CN114216982A - Vitamin B12Is detected by - Google Patents

Vitamin B12Is detected by Download PDF

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CN114216982A
CN114216982A CN202111531281.XA CN202111531281A CN114216982A CN 114216982 A CN114216982 A CN 114216982A CN 202111531281 A CN202111531281 A CN 202111531281A CN 114216982 A CN114216982 A CN 114216982A
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CN114216982B (en
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王象欣
解庆刚
李剑桥
张维
石红丽
蒋士龙
陈博
梁爱梅
冷友斌
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Heilongjiang Feihe Dairy Co Ltd
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Heilongjiang Feihe Dairy Co Ltd
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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
    • 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
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Abstract

The invention discloses a vitamin B12The method of (1). The invention provides four vitamin B in dairy products12The detection method of (2), the detection method comprising: sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain an initial extraction solution; two-dimensional liquid chromatography separation: performing chromatographic separation by using two-dimensional high performance liquid chromatography to obtain four vitamin B12Separating from the primary extract; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography; and (3) mass spectrum detection: separating four vitamin B after two-dimensional high performance liquid chromatography12And (5) carrying out mass spectrum detection.

Description

Vitamin B12Is detected by
Technical Field
The invention relates to the field of food, in particular to the technical field of analysis and detection of summarized nutrient substances in the field of food, and more particularly relates to four vitamin B in dairy products12The method of (1).
Background
Vitamin B12(Vitamin B12Abbreviated to VB12) Also known as cobalamins, generally refers to the cobalamin family with the same biological activity, which is also the only water-soluble vitamin containing metallic elements. Vitamin B12Is an important nutrient essential to the human body, which functions as a coenzyme for several key enzymes in single carbon (methyl) metabolism; has synergistic effect in many biochemical reactions, is an important coenzyme in the process of DNA synthesis, has promotion effect on the formation and growth of erythrocytes, participates in the regulation of nervous system and immune system and physiological activities such as metabolism of cells in human body, and is vital to the growth and development of human beings.
Vitamin B12It is produced only in microorganisms (mecobalamin, cobamamide and hydroxycobalt) and its food sources are therefore limited to non-vegetable sources such as meat or other animal sources. Theoretically, vitamin B12Deficiency symptoms are rare in normal healthy people, but pure vegetarians, pregnant women, infants and patients with eating disorders are easy to have vitamin B due to special eating habit requirements and physiological needs12Deficiency syndrome. In the absence of vitamin B12In cases of (a), increasing folate supply can cure anemia, but not neurological symptoms. If vitamin B is deficient12Although vitamin B can cause peripheral neuritis and anemia in children, and also can affect the development of infants and young children, vitamin B12The excessive supplementation can also cause adverse effects such as allergic reactions. So vitamin B12Is an important index of infant formula food nutrition, and the detection requirement is more accurate.
In addition, it is known that vitamin B12In the form of hydroxocobalamin, 5' -deoxyadenosylcobalamin, methylcobalamin and cyanocobalamin. Cyanocobalamin is relatively easy to produce in large quantities and has a high stability, so it is the vitamin B most used in food processing12However, cyanocobalamin is not biologically active until the cyanide group is enzymatically removed, and only after in vivo conversion to methylcobalamin and 5' -deoxyadenosylcobalamin does it work.
Vitamin B12The bioavailability of the synthesized form is inversely proportional to the amount administered, and is less than 4% in humans and animals receiving prophylactic or therapeutic supplements. The hydroxycobalamin, 5' -deoxyadenosylcobalamin and methylcobalamin are vitamin B12Is a naturally occurring form of (a). 5' -deoxyadenosylcobalamin and methylcobalamin have coenzyme activity in mammalian cells and have biological activity, while hydroxycobalamin is a product of their photolysis. Matte et al in a 2012 experiment using pigs as an Animal model in humans (J.J.Matte, M.Britten, C.L.Girard, Animal Frontiers, Volume 4, Issue 2, April 2014, Pages 32-37) indicated that the daily intake of vitamin B in the natural form compared to the daily intake of the synthetic form (cyanocobalamin) used in vitamin supplements12The resulting absorption is more efficient.
In addition, different forms of vitamin B were evaluated12The availability of intestinal absorption of vitamin B is also an important factor in assessing the quality of its source12Deficiency, essentially methylcobalamin and cobamamide deficiency in vivo. Thus, with the development of the dietary supplement and infant formula markets and the consumer demand, more and more new products contain "natural forms of vitamin B such as mecobalamin12", to respond to this market trend, all four vitamin B groups were measured individually12The detection method of (2) is of great importance.
At present, VB is measured12The method comprises the following steps of GB 5413.14-2010 national food safety standard vitamin B in infant food and dairy products12The method used in (1) determination of (1) is a microbiological method. American society of analytical chemists(AOAC) official method 2016.017, which utilizes cyanide to bind all VB12The format change to cyanocobalamin and the use of a single-use dedicated immuno-column in each assay, although increasing the sensitivity of the method, adds additional cost. There are also ICP-MS method using cobalt element introduction for quantification, enzyme linked immunosorbent assay method and the like. These methods all can only measure VB12Total amount of (2), no distinction between different forms of VB12。
