CN111855829B - Method for detecting lactulose in dairy product - Google Patents
Method for detecting lactulose in dairy product Download PDFInfo
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- CN111855829B CN111855829B CN202010209476.1A CN202010209476A CN111855829B CN 111855829 B CN111855829 B CN 111855829B CN 202010209476 A CN202010209476 A CN 202010209476A CN 111855829 B CN111855829 B CN 111855829B
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- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 title claims abstract description 52
- 229960000511 lactulose Drugs 0.000 title claims abstract description 52
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 235000013365 dairy product Nutrition 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- GMPKIPWJBDOURN-UHFFFAOYSA-N Methoxyamine Chemical compound CON GMPKIPWJBDOURN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000020200 pasteurised milk Nutrition 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 11
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 11
- NRKYWOKHZRQRJR-UHFFFAOYSA-N 2,2,2-trifluoroacetamide Chemical compound NC(=O)C(F)(F)F NRKYWOKHZRQRJR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000001212 derivatisation Methods 0.000 claims abstract description 9
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 23
- 150000002500 ions Chemical class 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 12
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 10
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 9
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 9
- 229960001763 zinc sulfate Drugs 0.000 claims description 9
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 235000013336 milk Nutrition 0.000 claims description 8
- 239000008267 milk Substances 0.000 claims description 8
- 210000004080 milk Anatomy 0.000 claims description 8
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000012071 phase Substances 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 239000012491 analyte Substances 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010813 internal standard method Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 235000020191 long-life milk Nutrition 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 235000016046 other dairy product Nutrition 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 229930182830 galactose Natural products 0.000 abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 10
- 239000008103 glucose Substances 0.000 abstract description 10
- 229930091371 Fructose Natural products 0.000 abstract description 9
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 abstract description 9
- 239000005715 Fructose Substances 0.000 abstract description 9
- 238000006146 oximation reaction Methods 0.000 abstract description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 abstract description 7
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 abstract description 7
- 239000008101 lactose Substances 0.000 abstract description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 6
- 238000012360 testing method Methods 0.000 abstract description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 abstract description 3
- 239000000376 reactant Substances 0.000 abstract 1
- 239000012085 test solution Substances 0.000 abstract 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 229940094989 trimethylsilane Drugs 0.000 description 5
- MSPCIZMDDUQPGJ-UHFFFAOYSA-N N-methyl-N-(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)N(C)C(=O)C(F)(F)F MSPCIZMDDUQPGJ-UHFFFAOYSA-N 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 150000002772 monosaccharides Chemical class 0.000 description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 4
- 235000019799 monosodium phosphate Nutrition 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 150000002231 fructose derivatives Chemical class 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 150000003741 xylose derivatives Chemical class 0.000 description 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8682—Group type analysis, e.g. of components having structural properties in common
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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Abstract
The invention provides a method for detecting lactulose in dairy products, which comprises the following steps: adding beta-D-galactosidase into a dairy product sample, carrying out enzymolysis reaction on lactose and lactulose to generate galactose, fructose and glucose, carrying out oximation reaction on the galactose, the glucose and the fructose with methoxyamine respectively, carrying out derivatization reaction on the generated reactant and N-methyl-N-trimethylsilane trifluoroacetamide, and finally obtaining the test solution. And analyzing the test sample liquid by adopting an ultra-high performance liquid chromatography tandem mass spectrometer to obtain the content of lactulose. The method can accurately and sensitively determine the lactulose content in pasteurized milk.
Description
Technical Field
The invention belongs to the field of lactulose detection, and particularly relates to a method for detecting lactulose in a dairy product.
Background
Liquid milk is usually sterilized during the production process by various heat treatments, which also destroy the nutrients. Many countries and international organizations select some indexes related to heat treatment intensity to estimate the heat damage degree of the liquid milk so as to evaluate the processing quality of the liquid milk, and lactulose is an important heat sensitive index generated in the processing process.
The existing analytical methods for lactulose mainly comprise an enzyme method, an ion chromatography and the like, and because pasteurized milk contains a lot of lactose, and the lactose and the lactulose are isomers, the difficulty of separation, qualification and quantification is increased in the measuring process of the lactulose. With the popularization of liquid chromatography tandem mass spectrometers and the continuous research and development of detection technologies in recent years, the use of liquid chromatography tandem mass spectrometers to measure lactulose has become a reality. However, the liquid chromatography tandem mass spectrometer has low sensitivity and high detection limit, and is easily interfered by lactose, and the lactulose has various optical isomers, so that the content of the lactulose cannot be accurately quantified by direct measurement.
