CN114216982B - Vitamin B12Is a detection method of (2) - Google Patents
Vitamin B12Is a detection method of (2) Download PDFInfo
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- CN114216982B CN114216982B CN202111531281.XA CN202111531281A CN114216982B CN 114216982 B CN114216982 B CN 114216982B CN 202111531281 A CN202111531281 A CN 202111531281A CN 114216982 B CN114216982 B CN 114216982B
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Classifications
-
- 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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a detection method of vitamin B 12. The invention provides a detection method of four vitamins B 12 in a dairy product, which comprises the following steps: sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain a primary extract; a two-dimensional liquid chromatography separation step: separating the four vitamins B 12 from the primary extract by chromatographic separation using two-dimensional high performance liquid chromatography; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography; mass spectrum detection: and carrying out mass spectrum detection on the four vitamins B 12 after the two-dimensional high performance liquid chromatography separation.
Description
Technical Field
The invention relates to the field of foods, in particular to the technical field of analysis and detection of total nutrient substances in the field of foods, and more particularly relates to a detection method of four vitamins B 12 in dairy products.
Background
Vitamin B 12(Vitamin B12, abbreviated as VB 12), also known as cobalamin, generally refers to the cobalamin family with the same biological activity, also the only water-soluble vitamins containing metallic elements. Vitamin B 12 is an important nutrient essential to the human body and functions as a coenzyme for several key enzymes in single carbon (methyl) metabolism; the preparation method plays a synergistic effect in various biochemical reactions, is an important coenzyme in the DNA synthesis process, has a promoting effect on the formation and growth of red blood cells, participates in the regulation of nervous system and immune system and the physiological activities such as the metabolism of cells in human bodies, and is vital to the growth and development of human beings.
Vitamin B 12 is produced only in microorganisms (mecobalamin, adenosylcobalamin and hydroxycobalamin) and therefore its food sources are limited to non-plant sources such as meat or other animal sources. Theoretically, vitamin B 12 deficiency is rare in normal healthy people, but pure vegetarian, pregnant women, infants and eating disorder patients are prone to vitamin B 12 deficiency due to their special dietary habit requirements and physiological needs. In the absence of vitamin B 12, increasing folate supply can cure anemia, but not neurological symptoms. Deficiency of vitamin B 12 may cause peripheral neuritis and anemia in children, and may affect development of infants, etc., but excessive supplementation of vitamin B 12 may cause adverse effects such as anaphylaxis. Therefore, the vitamin B 12 is an important index of nutrition of infant formula food, and the detection requirement is more accurate.
In addition, vitamin B 12 is known to exist in the form of hydroxycobalamin, 5' -deoxyadenosylcobalamin, mecobalamin, and cyanocobalamin. Cyanocobalamin is relatively easy to produce in large quantities and has high stability, so it is the form of vitamin B 12 that is most used in food processing, however, cyanocobalamin is not biologically active until the cyanide groups are enzymatically removed, and cyanocobalamin only works after conversion to mecobalamin and 5' -deoxyadenosylcobalamin in vivo.
The bioavailability of the synthetic form of vitamin B 12 is inversely proportional to the amount given, less than 4% in humans and animals receiving prophylactic or therapeutic supplements. Hydroxycobalamin, 5' -deoxyadenosylcobalamin, mecobalamin are naturally occurring forms of vitamin B 12. 5' -deoxyadenosylcobalamin and mecobalamin have coenzyme activity and biological activity in mammalian cells, while hydroxycobalamin is the product of their photolysis. Matte et al in 2012 (J.J.Matte, M.Britten, C.L.Girard, animal Frontiers, volume 4,Issue 2,April 2014,Pages 32-37) in an experiment using pigs as an animal model of humans indicated that daily intake of vitamin B 12 was more effective than the synthetic form (cyanocobalamin) used in daily intake of vitamin supplements.
In addition, assessing the availability of intestinal absorption of different forms of vitamin B 12 is also an important factor in assessing the quality of its source, vitamin B 12 deficiency, essentially mecobalamin and adenosylcobalamin deficiency in vivo. Therefore, with the development of the market for dietary supplements and infant formulas and consumer demand, more and more new products contain mecobalamin and other "natural forms of vitamin B 12", and in response to this market trend, a detection method for measuring all four vitamins B 12 alone is of great importance.
Currently, the method for measuring VB 12 is GB 5413.14-2010 national food safety standard (determination of vitamin B 12 in infant food and dairy products) and the adopted method is a microbiological method. The american society of analytical chemists (AOAC) official method 2016.017, which uses cyanide to change all of the VB 12 forms to cyanocobalamin and uses a single-use dedicated immunosorbent cartridge in each assay, increases the sensitivity of the method but adds additional cost. There are also ICP-MS method using cobalt element profile quantification, ELISA method, etc. These methods can only determine the total amount of VB 12 and cannot distinguish between different forms of VB 12.
As can be seen, there have been many approaches to the determination of vitamin B12 content in foods, fortified foods and dietary supplements. Most methods can only measure total VB 12 levels due to low vitamin B12 fortification. Since cyanocobalamin is the most stable form of all four VB 12. Thus, many current methods of detecting VB 12 in foods and dietary supplements utilize cyanide to change all VB 12 forms to cyanocobalamin, which reflects the overall level of cyanocobalamin.
