CN114166984A - Method for simultaneously detecting multiple acidic monosaccharides - Google Patents
Method for simultaneously detecting multiple acidic monosaccharides Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- Life Sciences & Earth Sciences (AREA)
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- Biochemistry (AREA)
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Abstract
The invention discloses a method for simultaneously detecting multiple acidic monosaccharides, which comprises the following steps: carrying out acidolysis treatment on a sample to be detected so as to obtain an acidolyzed sample; and detecting the sample after the acidolysis by using an ion chromatography-pulse amperometric detection system so as to obtain the content of the acidic monosaccharide. The method can be used for simultaneously detecting various monosaccharides, and has high detection sensitivity and good accuracy.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for simultaneously detecting multiple acidic monosaccharides.
Background
Polysaccharides are important naturally occurring forms of sugars, including structural, storage and bioactive polysaccharides. Polysaccharides in which the acidic groups are contained are generally referred to as acidic polysaccharides. The acidic polysaccharide has important biological activities of enhancing the body immunity, resisting tumors, viruses, oxidation, aging and the like, and is widely applied to the fields of functional foods, biological medicines, biological materials and the like. In order to analyze the composition of acidic polysaccharides, it is necessary to perform analysis after acidic polysaccharides are hydrolyzed into monosaccharides by using acids, and therefore, methods for analyzing a plurality of acidic monosaccharides simultaneously need to be studied.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for simultaneously detecting a plurality of acidic monosaccharides, which can simultaneously detect a plurality of monosaccharides, has high detection sensitivity and high detection accuracy, and is particularly suitable for detecting acidic monosaccharides in food, such as acidic monosaccharides in bird's nest.
Thus, according to one aspect of the invention, there is provided a method for simultaneously detecting a plurality of acidic monosaccharides. According to an embodiment of the invention, the method comprises: carrying out acidolysis treatment on a sample to be detected so as to obtain an acidolyzed sample; and detecting the sample after the acidolysis by using an ion chromatography-pulse amperometric detection system so as to obtain the content of the acidic monosaccharide.
According to the method for simultaneously detecting multiple acidic monosaccharides, provided by the embodiment of the invention, the sample is firstly subjected to acidolysis treatment to promote polysaccharide decomposition, and then the ion chromatography-pulse amperometric detection system is used for detection, so that the detection sensitivity and accuracy are high.
In addition, the method for simultaneously detecting multiple acidic monosaccharides according to the above embodiment of the present invention may further have the following additional technical features:
according to the embodiment of the present invention, the acid hydrolysis treatment is performed using trifluoroacetic acid, preferably, the concentration of the trifluoroacetic acid is 0.02 to 0.1mmol/L, preferably, 0.05 to 0.1 mmol/L.
According to the embodiment of the invention, the addition amount of the trifluoroacetic acid is 100-300 μ L based on 0.1g of the sample to be detected.
According to an embodiment of the present invention, the time of the acid hydrolysis treatment is 10 to 20 minutes.
According to the embodiment of the invention, the ion chromatography detection conditions of the ion chromatography-pulsed amperometric detection system are as follows: a chromatographic column: a Dionex CarboPac PA 20 chromatographic column with the specification of 3mm multiplied by 150mm and 6 μm; column temperature: 30 ℃; flow rate: 0.4 mL/min; sample introduction amount: 5 μ L.
According to the embodiment of the invention, the elution mode of the ion chromatograph is isocratic elution.
According to the embodiment of the invention, the eluent for isocratic elution is a mixed solution consisting of 10mM NaOH and 140mM sodium acetate.
According to the embodiment of the invention, the pulse ampere detection condition in the ion chromatography-pulse ampere detection system is integral pulse detection, and four-waveform potential sampling is adopted.
