mass spectrum detector is simple, quick, accurate and reliable to operate and good in repeatability.
3. The invention relates to a 2.1 x 150mm,1.7 mu m BEH Amide saccharide substance analysis chromatographic column, acetonitrile and 10mmol NH4HCO3Solution (containing 0.1% NH)4OH solution) mobile phase achieves excellent separation effect on 6 saccharides in the health care wine.
4. The invention does not need to use QDa detector
Purifying with C18 small column, and directly detectingAnd the detection cost is greatly reduced.
5. The method has the advantages of few pretreatment operation steps and better reproducibility and standard addition recovery, thereby reducing experimental errors and improving the accuracy of data.
6. The present invention improves the analytical selectivity of compound identification by combining retention time with mass analysis.
7. The present invention is not only able to distinguish co-eluting components by charge to mass ratio, but is also able to employ multiple methods on a single system and switch rapidly between different method conditions.
8. The sample introduction mode of the experiment is direct sample introduction, and the sample directly enters a mass spectrum detector after being separated by a BEH Amide carbohydrate analysis chromatographic column, so that the sensitivity of the method can be greatly improved, and the detection limit and the quantification limit of the method can be greatly reduced.
Drawings
FIG. 1 is a liquid chromatogram of a standard working solution of a mixture of six saccharides.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
1. Pretreatment
Measuring 5.0mL of a to-be-detected health-care wine sample into a 10mL plastic centrifuge tube, adding 10mL of acetonitrile solution with the volume concentration of 75%, performing vortex for 2min, centrifuging for 5-10 min at 1000 rpm, and filtering by using a 0.22-micrometer filter membrane needle filter to obtain a to-be-detected sample;
2. setting of detection conditions
The detection conditions of the ultra-high performance liquid chromatograph are set as follows:
a chromatographic column: a BEH Amide saccharide substance analytical column of 2.1X 150mm,1.7 μm; a vortex instrument and a pipette;
the column temperature was 40 ℃;
the temperature of the sample chamber is 15 ℃;
mobile phase: phase A is 75% acetonitrile water solution (A), phase B is 10mmol NH4HCO3Solution (containing 0.1% NH)4OH solution);
the flow rate is 0.15 mL/min;
the elution mode is isocratic elution, and the volume ratio of the mobile phase A to the mobile phase B is as follows: 15: 85;
the sample injection amount is 0.7 mu L;
the detection time is 17 min;
QDa the setting conditions of the detector are:
the system comprises the following steps: ACQUITY QDa Mass Spectroscopy Detector, Performance mode;
ionization mode: ESI-;
capillary voltage: 0.8V;
taper hole voltage: 4.0V;
temperature of the probe: 600 ℃;
acquisition rate: 1 HZ;
full scanning: 50-450 m/z;
the SIR mass numbers are shown in table 1 below:
TABLE 1
| Name of Compound | SIR(m/z) | Mode of ion formation |
| Fructose | 215.1 | [M+CL-]- |
| Glucose | 215.1 | [M+CL-]- |
| Inositol | 179.2 | [M-H+]- |
| Lactose | 377.2 | [M+CL-]- |
| Maltose | 377.2 | [M+CL-]- |
| Sucrose | 341.3 | [M-H+]- |
3. Drawing of standard curve
Preparing 1000mg/L stock solutions of five kinds of edible sugar (fructose, glucose, sucrose, maltose and lactose) and inositol by using water respectively, and then diluting by using acetonitrile aqueous solution (50:50, v/v) to prepare 50mg/L mixed stock solution (the concentration of each component in the mixed stock solution is 50 mg/L); diluting the obtained mixed stock solution with acetonitrile aqueous solution (50:50, v/v) to obtain mixed standard working solutions with different concentrations, detecting by using an ultra-high performance liquid chromatograph equipped with an QDa detector according to the set parameters in the step 2, and performing linear regression on the corresponding mass concentration of the substance to be detected according to the peak area of the substance to be detected to obtain linear regression equations of the six saccharide substances, wherein the curve corresponding to each linear regression equation is the standard curve of the corresponding saccharide substance;
the concentration of the mixed standard working solution is 5-1000 ng/mL, and the concentration is 5ng/mL, 50ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL respectively;
the linear regression equation and the correlation coefficient r corresponding to the six saccharides2See table 2 below.
TABLE 2 Linear regression equation, correlation coefficient, detection limit, and quantitation limit for six saccharides
4. Detection of health wine sample to be detected
Taking 1.0-2.0 mL of the sample to be detected obtained in the step 1, detecting by adopting the detection conditions in the step 2 to obtain a chromatogram of the sample to be detected, carrying out qualitative analysis according to the selected ions, and then carrying out quantitative analysis on the health wine sample to be detected according to the standard curves of the six saccharides. The data comparing the test data of the method with the test data of the existing method are shown in the following table 5.