Currently, these methods are successful in determining the total amount of VB 12, however, the use of cyanide in the laboratory presents safety issues. The microorganism detection method is a commonly used vitamin B 12 detection method, and the microorganism is commonly selected from Lactobacillus leishmaniasis. This approach has high sensitivity, but is poorly selective, sometimes overestimated, due to the lack of specificity of some inactive cobalamin precursors (corrines (Corrin), C 19H22N4) that interfere with the growth of microorganisms. Inactive cobalamin precursors interfere with the detection of active VB 12 and overestimate the overall VB 12 level.
Therefore, it is important to develop a method that can distinguish between inactive cobalamin precursors and can distinguish between the four VB 12.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problems that four vitamins B 12 cannot be distinguished in the existing vitamin B 12 detection method, cyanide is used, and interference detection results exist in cobalamin precursors, and the like, the invention provides a detection method of four vitamins B 12 in a dairy product, which adopts a two-dimensional high performance liquid chromatography-mass spectrometry technology, samples are digested in advance by pepsin, so that vitamin B 12 combined with proteins is released, primary extract passes through one-dimensional chromatography, size-exclusion chromatographic columns are used for eluting macromolecular proteins before 4 vitamins B 12, 4 vitamins B 12 are eluted after being eluted due to small molecular weight relative proteins, and eluent is switched to a two-dimensional reversed-phase analysis column according to elution time, so that 4 vitamins B 12 are separated, and 147.1 (m/z) is selected as quantitative ions for mass spectrometry detection, so that the interference of inactive cobalamin precursors can be effectively avoided, and the accuracy of the method is improved.
Solution for solving the problem
Through intensive researches of the inventor, the technical problems can be solved through the following scheme:
[1] The invention provides a detection method of four vitamins B 12 in a dairy product, wherein the detection method comprises the following steps:
Sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain a primary extract;
A two-dimensional liquid chromatography separation step: separating the four vitamins B 12 from the primary extract by chromatographic separation using two-dimensional high performance liquid chromatography; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography;
Mass spectrum detection: and carrying out mass spectrum detection on the four vitamins B 12 after the two-dimensional high performance liquid chromatography separation.
[2] The detection method according to [1], wherein the protease is one or more selected from the group consisting of pepsin, trypsin, cathepsin, papain and subtilisin.
[3] The detection method according to [1] or [2], wherein the quantitative ions of the four vitamins B 12 are 147.1m/z; and/or the four vitamins B 12 are hydroxycobalamin, 5' -deoxyadenosylcobalamin, mecobalamin and cyanocobalamin.
[4] The detection method according to any one of [1] to [3], wherein the conditions of the one-dimensional liquid phase size exclusion chromatography are: the mobile phase is an aqueous solution of 5-15% polar solvent by volume.
[5] The detection method according to any one of [1] to [4], wherein the conditions of the two-dimensional liquid phase inversion chromatography are: the mobile phase comprises an A phase and a B phase, and gradient elution is carried out, wherein the A phase is a buffer solution phase, and the B phase is a polar solvent phase.
[6] The detection method according to [5], wherein the gradient elution in volume percent comprises:
0min: 80-95% of phase A and 5-20% of phase B;
1min: 80-95% of phase A and 5-20% of phase B;
10min: 40-60% of phase A and 40-60% of phase B;
12min: 40-60% of phase A and 40-60% of phase B;
12.1min: 5-20% of phase A and 80-95% of phase B;
15min: 5-20% of phase A and 80-95% of phase B;
16min: 80-95% of phase A and 5-20% of phase B;
18min: 80-95% of phase A and 5-20% of phase B.
[7] The detection method according to any one of [1] to [6], wherein the conditions for mass spectrometry detection include: a charged spray ion source, positive ion scan mode, is employed.
[8] The detection method according to [7], wherein the collection of quantitative ion pairs in the mass spectrometry detection is as follows:
The parent ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
the parent ion m/z 672.80 of mecobalamin and the quantitative ion m/z 147.1;
The parent ion m/z 673.79 of hydroxycobalamin and the quantitative ion m/z 147.1;
The parent ion of 5' -deoxyadenosylcobalamin, m/z 790.34, was quantified at ion m/z 147.1.
[9] The detection method according to any one of [1] to [8], wherein the detection method further comprises a step of constructing a standard curve, comprising: and respectively adopting four standard substances of vitamin B 12 to manufacture a standard curve through the two-dimensional liquid chromatography and mass spectrometry detection.
[10] The detection method according to any one of [1] to [9], wherein the step of performing the two-dimensional liquid chromatography separation further comprises the step of adding a stable isotope internal standard.
ADVANTAGEOUS EFFECTS OF INVENTION
Through implementation of the technical scheme, the invention can obtain the following technical effects:
(1) The detection method of the four vitamins B 12 in the dairy product provided by the invention can accurately detect the four forms of the vitamin B 12.
(2) The detection method of the four vitamins B 12 in the dairy product provided by the invention avoids the safety and environmental protection problems caused by cyanide.