According to an embodiment of the present invention, the acidic monosaccharide is at least one selected from the group consisting of N-acetylneuraminic acid (N-acetyl neuraminic acid), N-glycolylneuraminic acid (N-glycolylneuraminic acid), D-mannuronic acid (manuuronic acid), Glucuronic acid (Glucuronic acid) and D- (+) -Galacturonic acid (D- (+) -Galacturonic acid).
According to an embodiment of the invention, the plurality is at least 5.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows an experimental graph of the HPLC-MS/MS analysis of sialic acid chromatography results using different chromatography columns according to one embodiment of the invention, wherein A is a reversed phase C18 chromatography column; b is a hydrophilic Hilic chromatographic column;
FIG. 2 is a schematic representation of the ion chromatography results of 5 sialic acid-based acidic monosaccharide chromatograms, wherein 1 is N-acetylneuraminic acid; 2 is N-glycolylneuraminic acid; 3 is D- (+) -galacturonic acid; 4 is glucuronic acid; 5 is D-mannuronic acid;
FIG. 3 shows ion chromatograms of bird's nest samples subjected to acid hydrolysis with different substances, wherein A is 0.02M and 0.05M hydrochloric acid; b is 0.05M and 0.1M trifluoroacetic acid; c is acidolysis of acetic acid, hydrochloric acid and trifluoroacetic acid.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
According to one aspect of the invention, a method for simultaneously detecting a plurality of acidic monosaccharides is provided. According to the method for simultaneously detecting multiple acidic monosaccharides, provided by the embodiment of the invention, the sample is firstly subjected to acidolysis treatment to promote polysaccharide decomposition, and the detection is carried out by using an ion chromatography-pulse amperometric detection system, so that the detection sensitivity and accuracy are high.
The embodiment of the invention selects an ion chromatography-pulse amperometric detection system for detection, has higher sensitivity and accuracy compared with a liquid chromatography-tandem system, adopts the liquid chromatography-tandem mass spectrometry to detect the acid monosaccharide, and has no acid monosaccharide retention on the liquid chromatography. To facilitate an understanding of the method for simultaneously detecting a plurality of acidic monosaccharides, which is explained herein, the method comprises, according to an embodiment of the present invention:
s100 acid hydrolysis treatment
According to the embodiment of the invention, the sample to be detected is subjected to acidolysis treatment so as to obtain the sample after acidolysis. Since acidic monosaccharides in biological samples are covalently bonded to proteins or other monosaccharides in the form of glycosidic bonds, they need to be subjected to acidolysis before detection, and then to be converted into monosaccharides for detection.
According to an embodiment of the present invention, the acid hydrolysis treatment is performed using trifluoroacetic acid. The trifluoroacetic acid is organic acid, can protect the hydroxyl of monosaccharide in the acidic hydrolysis process, is strong in acidity, is far larger than acetic acid and is only slightly smaller than hydrochloric acid, has strong acidolysis capacity, is favorable for realizing the dissociation of acidic monosaccharide in glycoprotein, and is suitable for acidolysis treatment of food. According to a preferred embodiment of the invention, the concentration of trifluoroacetic acid is between 0.02 and 0.1mmol/L, preferably between 0.05 and 0.1 mmol/L. Thus, the acid hydrolysis effect of trifluoroacetic acid is more excellent in this concentration range.
According to the embodiment of the invention, the addition amount of the trifluoroacetic acid is 100-300 μ L based on 0.1g of the sample to be detected. Therefore, the method is beneficial to the full acidolysis of the polysaccharide in the sample to be detected by the trifluoroacetic acid, and the influence of the excessive trifluoroacetic acid on the subsequent detection is avoided.
According to an embodiment of the present invention, the time of the acid hydrolysis treatment is 10 to 20 minutes. Therefore, the acidolysis treatment is fully performed, the polysaccharide acidolysis is more thorough, and the reaction time process and the reaction efficiency are prevented from being influenced.