TABLE 5 comparison of the test data of the present method with the test data of the prior art method
As can be seen from the data in Table 5, the method has lower detection limit compared with the existing method for the same sample, the content which cannot be detected by the existing method can be detected by using the method, the content which is above the detection limit can be detected by using the two methods, the data detected by the two methods are in a reasonable error range, and the method can be fully proved to be superior to the existing method by detecting the data, so that the saccharide substances in the 6 can be accurately quantified.
Claims (3)
1. A method for simultaneously and rapidly detecting six saccharides in health wine by connecting QDa in series by using ultra-high performance liquid chromatography is characterized by comprising the following steps:
step 1: pretreatment
Measuring 1.0-5.0 mL of a to-be-detected health-care wine sample into a 10mL plastic centrifuge tube, adding 8-10 mL of acetonitrile solution with the volume concentration of 75%, swirling for 1-5 min, centrifuging for 5-10 min at 1000 rpm, and filtering by a 0.22-micrometer filter membrane needle filter to obtain a to-be-detected sample;
step 2: drawing of standard curve
Preparing 1000mg/L stock solutions of fructose, glucose, sucrose, maltose, lactose and inositol by using water respectively, and then diluting by using acetonitrile aqueous solution to prepare 50mg/L mixed stock solution; diluting the obtained mixed stock solution with an acetonitrile aqueous solution to obtain mixed standard working solutions with different concentrations, detecting by using an ultra-high performance liquid chromatograph equipped with an QDa detector, performing linear regression on the corresponding mass concentration of the substance to be detected by using the peak area of the substance to be detected to obtain linear regression equations of the six saccharide substances, wherein the curve corresponding to each linear regression equation is the standard curve of the corresponding saccharide substance;
and step 3: detection of health wine sample to be detected
Taking 1.0-2.0 mL of the sample to be detected obtained in the step 1, detecting by adopting the same detection conditions in the step 2 to obtain a chromatogram of the sample to be detected, carrying out qualitative analysis according to the selected ions, and then carrying out quantitative analysis on the health wine sample to be detected according to the standard curves of the six saccharides;
in the step 2, the detection conditions of the ultra-high performance liquid chromatograph are set as follows:
a chromatographic column: a BEH Amide saccharide substance analytical column of 2.1X 150mm,1.7 μm; a vortex instrument and a pipette;
the column temperature is 30-50 ℃;
the temperature of the sample chamber is 10-20 ℃;
mobile phase: phase A is 75% acetonitrile water solution, phase B is 10mmol NH4HCO3Solution of 0.1% NH4OH solution;
the flow rate is 0.15-0.30 mL/min;
the elution mode is isocratic elution, and the elution volume ratio of the mobile phase A to the mobile phase B is as follows: 15: 85;
the sample injection amount is 0.5-2 mu L;
the detection time is 15-20 min;
in step 2, the setting conditions of the QDa detector are as follows:
the system comprises the following steps: ACQUITY QDa Mass Spectroscopy Detector, Performance mode;
ionization mode: ESI-;
capillary voltage: 0.8V;
taper hole voltage: 4.0V;
temperature of the probe: 600 ℃;
acquisition rate: 1 HZ;
full scanning: 50-450 m/z;
SIR mass numbers are as follows:
the six saccharides are glucose, fructose, sucrose, lactose, maltose and inositol.
2. The method of claim 1, wherein:
in step 2, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 50: 50.
3. The method of claim 1, wherein:
in the step 2, the concentration of the mixed standard working solution is 5-1000 ng/mL, and at least 5 point values with different concentrations are prepared.
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| CN110554114B (en) * | 2019-09-13 | 2022-06-28 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for analyzing oligomeric isomaltose and isomers thereof in yoghourt |
| CN112578067A (en) * | 2019-09-29 | 2021-03-30 | 山东绿叶制药有限公司 | Method for measuring content of glucose in star-shaped PLGA |
| CN111610275A (en) * | 2020-06-08 | 2020-09-01 | 山东省分析测试中心 | Method for determining malto-oligosaccharide and application of method in field of product quality control |
| CN112903839B (en) * | 2021-01-15 | 2023-02-17 | 中国检验检疫科学研究院 | Method for qualitatively detecting alpha and beta configurational isomers of monosaccharide and method for detecting proportion of monosaccharide isomers |
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