(3) Compared with the existing microbial method, the detection method of the four vitamins B 12 in the dairy product can avoid the problems of poor selectivity and overestimated vitamin B 12 content caused by the interference of inactive cobalamin precursors on the growth of microorganisms.
Drawings
Fig. 1 is a two-dimensional high performance liquid chromatography-mass spectrum diagram of detecting 4 vitamins B 12 by adopting the detection method of four vitamins B 12 in the dairy product. Wherein a of fig. 1 is a total ion flow diagram; b in FIG. 1 is 5' -deoxyadenosylcobalamin; c in fig. 1 is cyanocobalamin; d in fig. 1 is mecobalamin; e in FIG. 1 is hydroxycobalamin.
Fig. 2A-2C are schematic diagrams of flow path states of a six-way valve switching control instrument adopted in a two-dimensional high performance liquid chromatography-mass spectrometry in the detection method of four vitamins B 12 in a dairy product according to the present invention; wherein, FIG. 2A is a one-dimensional chromatographic column purification; FIG. 2B shows the valve switched to the enrichment ring, 4 vitamins B 12 being enriched in the ring; fig. 2C shows the valve switched to the analytical column, through which 4 vitamins B 12 were separated and detected by mass spectrometry.
FIGS. 3A-3D are ion scans of 4 vitamins B 12; wherein FIG. 3A is 5' -deoxyadenosylcobalamin; FIG. 3B is mecobalamin; FIG. 3C is hydroxycobalamin; fig. 3D is cyanocobalamin.
FIG. 4 is a schematic diagram of the comparative example without one-dimensional liquid phase size exclusion chromatography, with two-dimensional liquid phase reversed phase chromatography after pretreatment and mass spectrometry detection. Wherein a in fig. 4 is 5' -deoxyadenosylcobalamin; b in fig. 4 is cyanocobalamin; c in fig. 4 is mecobalamin; d in FIG. 4 is hydroxycobalamin.
Detailed Description
The following describes the present invention in detail. The following description of the technical features is based on the representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:
In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.
In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
The detection method adopts a two-dimensional high performance liquid chromatography-mass spectrometry technology, a dairy product sample to be detected is digested by protease in advance, so that vitamin B 12 combined with protein in the dairy product sample to be detected is released, the obtained primary extract is eluted before four vitamins B 12 by using one-dimensional chromatography of size-array chromatographic columns, the four vitamins B 12 are eluted after the elution due to small molecular weight relative to the protein (polypeptide), and the eluent is switched to the two-dimensional chromatography, namely an inverse analytical column according to the elution time, so that the four vitamins B 12 are separated and detected by mass spectrometry, wherein the mass spectrometry detection can effectively avoid the interference of inactive cobalamin precursors, the accuracy of the method is improved, and cyanide is not needed.
Specific information for the four vitamins B 12 and inactive cobalamin precursors is given in table 1 below.
Table 1:
The method for detecting the four vitamins B 12 in the dairy product of the invention will be specifically described below.
< Sample Source and pretreatment >
In the detection method of the invention, the sample to be detected is a dairy product.
There is no particular limitation in the present invention regarding the "dairy product" or source of the test sample, which may refer to food products produced by animals such as cows, goats, sheep, yaks, horses, camels, and other mammals.
Examples of dairy products are low fat milk (e.g. 0.1%, 0.5% or 1.5% fat), non-fat milk, milk powder, whole milk products, butter, buttermilk products, skim milk products, high milk fat products, rendered milk, fresh cream, cheese, ice cream and confectionery products, probiotic beverages or probiotic yoghurt-type beverages. Wherein "milk powder" refers to an artificial dairy product made by evaporating milk to dryness. In some preferred embodiments of the invention, the sample in the detection method of the invention is derived from dairy products of cattle or sheep.
Further, since vitamin B 12 is an important indicator of nutrition in infant formulas, especially formula milk products. Therefore, the detection method of the four vitamins B 12 in the dairy product is particularly suitable for infant formula dairy products. In some embodiments of the invention, the four vitamins B 12 include hydroxycobalamin, 5' -deoxyadenosylcobalamin, mecobalamin, and cyanocobalamin.
In some embodiments of the invention, prior to performing a two-dimensional high performance liquid chromatography-tandem mass spectrometry test, the dairy sample to be tested is subjected to a pretreatment step to render the sample suitable for testing and preliminary enrichment of the test object.
Specifically, the sample pretreatment step adopts protease to carry out enzymolysis reaction on a dairy product sample to be detected, and supernatant fluid is obtained by centrifugation, thus obtaining primary extract. Through the sample pretreatment step, the combined action of the buffer solution and the protease is used, so that the vitamin B 12 combined with the protein in the dairy product sample to be detected is released, and the subsequent two-dimensional high performance liquid chromatography separation and mass spectrum detection are performed, so that the detection is more accurate.
In some embodiments of the invention, the protease is selected from one or more of pepsin, trypsin, cathepsin, papain, and subtilisin. In some preferred embodiments of the invention, the protease is pepsin. In some embodiments of the invention, the amount of enzyme may be adjusted according to the amount of added dairy sample to be tested, e.g. 50000-100000U protease is added to 5-10 g of dairy sample to be tested.