S200 detection analysis
According to the embodiment of the invention, the sample after acidolysis is detected by an ion chromatography-pulse amperometric detection system, so as to obtain the content of the acidic monosaccharide. Since the acidic monosaccharide is rich in a plurality of hydroxyl groups and a carboxyl group, the acidic monosaccharide is extremely large and has similar structure, so that no matter a common C18 chromatographic column or a Hilic hydrophilic chromatographic column is used on liquid chromatography, the acidic monosaccharide cannot be separated, but the acidic polysaccharide can be detected by using an ion chromatography-pulse amperometric detection system, and a plurality of acidic monosaccharides can be detected.
According to the embodiment of the invention, the ion chromatography detection conditions of the ion chromatography-pulsed amperometric detection system are as follows: a chromatographic column: a Dionex CarboPac PA 20 chromatographic column with the specification of 3mm multiplied by 150mm and 6 μm; column temperature: 30 ℃; flow rate: 0.4 mL/min; sample introduction amount: 5 μ L. Thereby, separation of various acidic monosaccharide components is facilitated.
Since the acidic monosaccharides, especially the five sugars separated in the examples of the present invention, have similar structures, the separation mode of isocratic elution is selected to ensure the full separation of the five sugars. According to the embodiment of the invention, the elution mode of the ion chromatograph is isocratic elution.
According to the embodiment of the invention, the eluent for isocratic elution is a mixed solution consisting of 10mM NaOH and 140mM sodium acetate. For the separation of neutral and basic monosaccharides, a mobile phase of a NaOH system is generally used, but the inventor researches and discovers that the elution of acidic monosaccharides cannot be realized only by the NaOH system, the mobile system is optimized, the elution of acidic monosaccharides can be realized by adding CH3COONa containing acetate, and the elution effect is better when the concentration of NaOH is 10mM and the concentration of sodium acetate is 140 mM.
According to the embodiment of the invention, the pulse ampere detection condition in the ion chromatography-pulse ampere detection system is integral pulse detection, and four-waveform potential sampling is adopted. Thus, the sensitivity and accuracy of the detection is higher.
According to an embodiment of the present invention, the acidic monosaccharide is at least one selected from the group consisting of N-acetylneuraminic acid (N-acetyl neuraminic acid), N-glycolylneuraminic acid (N-glycolylneuraminic acid), D-mannuronic acid (manuuronic acid), Glucuronic acid (Glucuronic acid) and D- (+) -Galacturonic acid (D- (+) -Galacturonic acid).
According to an embodiment of the invention, the plurality is at least 5. That is, according to the method for simultaneously detecting a plurality of acidic monosaccharides of the embodiment of the present invention, at least 5 acidic monosaccharides can be simultaneously detected.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.
Example 1
The method for simultaneously detecting various acidic monosaccharides of the embodiment of the invention is used for detecting acidic polysaccharides in edible bird's nests sold in the market, and specifically comprises the following steps:
1. materials and methods
1.1 instruments and reagents
ICS-6000 ion chromatograph (seimer feishell science, usa); ExionLC AD 6500 ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer (AB SCIEX, USA); analyst workstation (AB SCIEX, usa); PL203 electronic balance (mettler-toledo instruments (shanghai) ltd); water purifiers (beijing de quan business limited); KQ-500DE digital control ultrasonic cleaner (Xiamen Ruisijie scientific instruments Co., Ltd.).
5 kinds of acidic monosaccharides: n-acetylneuraminic acid, N-glycolylneuraminic acid, D-mannuronic acid, Glucuronic acid, D- (+) -Galacturonic acid (D- (+) -Galactinic acid) were purchased from Sigma.
Preparing a standard substance stock solution: accurately weighing 10.0mg of each monosaccharide in a 10.0mL volumetric flask, dissolving with ultrapure water, diluting to constant volume, preparing into standard stock solution with concentration of 1mg/mL, storing at-20 deg.C, and using within one month.