Disclosure of Invention
The invention aims to solve the technical problem of researching and providing a method for detecting lactulose in dairy products, and the method can accurately and sensitively detect the lactulose content in the dairy products.
The invention provides the following technical scheme:
a method for detecting lactulose in dairy products comprises the following steps
(1) Taking a dairy product sample, diluting with water, adding a potassium ferrocyanide solution, a zinc sulfate solution and a phosphate buffer solution to form a sample diluent, diluting, adding beta-D-galactosidase for constant-temperature culture to obtain an enzymolysis solution, adding methanol for constant volume, adding an internal standard substance, and drying with nitrogen; adding methoxyamine solution for reaction, adding N-methyl-N-trimethylsilane trifluoroacetamide, cooling, diluting to constant volume, and removing precipitate to obtain solution to be detected;
(2) And (3) detecting and analyzing the liquid to be detected obtained in the step (1) of the detection method by using an ultra-high performance liquid chromatography tandem mass spectrometer, and calculating to obtain the lactulose content.
According to the invention, the dairy product in step (1) is pasteurized milk, UHT milk, raw milk or other dairy products; for example the milk product is pasteurized milk in liquid form.
According to the invention, in step (1), the reaction temperature after the addition of beta-D-galactosidase is 45 ℃ to 55 ℃, for example 50 ℃; the culture time is 0.5-2h, such as 1h and 1.5 h.
According to the invention, in step (1), the reaction temperature after the methoxyamine is added is 30 ℃ to 40 ℃, for example 35 ℃, 37 ℃ and the reaction time is 1 to 1.5 h.
According to the invention, in step (1), the reaction temperature after the addition of N-methyl-N-trimethylsilane trifluoroacetamide is from 30 ℃ to 40 ℃, for example 35 ℃ at 37 ℃; the reaction time is 0.5-1.5h, such as 0.5h, 1h, 1.2 h.
According to the invention, the concentration of the potassium ferrocyanide solution in the step (1) is 100-150g/L, such as 110 g/L, 120 g/L and 130 g/L, the volume ratio of the potassium ferrocyanide solution to the dairy product sample is 2 (1-5), the concentration of the zinc sulfate solution is 168 g/L, and the volume ratio of the zinc sulfate solution to the dairy product sample is 2 (1-5).
According to the invention, the volume ratio of the beta-D-galactosidase to the sample diluent in the step (1) is 1 (1-5).
According to the invention, after the methanol diluent is dried by nitrogen in the step (1), the concentration of the added methoxyamine is 10-20 mg/mL, and the volume ratio of the methoxyamine solution to the dairy product sample is (1-3): 100. in the present invention, the amount of methoxyamine added is excessive relative to lactulose in the liquid sample to be tested to ensure that the sugars in the sample are fully reacted.
According to the invention, the volume ratio of the derivatization reagent N-methyl-N-trimethyl silane trifluoroacetamide added in the step (1) to the dairy product sample is 1: 50-100.
According to the invention, the internal standard substance added in the step (1) is similar to lactulose, but does not contain substances in the sample; for example, the internal standard is xylose. In one embodiment, the amount of the internal standard is not particularly limited, and can be determined reasonably with reference to the fact that the response intensity of the internal standard on a liquid chromatography tandem mass spectrometry instrument can reach the detection limit, for example, the concentration of the internal standard in the solution to be detected is 1 to 5 μ g/mL.
According to the invention, the precipitate is removed in step (1) by filtration using a 0.22 μm organic phase filter.
In one embodiment of the present invention, the analysis conditions of the hplc-tandem mass spectrometer in step (2) are:
mobile phase: a-0.1 aqueous formic acid solution; b-acetonitrile. A: B =5: 95;
flow rate: 0.5 mL/min;
a chromatographic column: c 18 A chromatographic column;
sample introduction volume: 2 mu L of the solution;
target analyte ion pair information: 570.4 > 319.3, 570.4 > 103.1;
an ion source: electrospray (ESI +);
the scanning mode is as follows: multiple Reaction Monitoring (MRM);
spraying voltage: 4500V;
ionization temperature: 500 ℃;
de-clustering voltage: 56V.
The specification of the chromatographic column is as follows: length 75 mm x inner diameter 2.0 mm.
According to the invention, the content of lactulose in step (2) is quantified by an internal standard method.