The enzymatic hydrolysis reaction is usually carried out in a buffer solution, which may be sodium acetate buffer solution. In some preferred embodiments of the invention, the buffer is sodium acetate buffer. The concentration of the sodium acetate buffer is 40 to 60mmol/L, preferably 40 to 55mmol/L, more preferably 45 to 55mmol/L, and even more preferably 50mmol/L. In some preferred embodiments of the invention, the pH of the sodium acetate buffer is 4.0.+ -. 0.5, preferably 4.0.+ -. 0.4, 4.0.+ -. 0.3, 4.0.+ -. 0.2, more preferably 4.0.+ -. 0.1. In some embodiments of the invention, the amount of buffer may be adjusted according to the amount of dairy sample to be added, for example, 5-10 g of dairy sample to be tested is added per 100mL of buffer. In other embodiments of the present invention, the buffer may be phosphate buffer or citrate buffer, and the ionic strength and pH of the buffer may be equivalent to those of the sodium acetate buffer described above.
In some preferred embodiments of the invention, the reaction solution may be diluted to a final volume (fixed volume) by adding buffer after the enzymatic hydrolysis reaction is performed in a buffer smaller than the final volume (fixed volume) used for the buffer. Under a smaller volume, the protease can be more fully contacted with the dairy product sample to be tested so as to exert enzymolysis effect and improve detection efficiency. For example, when the final volume of the buffer solution is 100mL, the dairy product sample to be tested and the protease may be added to 30mL, 40mL, 50mL, 60mL or 70mL of the buffer solution for enzymolysis, and then the volume is fixed to 100mL.
The temperature and time of the enzymatic hydrolysis reaction may be adjusted according to the type of protease selected, for example, in some embodiments of the present invention, the enzymatic hydrolysis reaction is carried out at 35 to 39℃for 15 to 50 minutes. Preferably, heating in a 37 ℃ water bath for 15-50 min; more preferably, the heating in a water bath at 37℃is carried out for 20 to 40 minutes, and still more preferably, the heating in a water bath at 37℃is carried out for 30 minutes. The heating method is not particularly limited, and may be, for example, water bath heating or the like.
In some embodiments of the invention, the enzymatic hydrolysis reaction is followed by a centrifugation step. In some embodiments of the invention, the enzymatic reaction product is centrifuged after shaking up at 7000 to 15000 rpm for 5 to 15 minutes. Preferably, the centrifugation conditions are 10000 revolutions per minute for 10min. After centrifugation, the supernatant was taken as the primary extract of vitamin B 12.
In some embodiments of the invention, a stable isotope internal standard is also added to the buffer. In other embodiments of the invention, stable isotope internal standards may also be added prior to performing a two-dimensional high performance liquid chromatography separation step. The addition amount of the stable isotope internal standard is 5-20 ng of the stable isotope internal standard added into each 5-10 g of dairy product sample to be detected. The use of stable isotope internal standard can avoid potential matrix effects in subsequent two-dimensional high performance liquid chromatography-tandem mass spectrometry detection.
< Two-dimensional liquid chromatography separation >
In the present invention, two-dimensional liquid chromatography is used to separate the individual components of vitamin B 12 in the sample. In the present invention, the two-dimensional liquid chromatography may also be referred to as two-dimensional high performance liquid chromatography.
In some embodiments of the invention, the primary extract obtained in the sample pretreatment step is chromatographically separated using two-dimensional high performance liquid chromatography, such that the four vitamins B 12 are separated from the primary extract, reducing mass spectral interference. The two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography, which can be linked through valve switching, and the valve can be a manual valve or an automatic switching valve, preferably an automatic switching six-way valve.
In the invention, the macromolecular proteins (polypeptides) in the primary extract are eluted before 4 vitamins B 12 (liquid phase size exclusion method is used for purification) by the one-dimensional chromatography, 4 vitamins B 12 are eluted later due to the small molecular weight relative to the proteins (polypeptides), and the eluent is switched to a two-dimensional liquid phase reversed phase analytical column according to the elution time (for example, an online flow component switching method switched by a six-way valve) so that 4 vitamins B 12 are separated.
In the present invention, the column used for one-dimensional liquid size exclusion chromatography may be a size exclusion column suitable for separating proteins/polypeptides from vitamins, preferably a water-soluble size exclusion column, e.g. having a pore size ofSize exclusion chromatography column of (2), preferablyMore preferablyToo small pore size can not be separated, is easy to block, and too large pore size can easily cause synchronous outflow of vitamin B 12 and protein/polypeptide, thereby increasing interference. Size exclusion chromatography columns are commercially available, for example, commercially available Agilent Bio SEC-5 can be used: 5190-2523 chromatographic column with inner diameter of 4.6mm, length of 300mm, particle size of 5 μm and pore size
In some embodiments of the invention, the chromatographic conditions of one-dimensional liquid phase size exclusion chromatography include: the mobile phase is a polar solvent aqueous solution with a volume percentage of 5-15%, and acetonitrile is a preferred polar solvent from the viewpoints of compatibility with reverse phase chromatography and elution purification. In some preferred embodiments, the mobile phase is an aqueous acetonitrile solution of 5 to 10% by volume, more preferably an aqueous acetonitrile solution of 10% by volume; the flow rate is 0.4.+ -. 0.2mL/min, preferably 0.4.+ -. 0.1mL/min, more preferably 0.4mL/min. The column temperature of the chromatographic column is 40+ -3deg.C, preferably 40+ -2deg.C, and 40+ -1deg.C. The sample injection volume in the one-dimensional liquid phase size exclusion chromatography is 500+ -100 μL, preferably 500+ -50 μL, more preferably 500+ -20 μL, 500+ -10 μL.