1.2 chromatographic conditions
1.2.15 analysis of sialic acid monosaccharide
A chromatographic column: dionex CarboPac PA 20(3 mm. times.150 mm,6 μm) column; column temperature: 30 ℃; 10mM NaOH +140mM sodium acetate isocratic elution, flow rate of 0.4mL/min, sample volume of 5 μ L, analysis time of 30min, and chromatographic column equilibrium time of 10 min. The ampere detector adopts integral pulse detection and selects four-waveform potential sampling.
1.3 sample treatment
The sample bird's nest used in the experiment is Indonesia white bird's cup, and the specific sample processing mode refers to the national standard sample processing mode, which is briefly described as follows: grinding 200g of cubilose sample into fine powder by using a mortar, uniformly mixing, and sieving by using a 40-mesh sieve for later use.
1.3.1 determination of total sialic acid and other monosaccharides in dried bird's nest:
(1) accurately weighing 0.1g, placing in a 25mL colorimetric tube with a plug, adding 25mL ultrapure water, standing overnight, adding 200 μ L of trifluoroacetic acid into the 25mL colorimetric tube with a plug, covering with a glass plug, and performing acidolysis at 85 ℃ for 30 min.
(2) And cooling the hydrolysate to room temperature, transferring the hydrolysate into a 100mL colorimetric tube, washing the 25mL colorimetric tube for 3 times by using ultrapure water, transferring the washing solution into the 100mL colorimetric tube, metering the volume to 100mL, uniformly mixing, and standing for 5 min.
(3) Sucking 1mL to 25mL of colorimetric tube with plug, adding 9mL of ultrapure water, mixing uniformly, filtering the constant volume solution with a 0.22 μm nylon filter membrane, and measuring by ion chromatography.
1.3.2 determination of free sialic acid in dried bird's nest:
accurately weighing 0.1g, placing in a 50mL centrifuge tube, adding 40mL ultrapure water, covering, standing overnight, centrifuging at 5000r/m for 10min, collecting 1mL supernatant, filtering with 0.22 μm nylon filter membrane, and measuring by ion chromatography.
2. Results and discussion
2.1 selection of the conditions of the apparatus
This example examined the separation of sialic acid by HPLC-MS/MS and ion chromatography-pulsed amperometric detection, respectively, and showed that sialic acid was not retained on either of the Acquity UPLC BEH Hilic (2.1 × 50mm, 1.7 μm) or Acquity UPLC HSS C18(2.1 × 100mm, 1.8 μm) columns (as shown in FIG. 1), and therefore, 5 acidic sialic acids and 10 neutral and basic monosaccharides were preferably analyzed by ion chromatography-pulsed amperometric detection, and the separation chromatogram for 5 acidic sialic acids is shown in FIG. 2.
3.2 optimization of sample treatment conditions
For the treatment of the bird's nest sample, the national standard of detection of sialic acid in the bird's nest and products thereof-liquid chromatography (GB/T30636-2014) adopts a mode of hydrolysis for 10min by 50% acetic acid solution at 100 ℃, after a large amount of experiments are carried out, and the hydrolysis time is tried to be prolonged to 20min, the experimental results show that when 50% acetic acid is used for hydrolysis, the hydrolysis effects of 10min and 20min are basically the same, but the measured sialic acid content in the dry bird's nest is only 7.8%, which is obviously lower than the reports of related documents. For this reason, in this example, the hydrolysis ability of 0.02M, 0.05M hydrochloric acid and 0.05M, 0.1M trifluoroacetic acid to sialic acid in dry bird's nest were examined, and the results of the chromatogram of analysis of the content of sialic acid in bird's nest under different acid hydrolysis conditions are shown in fig. 3, which revealed that the hydrolysis ability of 0.05M hydrochloric acid to dry bird's nest was superior to that of 0.02M hydrochloric acid to sialic acid in dry bird's nest, that the content of sialic acid in dry bird's nest was 10.3% when 0.02M hydrochloric acid was used, that the content of sialic acid in dry bird's nest was only 13.1% when 0.05M hydrochloric acid was used, that the content of sialic acid in dry bird's nest was 13.8% when 0.05M trifluoroacetic acid was used, and that the content of sialic acid in dry bird's nest was 14.3% when 0.1M trifluoroacetic acid was used, respectively. Therefore, 0.1M trifluoroacetic acid is preferred for hydrolysis of the bird's nest sample.