The invention uses a high performance liquid chromatography tandem mass spectrometer to determine the content of lactulose in pasteurized milk based on the basic principle that: after proteins and fat in pasteurized milk are precipitated by inorganic salt reaction, lactose and lactulose are respectively enzymolyzed by beta-D-galactosidase to generate glucose and galactose, galactose and fructose, inorganic salt and part of glucose and galactose are separated out by methanol, internal standard is added, oximation reaction is carried out on the internal standard and methoxyamine at 37 ℃, N-methyl-N-trimethylsilane trifluoroacetamide is added for derivatization reaction, and qualitative and quantitative analysis is carried out on derivatives after the oximation reaction of fructose, so that the content of lactulose in the original pasteurized milk is obtained. Because the monosaccharide is derived Structural interconversion occurs biochemically, and to minimize or reduce this, the spatial structural distortion of the monosaccharide is reduced by an oximation reaction prior to derivatization, resulting in a relatively stable derivatized product. The derivative products of xylose, glucose, galactose and fructose in the solution to be tested are processed by C 18 After the chromatographic column is separated, the ion source and the mass analyzer are sequentially fed into the ion source and the mass analyzer, so that each substance has specific retention time and corresponding characteristic ion abundance ratio, and the purposes of separating and determining the nature of each substance are achieved. And (3) carrying out quantitative analysis by an internal standard method through scanning and integrating chromatographic peaks of the fructose derivative and the xylose derivative to obtain the concentration of the fructose derivative in the liquid to be detected, so as to obtain the content of lactulose in the pasteurized milk sample.
The invention has the beneficial effects that:
the method uses potassium ferrocyanide and zinc sulfate solution with certain concentration as a precipitating agent of protein and fat in pasteurized milk, effectively separates the protein and fat, and does not change the water phase property of the original sample.
The invention uses beta-D-galactosidase to carry out enzymolysis on the disaccharide into monosaccharide, and avoids the reality that the lactose content in pasteurized milk is too high and the lactose and lactulose can not be completely separated. By utilizing the difference of the solubility of the glucose, the galactose and the fructose in the organic reagent, partial glucose and galactose are removed, and the interference of the glucose and the galactose on the fructose in the instrumental analysis is reduced.
According to the invention, N-methyl-N-trimethylsilane trifluoroacetamide is adopted to carry out derivatization reaction on a target compound, the molecular weight of a target object to be detected is increased, a liquid to be detected is scanned by using a liquid chromatography tandem mass spectrometer, the derivative provides a larger mass-to-charge ratio, the sensitivity of parent ions is increased, the selection range of daughter ions is expanded, and qualitative and quantitative analysis on the compound is facilitated.
The oximation reaction is carried out on the methoxylamine solution dissolved in the pyridine and glucose, galactose and fructose generated after enzymolysis. The method inhibits the spatial isomerization phenomenon of monosaccharide by increasing the using amount of methoxyamine, increasing the reaction time and strictly controlling the reaction conditions, so that the products of subsequent derivatization reaction are more concentrated, and the inconvenience in detection caused by excessive derivative types is avoided.
The invention adopts xylose as an internal standard to carry out quantitative analysis on the substance to be detected. Free xylose is not found in nature, so that the pasteurized milk sample does not contain xylose background, and xylose is introduced as an internal standard substance in the detection process, so that the lactulose can be quantified more accurately.
Drawings
FIG. 1 is a chromatogram of a derivative of a glucose standard.
FIG. 2 is a chromatogram of a galactose standard substance derivative.
FIG. 3 is a chromatogram of a fructose standard derivative.
FIG. 4 is a chromatogram of a xylose standard derivative.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The materials are commercially available from the open literature unless otherwise specified.
Example 1: and adding the lactulose standard solution into the weighed fresh milk sample, and uniformly mixing to obtain the lactulose standard sample of the embodiment.
(1) Sample pretreatment
5.00 mL of lactulose spiked sample was pipetted into a 50 mL centrifuge tube, followed by addition of 5.0 mL of water, 2.0 mL of 130 g/L potassium ferrocyanide solution, 2.0 mL of 168 g/L zinc sulfate solution, and 6.0 mL of phosphate buffer (48 g/L disodium hydrogen phosphate, 8.6 g/L sodium dihydrogen phosphate, 1 g/L magnesium sulfate, pH adjusted to 7.5) in that order, and each reagent was added and mixed thoroughly by vortexing. After mixing well, centrifuging at 8000 r/min for 5min at 4 deg.C, collecting 1.0 mL supernatant, adding 4.0 mL phosphate buffer (48 g/L disodium hydrogen phosphate, 8.6 g/L sodium dihydrogen phosphate, 1 g/L magnesium sulfate, adjusting pH to 7.5), and mixing well to obtain diluted solution. Sucking 1.00 mL of diluent, adding 200 μ L of beta-D-galactosidase suspension (enzyme concentration is 40 mg/mL), slightly shaking, and performing enzymolysis reaction at 50 ℃ for 1 h to obtain an enzymolysis solution.