In some embodiments of the invention, the detection wavelength employed in one-dimensional liquid phase size exclusion chromatography is 550nm. In some more specific embodiments of the invention, the wavelength at 550nm of the mobile phase can be detected by an ultraviolet detector to determine the retention time of one-dimensional liquid phase size exclusion chromatography, as well as to detect missed-out, migration time drift.
In the present invention, the two-dimensional liquid phase reversed phase chromatography may employ any reversed phase chromatography column suitable for separating vitamins, such as a nonpolar chromatography column, typically a C18 reversed phase chromatography column. Reverse phase chromatography columns are commercially available, for example from Zorbax Plus-C18 RRHD, having an inner diameter of 2.1mm, a length of 150mm and a particle size of 1.8. Mu.m.
In some embodiments of the invention, the chromatographic conditions of two-dimensional liquid phase reversed phase chromatography include: the mobile phase comprises a phase A and a phase B, wherein the phase A is a buffer solution phase, so that on one hand, the separation property of the reversed phase chromatography can be improved, and on the other hand, the ionization property of electrospray can be improved when the mobile phase is compatible with mass spectrum. In some embodiments, phase A may be 7 to 15mmol/L aqueous ammonium formate, preferably 9 to 12mmol/L aqueous ammonium formate, more preferably 10mmol/L aqueous ammonium formate; the phase B is a polar solvent, preferably the same as the polar solvent in one-dimensional chromatography, and may be acetonitrile to increase the operation convenience of automatic detection. The two-dimensional liquid phase reversed phase chromatography adopts gradient elution, flow rate: 0.4.+ -. 0.2mL/min, preferably 0.4.+ -. 0.1mL/min. The column temperature of the chromatographic column is 35+ -3deg.C, preferably 35+ -2deg.C and 35+ -1deg.C.
In some more specific embodiments of the invention, the gradient elution comprises:
0min: 80-95% of phase A and 5-20% of phase B;
1min: 80-95% of phase A and 5-20% of phase B;
10min: 40-60% of phase A and 40-60% of phase B;
12min: 40-60% of phase A and 40-60% of phase B;
12.1min: 5-20% of phase A and 80-95% of phase B;
15min: 5-20% of phase A and 80-95% of phase B;
16min: 80-95% of phase A and 5-20% of phase B;
18min: 80-95% of phase A and 5-20% of phase B.
In some preferred embodiments of the invention, the gradient elution comprises:
0min: 90% of phase A and 10% of phase B;
1min: phase a 90%, phase B10;
10min: 50% of phase A and 50% of phase B;
12min: 50% of phase A and 50% of phase B;
12.1min: 10% of phase A and 90% of phase B;
15min: 10% of phase A and 90% of phase B;
16min: 90% of phase A and 10% of phase B;
18min: 90% of phase A and 10% of phase B.
< Mass Spectrometry detection >
In some embodiments of the invention, mass spectrometry is performed on four vitamins B 12 after two-dimensional high performance liquid chromatography.
The conditions for mass spectrometry detection include: using a charged spray ion source, positive ion scan mode, capillary temperature: 300+/-50 ℃; capillary voltage: +3.5.+ -. 0.5KV.
The mass spectrum detection collection quantitative ion pairs are as follows:
The parent ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
the parent ion m/z 672.80 of mecobalamin and the quantitative ion m/z 147.1;
The parent ion m/z 673.79 of hydroxycobalamin and the quantitative ion m/z 147.1;
The parent ion of 5' -deoxyadenosylcobalamin, m/z 790.34, was quantified at ion m/z 147.1.
In the quantitative test, the quantitative ions of each component of vitamin B 12 are determined to be 147.1m/z, so that the interference of interference peaks can be avoided more easily, the interference of inactive cobalamin on the premise can be avoided, and the accuracy of quantitative analysis is improved effectively.
< Construction of Standard Curve >
The standard curve is established to determine the basis of quantitative analysis comparison, and the detection limit and the quantitative limit of a detection system or a detection method can be determined.
The detection method of the four vitamins B 12 in the dairy product also comprises a step of constructing a standard curve, specifically, a series of standard substance working solutions with different concentrations are respectively prepared by adopting standard substances of the four vitamins B 12, the two-dimensional high performance liquid chromatography separation step and the mass spectrum detection step are carried out, and a standard curve regression equation is obtained according to the mapping of the peak area corresponding concentration of the parent ion chromatographic peak of each target compound (namely four vitamins B 12) of the detected standard substance working solution.
In addition, in the establishment of the standard curve, the accuracy of the standard curve is preferably verified by an isotope internal standard method.