2.3 Linear Range of the method
(1) Detection and quantitation limits of methods
And (3) carrying out a standard adding experiment according to the verification conditions, carrying out sample injection for six times under the optimized conditions, and respectively obtaining signal-to-noise ratios of the six sample injections, wherein the mass concentration of the target object corresponding to 3 times of the signal-to-noise ratio is taken as the detection Limit (LOD) of the method, and the mass concentration of the target object corresponding to 10 times of the signal-to-noise ratio is taken as the quantitative Limit (LOQ) of the method, so that the detection limit of the solid bird nest method is determined to be 0.1g/kg, and the quantitative limit of the method is 0.5 g/kg.
TABLE 1 ion chromatography linear equation and correlation coefficient
(2) Accuracy and precision of the method (repeatability)
In this example, solid bird's nest was used as a sample, and the recovery rate and precision of the added sample were measured by a standard addition method. In the experimental verification: the solid edible bird's nest is added with sialic acid standard substances which are 0.5 time, 1 time and 2 times of the background value respectively, then the measurement is carried out (if the measurement value of the standard sample exceeds the curve range, the standard sample is diluted and then the measurement is carried out on the machine), the standard sample of each concentration is measured for 3 times respectively, the recovery rate is between 83.96% and 108.3%, the Relative Standard Deviation (RSD) is between 0.52% and 8.426%, and the table 2 shows the recovery rate and precision experimental results of the solid edible bird's nest in different standard concentrations.
TABLE 2 recovery of solid sialic acid from cubilose
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A method for simultaneously detecting a plurality of acidic monosaccharides, comprising:
carrying out acidolysis treatment on a sample to be detected so as to obtain an acidolyzed sample; and
and detecting the sample after the acidolysis by using an ion chromatography-pulse amperometric detection system so as to obtain the content of the acidic monosaccharide.
2. The method according to claim 1, characterized in that the acid hydrolysis treatment is carried out with trifluoroacetic acid, preferably at a concentration of 0.02-0.1mmol/L, preferably 0.05-0.1 mmol/L.
3. The method as claimed in claim 2, wherein the amount of the trifluoroacetic acid added is 100-300 μ L based on 0.1g of the sample to be tested.
4. The method as claimed in claim 2, wherein the time for the acid hydrolysis treatment is 10 to 20 minutes.
5. The method of claim 1, wherein the ion chromatography-pulsed amperometric detection system has ion chromatography detection conditions of:
a chromatographic column: a Dionex CarboPac PA 20 chromatographic column with the specification of 3mm multiplied by 150mm and 6 μm;
column temperature: 30 ℃;
flow rate: 0.4 mL/min;
sample introduction amount: 5 μ L.
6. The method of claim 5, wherein the ion chromatograph is eluted in an isocratic manner.
7. The method according to claim 6, wherein the eluent for isocratic elution is a mixed solution of 10mM NaOH and 140mM sodium acetate.
8. The method of claim 1, wherein the pulsed amperometric detection condition in the ion chromatography-pulsed amperometric detection system is integrated pulse detection and four-waveform potential sampling is selected.
9. The method according to claim 1, wherein the acidic monosaccharide is at least one selected from the group consisting of N-acetylneuraminic acid, N-glycolylneuraminic acid, D-mannuronic acid, glucuronic acid and D- (+) -galacturonic acid.
10. The method of claim 1, wherein said plurality is at least 5.
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