Taking 0.60 mL of enzymolysis liquid, diluting methanol to 5 mL, performing ultrasonic treatment for 5 min, and centrifuging at 4 deg.C at 8000 r/min for 5 min. 2.0 mL of the supernatant was added with 3.0 mL of methanol, and centrifuged at 8000 r/min at 4 ℃ for 5 min. 1.0 mL of the supernatant was taken, 50. mu.L of xylose internal standard (final xylose concentration 1. mu.g/mL) was added, and nitrogen was purged to dryness at 35 ℃. 150 μ L of a 20 mg/mL methoxyamine solution was added to the centrifuge tube and reacted at 37 ℃ for 1.5 h. After the oximation reaction, 50. mu. L N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added and reacted at 37 ℃ for 0.5 h. After derivatization reaction, 0.8 mL of acetonitrile is added, the mixture is filtered through a 0.22-micron organic filter membrane, and the filtrate is collected to be detected.
(2) The analysis conditions of the ultra-high performance liquid chromatography tandem mass spectrometer are as follows:
chromatographic conditions are as follows:
mobile phase: a-0.1 aqueous formic acid; b-acetonitrile. A: B = 5: 95;
flow rate: 0.5 mL/min; a chromatographic column: c 18 A chromatographic column; sample introduction volume: 2 mu L of the solution;
mass spectrum conditions:
target analyte ion pair information: 570.4 > 319.3, 570.4 > 103.1;
an ion source: electrospray (ESI +); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
spraying voltage: 4500V; ionization temperature: 500 ℃; de-clustering voltage: 56V.
The sample is subjected to a labeling recovery test by the analysis method, accurate maps and corresponding information can be obtained in each analysis, the lactulose content and related recovery information in the sample are calculated according to a standard curve obtained by analysis of a standard strain column gradient instrument (determination of the standard curve: preparation of lactulose standard solution with the concentration of 0.2, 0.4, 1.0, 2.0 and 10.0 mug/mL, the processing method is the same as that of the sample, finally, the concentration measured in the on-machine process is 8, 16, 40, 80 and 400 ng/mL through reaction and derivation, and the table 1 shows.
TABLE 1 standard recovery test results of lactulose in fresh milk
Example 2:
a pasteurized milk sample was prepared.
(1) Sample pretreatment
5.00 mL of pasteurized milk sample was taken out of a 50 mL centrifuge tube, and 5.0 mL of water, 2.0 mL of 130 g/L potassium ferrocyanide solution, 2.0 mL of 168 g/L zinc sulfate solution, and 6.0 mL of phosphate buffer (48 g/L disodium hydrogen phosphate, 8.6 g/L sodium dihydrogen phosphate, 1 g/L magnesium sulfate, pH adjusted to 7.5) were added in this order, and each reagent was added and mixed thoroughly by vortexing. After mixing, the mixture was centrifuged at 8000 r/min for 5 min at 4 ℃ to obtain 1.0 mL of supernatant, and 4.0 mL of phosphate buffer (48 g/L disodium hydrogenphosphate, 8.6 g/L sodium dihydrogenphosphate, 1 g/L magnesium sulfate, pH adjusted to 7.5) was added thereto and mixed well. 1.0 mL of the diluted solution was aspirated, 0.2 mL of a beta-D-galactosidase suspension (enzyme concentration: 40 mg/mL) was added thereto, and the mixture was gently shaken and subjected to an enzymatic reaction at 50 ℃ for 1 hour.
Taking 0.60 mL of enzymolysis liquid, diluting methanol to 5 mL, performing ultrasonic treatment for 5 min, and centrifuging at 4 deg.C at 8000 r/min for 5 min. 2.0 mL of the supernatant was added with 3.0 mL of methanol, and centrifuged at 8000 r/min at 4 ℃ for 5 min. 1.0 mL of the supernatant was taken, 50. mu.L of xylose internal standard (final xylose concentration 1. mu.g/mL) was added, and nitrogen was purged to dryness at 35 ℃. 150 mu.L of methoxyamine solution with the concentration of 20 mg/mL is added into a centrifuge tube, and oximation reaction is carried out for 1.5 h at 37 ℃. After the oximation reaction, 50. mu. L N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added and reacted at 37 ℃ for 0.5 h. After derivatization reaction, 0.8 mL of acetonitrile is added, the mixture is filtered through a 0.22-micron organic filter membrane, and the filtrate is collected to be detected.