The R value (linear correlation coefficient) of the standard curve in the present invention should be 0.99 or more.
Further, according to the constructed standard curve regression equation, the concentrations of the four vitamins B 12 in the dairy product sample to be detected are respectively obtained, and then the content of the four vitamins B 12 in the dairy product sample to be detected is calculated.
The detection method of each component of vitamin B 12 provided by the invention can be used for detecting dairy product terminal commodity, and can also be used as an online detection and monitoring method for dairy product production through automatic setting.
Examples
The invention is further illustrated by the following examples, which are not intended to be limiting. Specific materials and sources thereof used in embodiments of the present invention are provided below. It will be understood that these are merely exemplary and are not intended to limit the invention, as materials identical or similar to the type, model, quality, nature or function of the reagents and instruments described below may be used in the practice of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
1. Reagents and materials
1.1 Reagents
Unless otherwise specified, only analytically pure reagents were used.
1.1.1 Water, GB/T6682, first order.
1.1.2 Acetonitrile (CH 3 CN, chromatographically pure).
1.1.3 Methanol (CH 3 OH, chromatographically pure).
1.1.4 Formic acid (HCOOH, chromatographic purity).
1.1.5 Acetic acid (CH 3 COOH, chromatographically pure).
1.1.6 Sodium acetate (CH 3 COONa).
1.1.7 Ammonium formate (HCOONH 4, chromatographic purity).
1.1.8 Pepsin (vitality, 38U/mg).
1.1.9 Internal standard of cyanocobalamin-dimethylbenzimidazole- 13C7 isotope: the purity is more than or equal to 95 percent and 1 mug/mL.
1.2 Preparation of reagents
1.2.1 Sodium acetate buffer (50 mmol/L): 4.1g of anhydrous sodium acetate (1.1.6) was weighed, dissolved in 950mL of water and adjusted to pH 4.0.+ -. 0.1 with acetic acid (1.1.5) and then fixed to 1000mL of water.
1.2.2 Acetonitrile-water solution (10% by volume): 100mL of acetonitrile (1.1.2) was accurately measured, dissolved in water and the volume was set to 1000mL.
1.2.3 Ammonium formate aqueous solution (10 mmol/L): 0.63g of ammonium formate (1.1.7) was weighed, dissolved in 950mL of water, adjusted to pH 4.0.+ -. 0.1 with formic acid (1.1.4) and then fixed to volume 1000mL with water.
1.3 Standard substance
The standard used was all from Sigma-Aldrich and the details are shown in Table 2.
Table 2: standard substance information used in the embodiment of the invention
1.4 Preparation of Standard solution
1.4.1 Standard stock (1 mg/mL): weighing 10mg (accurate to 0.01 mg) of 4 VB12 standard substances respectively, dissolving with water, transferring to a 10mL volumetric flask, adding 5mL of methanol (1.1.3), metering to scale with water, subpackaging each 0.5mL of solution, transferring to a 1mL jaw sample bottle, sealing, and storing in dark at-20deg.C for use.
1.4.2 Standard working fluid (1. Mu.g/mL): accurately sucking 10 mu L (1.4.1) of 4 VB12 standard stock solutions into a 10mL volumetric flask, calibrating the volume with water, and storing in a dark place at 4 ℃.
1.4.3 Isotope internal standard working fluid: 0.5mL (1. Mu.g/mL) of VB12 isotope internal standard solution was taken and diluted to 10mL with water, and the concentration of the solution was 50ng/mL. Stored at 4℃in the absence of light.
1.4.4 Standard series of working solutions: 5 mu L, 10 mu L, 20 mu L, 50 mu L, 80 mu L, 100 mu L and 200 mu L of standard working solution (1.4.2) are respectively and accurately removed, and 200 mu L of isotope internal standard solution (1.4.3) is respectively added at the same time, and the volume is fixed to 10mL by using 10% acetonitrile aqueous solution (1.2.2). The concentrations are respectively as follows: 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, and the isotopic internal standard concentration is 1ng/mL. The standard series of solutions are formulated prior to use.
1.5 Materials
1.5.1 Microporous filtration membrane: aqueous phase, 0.22 μm.
1.5.2 Size exclusion chromatography column (one-dimensional chromatography): 4.6X103 mm,5 μm,Agilent Bio SEC-5：5190-2523。
1.5.3 Reverse phase chromatography column (two-dimensional chromatography): zorbax Plus-C18 RRHD, 2.1X105 mm,1.8 μm.
2. Apparatus and device
2.1 Two-dimensional high performance liquid chromatography-high resolution mass spectrometer: a charged spray ion source.
2.2 Balance: the sensing amount is 0.01g and 0.1mg.
2.3 Centrifuge: the rotating speed is not lower than 10000 revolutions/min.
2.4 Quantitative pipettor: 10 mu L-1 mL.
2.5 Centrifuge tube: 15mL.
2.6 Constant temperature water bath shaking table.
3. Sample pretreatment
5-10 G (accurate to 0.01 g) of the sample is accurately weighed in a 150mL brown conical flask, 200 mu L of isotope internal standard solution (1.4.3), 50mL of sodium acetate buffer solution (1.2.1) and 2g of pepsin (1.1.8) are sequentially added, and the mixture is fully mixed to fully dissolve the sample, and water bath is carried out at 37 ℃ for 30min. After cooling to room temperature, the sample solution was transferred to a 100mL volumetric flask, the volume was fixed to the scale with sodium acetate buffer (1.2.1), and after shaking up, centrifugation was performed for 10min at 10000 rpm, and 5mL of supernatant was aspirated for use.
4. Liquid chromatography-tandem mass spectrometry reference conditions
Fig. 2A to 2C are schematic views of an apparatus connection system for combined use of liquid chromatography and mass spectrometry according to an embodiment of the present invention.
4.1 Liquid chromatography reference conditions
4.1.1 One-dimensional chromatographic reference conditions
Chromatographic column: 4.6X103 mm,5 μm,Or equivalent.
Mobile phase: 10% acetonitrile in water (1.2.2).
Flow rate: 0.4mL/min.
Detection wavelength: 550nm.
Column temperature of chromatographic column: 40 ℃;
Sample injection volume: 500. Mu.L;
the six-way valve switching control schematic diagram is shown in fig. 2A to 2C, and the switching time is set according to the detection condition of the ultraviolet detector.
4.1.2 Two-dimensional chromatography (gradient Pump) chromatographic reference conditions
Chromatographic column: 2.1X105 mm,1.8 μm.
Mobile phase: phase A, 10mmol/L ammonium formate aqueous solution (1.2.3); phase B, acetonitrile (1.1.2)
Flow rate: 0.4mL/min.
Column temperature of chromatographic column: 35 ℃.
Gradient elution: see table 3.
Table 3: gradient elution conditions
| Sequence number | Time (min) | A(％) | B(％) | Curve of curve |
| 1 | 0 | 90 | 10 | 6 |
| 2 | 1 | 90 | 10 | 6 |
| 3 | 10 | 50 | 50 | 6 |
| 4 | 12 | 50 | 50 | 6 |
| 5 | 12.1 | 10 | 90 | 6 |
| 6 | 15 | 10 | 90 | 6 |
| 7 | 16 | 90 | 10 | 6 |
| 8 | 18 | 90 | 10 | 6 |
4.2 Mass Spectrometry reference Condition (ESI+)
The mass spectrum reference conditions for the 4 vitamins B 12 are shown in table 4. The instrument conditions are as follows: scanning mode: a positive ion; capillary temperature: 300 ℃; capillary voltage: +3.5KV.
Table 4: mass spectrum reference condition of 4 vitamins B 12
| Sequence number | Names of Compounds | Ionized form | Parent ion (m/z) | Quantitative ion 1 (m/z) |
| 1 | CN-Cbl | [M+2H]2+ | 678.29 | 147.1 |
| 2 | 13C7-CN-Cbl | [M+2H]2+ | 681.80 | 154.1 |
| 3 | Me-Cbl | [M+2H]2+ | 672.80 | 147.1 |
| 4 | OH-Cbl | [M+2H]2+ | 673.79 | 147.1 |
| 5 | ADO-Cbl | [M+2H]2+ | 790.34 | 147.1 |
Wherein, the scanning chart of the ions of 4 vitamins B 12 is shown in figures 3A to 3D.
4.3 Qualitative determination
The retention time of the chromatographic peak of the target compound in the sample is compared with the retention time of the corresponding standard chromatographic peak, and the variation range is within +/-2.5 percent.
The mass spectrometric qualitative ions of each compound should be present, at least including one parent ion and two daughter ions, and the relative abundance ratio of the two daughter ions of the target compound in the sample should not exceed the ranges specified in table 5 for the same compound for the same test batch as compared to standard solutions of comparable concentration.
Table 5: maximum allowable deviation of relative ion abundance in qualitative
| Relative ion abundance | >50％ | 20％～50％ | 10％～20％ | ≤10％ |
| Allowable relative deviation | ±20％ | ±25％ | ±30％ | ±50％ |
4.4 Preparation of standard curve
Under the conditions of liquid chromatography-mass spectrometry of 4.1 and 4.2, the standard series solution (1.4.4) is detected by sampling from low concentration to high concentration, and the peak area of the parent ion chromatographic peak of each target compound is plotted with the corresponding concentration to obtain a standard curve regression equation, wherein the linear correlation coefficient is larger than 0.99.
4.5 Measurement of sample solution
Taking the solution to be detected obtained in the sample pretreatment, looking up a standard curve to obtain the concentration of 4 vitamins B 12 in the test solution, and calculating the content of the substance to be detected in the sample according to the test data treatment. The response value of the object to be detected in the test solution should be within the linear range of the standard curve, and should be re-measured after being diluted properly beyond the linear range.
5. Test data processing
The mass fraction X of 4 vitamins B 12 in the sample is expressed in micrograms per kilogram (mug/kg), and is calculated according to the following formula:
Wherein:
x-the content of the component to be measured in the sample in micrograms per kilogram (μg/kg).
C-the concentration of the component to be measured in the standard solution in nanograms per milliliter (ng/mL).
C i -the concentration of the component to be measured in nanograms per milliliter (ng/mL) in the measurement solution.