(2) The analysis conditions of the ultra-high performance liquid chromatography tandem mass spectrometer are as follows:
chromatographic conditions are as follows:
mobile phase: a-0.1 aqueous formic acid solution; b-acetonitrile. A: B = 5: 95;
flow rate: 0.5 mL/min; a chromatographic column: c 18 ChromatographyA column; sample introduction volume: 2 mu L of the solution;
mass spectrum conditions:
target analyte ion pair information: 570.4>319.3, 570.4> 103.1;
an ion source: electrospray (ESI +); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
spraying voltage: 4500V; ionization temperature: 500 ℃; de-clustering voltage: 56V.
The data of the sample is analyzed by the analysis method, and the content of lactulose in the sample is calculated according to a standard curve obtained by the analysis of a standard substance series gradient instrument, and is shown in table 2.
TABLE 2 lactulose content test results in fresh milk
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A method for detecting lactulose in dairy products comprises the following steps:
(1) taking a dairy product sample, diluting the dairy product sample with water, adding a potassium ferrocyanide solution, a zinc sulfate solution and a phosphate buffer solution to form a sample diluent, diluting the sample diluent, adding beta-D-galactosidase to culture at constant temperature, adding methanol, centrifugally separating, taking supernatant, adding an internal standard substance, and drying by nitrogen; adding methoxyamine solution for reaction, adding N-methyl-N-trimethylsilane trifluoroacetamide, cooling, diluting to constant volume, and removing precipitate to obtain solution to be detected;
(2) detecting and analyzing the liquid to be detected obtained in the step (1) of the detection method by using an ultra-high performance liquid chromatography tandem mass spectrometer to obtain the content of lactulose;
The analysis conditions of the ultra-high performance liquid chromatography tandem mass spectrometer in the step (2) are as follows:
mobile phase: a-0.1 aqueous formic acid solution; b-acetonitrile; a: B =5: 95;
flow rate: 0.5 mL/min;
a chromatographic column: c 18 A chromatographic column;
sample introduction volume: 2 mu L of the solution;
target analyte ion pair information: 570.4 > 319.3, 570.4 > 103.1;
an ion source: electrospray (ESI +);
the scanning mode is as follows: multiple Reaction Monitoring (MRM);
spraying voltage: 4500V;
ionization temperature: 500 ℃;
de-clustering voltage: 56V;
the specification of the chromatographic column is as follows: length 75 mm x inner diameter 2.0 mm.
2. The method for detecting lactulose in dairy product according to claim 1, wherein the dairy product in step (1) is pasteurized milk, UHT milk, fresh milk or other dairy products.
3. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein in step (1), the volume ratio of beta-D-galactosidase to sample diluent is 1: 1-5.
4. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein the reaction temperature is 45-55 ℃ after the beta-D-galactosidase is added in the step (1); the culture time is 0.5-2 h.
5. The method for detecting lactulose in dairy product as claimed in claim 1 or 2, wherein the concentration of the potassium ferrocyanide solution in the step (1) is 100-150g/L, and the volume ratio of the potassium ferrocyanide solution to the dairy product sample is 2: 1-5.
6. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein the concentration of the zinc sulfate solution is 168 g/L, and the volume ratio of the zinc sulfate solution to the dairy product sample is 2: 1-5.
7. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein the concentration of the added methoxyamine after the methanol diluent is dried by nitrogen in step (1) is 10-20 mg/mL, and the amount of methoxyamine solution is excessive relative to the amount of lactulose in the solution.
8. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein in the step (1), the reaction temperature is 30-40 ℃ after the methoxylamine is added, and the reaction time is 1-1.5 h.
9. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein in the step (1), the volume ratio of the derivatization reagent N-methyl-N-trimethylsilane trifluoroacetamide added in the step (1) to the dairy product sample is 1: 50-100.
10. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein the reaction temperature is 30-40 ℃ after the N-methyl-N-trimethylsilane trifluoroacetamide is added, and the reaction time is 0.5-1.5 h.
11. The method for detecting lactulose in dairy product according to claim 1, wherein the internal standard substance added in the step (1) is xylose.
12. The method for detecting lactulose in dairy product according to claim 1, wherein the step (1) is performed by filtering to remove precipitate, and the filtering is performed by using a 0.22 μm organic phase filter membrane.
13. The method for detecting lactulose in dairy product according to claim 1 or 2, wherein the content of lactulose in step (2) is quantified by an internal standard method.
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