A is the peak area of the component to be measured in the measuring solution.
A si -peak area of internal standard substance in standard solution.
V-constant volume in milliliters (mL).
C si -concentration of internal standard substance in standard solution in nanograms per milliliter (ng/mL).
A i -peak area of internal standard substance in measurement solution.
A s -peak area of the component to be measured in the standard solution.
M-sample weight in grams (g).
The result of the calculation retains two significant digits.
6. Precision of
The absolute difference between the two independent test results obtained under reproducible conditions is not more than 10% of the arithmetic mean.
Example 1
The actual measurement results according to the invention are as follows:
The error range accords with the related requirements of an attached table F1 in GB/T27404-2008 'laboratory quality control Specification food physicochemical detection Standard'. The actual measurement result of the method meets the standard, and can accurately detect the four vitamins B 12.
Comparative example 1
In this comparative example, the sample was not subjected to one-dimensional chromatography, and after pretreatment, was directly subjected to two-dimensional chromatography and mass spectrometry (conditions were the same as in the example), and the detection results are shown in fig. 4. As shown in the mass spectrum chart of fig. 4, the detection result has large interference, and four vitamins B 12 cannot be distinguished.
In summary, the detection method of the four vitamins B 12 in the dairy product provided by the invention extracts 4 vitamins B 12 from a sample under the combined action of sodium acetate buffer solution (pH=4.0) and pepsin under the light-shielding condition. Purifying the extract by one-dimensional liquid phase size elimination method, separating the purified sample liquid by two-dimensional liquid phase inversion with on-line flow splitting and conversion method, and measuring the content of 4 vitamins B 12 by mass spectrometry. The invention is especially suitable for measuring 4 kinds of vitamin B 12 in infant formula milk powder. The detection limit of 4 kinds of vitamin B 12 in the infant formula milk powder is 0.2 mug/Kg, and the quantitative limit is 0.5 mug/Kg.
The description of the exemplary embodiments presented above is merely illustrative of the technical solution of the present invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those of ordinary skill in the art. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable others skilled in the art to understand, make and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.
Industrial applicability
The detection method of the four vitamins B12 in the dairy product provided by the invention can be applied to detection and identification of the content of the four vitamins B12 in the dairy product, especially infant dairy products.
Claims (6)
1. The detection method of the four vitamins B 12 in the dairy product is characterized by comprising the following steps of:
sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain an initial extracting solution, wherein the enzymolysis reaction is carried out in a buffer solution, and the buffer solution is sodium acetate buffer solution;
A two-dimensional liquid chromatography separation step: separating the four vitamins B 12 from the primary extract by chromatographic separation using two-dimensional high performance liquid chromatography; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography;
mass spectrum detection: performing mass spectrum detection on the four vitamins B 12 after the two-dimensional high performance liquid chromatography separation;
the quantitative ions of the four vitamins B 12 are 147.1m/z, and the four vitamins B 12 are hydroxycobalamin, 5' -deoxyadenosylcobalamin, mecobalamin and cyanocobalamin;
the protease is pepsin;
The concentration of the sodium acetate buffer solution is 40-60 mmol/L, and the pH value of the sodium acetate buffer solution is 4.0+/-0.5;
the conditions of the one-dimensional liquid phase size exclusion chromatography are as follows: the mobile phase is acetonitrile water solution with the volume percentage of 5-10%;
The conditions of the two-dimensional liquid phase reversed phase chromatography are as follows: the mobile phase comprises a phase A and a phase B, and gradient elution is carried out, wherein the phase A is a buffer solution phase, the phase B is a polar solvent phase, the phase A is 7-15 mmol/L ammonium formate aqueous solution, and the phase B is acetonitrile.
2. The method of claim 1, wherein the gradient elution in volume percent comprises:
0min: 80-95% of phase A and 5-20% of phase B;
1min: 80-95% of phase A and 5-20% of phase B;
10min: 40-60% of phase A and 40-60% of phase B;
12min: 40-60% of phase A and 40-60% of phase B;
12.1min: 5-20% of phase A and 80-95% of phase B;
15min: 5-20% of phase A and 80-95% of phase B;
16min: 80-95% of phase A and 5-20% of phase B;
18min: 80-95% of phase A and 5-20% of phase B.
3. The method of claim 1, wherein the conditions for mass spectrometry comprise: a charged spray ion source, positive ion scan mode, is employed.
4. A method according to claim 3, wherein the collection of quantitative ion pairs in the mass spectrometry is as follows:
The parent ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
the parent ion m/z 672.80 of mecobalamin and the quantitative ion m/z 147.1;
The parent ion m/z 673.79 of hydroxycobalamin and the quantitative ion m/z 147.1;
The parent ion of 5' -deoxyadenosylcobalamin, m/z 790.34, was quantified at ion m/z 147.1.
5. The method of claim 1, further comprising the step of constructing a standard curve, comprising: and respectively adopting four standard substances of vitamin B 12 to prepare a standard curve through the two-dimensional liquid chromatography and mass spectrometry detection.
6. The method of claim 1, further comprising the step of adding a stable isotope internal standard prior to the step of performing the two-dimensional liquid chromatography separation.
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