As can be seen, there have been many methods for determining the vitamin B12 content in food products, fortified food products and dietary supplements. Due to the low level of vitamin B12 enhancement, most methods are only capable of measuring total VB12And (4) horizontal. Since cyanocobalamin is all four VB12The most stable form of (c). Thus, VB is currently being detected in food and dietary supplements12In many cases, all VB is separated from the cyanide12The form was changed to cyanocobalamin, and the level of the total content was reflected by the content of cyanocobalamin.
Currently, these methods are used to determine VB12The total amount of (a) was successful, however, there were safety problems with the use of cyanide in the laboratory. The microbiological assay is a commonly used vitamin B12The detection method comprises selecting microorganism from Lactobacillus Leishmania. This method possesses high sensitivity, but due to some inactive cobalamin precursor (Corrin, C)19H22N4) It interferes with the growth of the microorganism and causes it to lack specificity, resulting in poor selectivity and sometimes in overestimation. Inactive cobalamin precursors interfere with the active VB12Over-estimation of total VB12And (4) horizontal.
Thus, the development of a precursor of cobalamin which can distinguish between inactive and four VB' s12The method of (2) is of great importance.
Disclosure of Invention
Problems to be solved by the invention
For solving the problem of the existing vitamin B12In the detection method, four vitamin B can not be distinguished12And the presence of cyanide, and cobalaminThe invention provides four vitamin B in dairy products and solves the problems of interference detection result and the like of the existence of the precursor12The detection method of (1) is a two-dimensional high performance liquid chromatography-mass spectrometry technique, wherein a sample is digested with pepsin in advance to bind vitamin B to a protein12Is released, and the primary extract is passed through one-dimensional chromatography using size-exclusion chromatography column to make macromolecular protein prior to 4 vitamin B12Is eluted, 4 kinds of vitamin B12Because the molecular weight is relatively small, the protein is eluted later, and the eluent is switched to a two-dimensional reverse phase analysis column according to the elution time, so that 4 kinds of vitamin B12And 147.1(m/z) is selected as a quantitative ion for mass spectrum detection, so that the interference of an inactive cobalamin precursor can be effectively avoided, and the accuracy of the method is improved.
Means for solving the problems
Through the intensive research of the inventor, the technical problem can be solved by the following scheme:
[1]the invention provides four vitamin B in a dairy product12The detection method of (2), wherein the detection method comprises:
sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain an initial extraction solution;
two-dimensional liquid chromatography separation: performing chromatographic separation by using two-dimensional high performance liquid chromatography to obtain four vitamin B12Separating from the primary extract; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography;
and (3) mass spectrum detection: separating four vitamin B after two-dimensional high performance liquid chromatography12And (5) carrying out mass spectrum detection.
[2] The detection method according to [1], wherein the protease is one or more selected from pepsin, trypsin, cathepsin, papain and subtilisin.
[3]According to [1]]Or [2]]The detection method described above, wherein the four vitamin B compounds12The quantitative ions are all 147.1 m/z; and/orThe four vitamins B12Is hydroxycobalamin, 5' -deoxyadenosylcobalamin, methylcobalamin 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% by volume of a polar solvent.
[5] The detection method according to any one of [1] to [4], wherein the conditions of the two-dimensional liquid reverse phase 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 percentage comprises:
0 min: 80-95% of phase A and 5-20% of phase B;
1 min: 80-95% of phase A and 5-20% of phase B;
10 min: 40-60% of phase A and 40-60% of phase B;
12 min: 40-60% of phase A and 40-60% of phase B;
12.1 min: 5-20% of phase A and 80-95% of phase B;
15 min: 5-20% of phase A and 80-95% of phase B;
16 min: 80-95% of phase A and 5-20% of phase B;
18 min: 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: charged spray ion source and positive ion scanning mode are adopted.
[8] The detection method according to [7], wherein the collection of quantitative ion pairs in the mass spectrometric detection is as follows:
mother ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
mother ion m/z 672.80 of methylcobalamin, quantitative ion m/z 147.1;
mother ion m/z 673.79 of hydroxycobalamin, quantitative ion m/z 147.1;
the parent ion m/z 790.34 of 5' -deoxyadenosylcobalamin, and the quantitative ion m/z 147.1.
[9]According to [1]]～[8]The detection method of any one of the above, wherein the detection method further comprises a step of constructing a standard curve, which comprises: respectively adopting four vitamins B12Making a standard curve via the two-dimensional liquid chromatography and mass spectrometry detection.
[10] The detection method according to any one of [1] to [9], further comprising a step of adding a stable isotope internal standard substance before the step of performing the two-dimensional liquid chromatography.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, the invention can obtain the following technical effects:
(1) the invention provides four vitamin B in dairy products12The detection method of (A) can be applied to vitamin B12The four forms of (2) for accurate detection.
(2) The invention provides four vitamin B in dairy products12The detection method avoids the problems of safety and environmental protection caused by using cyanide.
(3) The invention provides four vitamin B in dairy products12Compared with the existing microbiological method, the detection method can avoid poor selectivity caused by the interference of inactive cobalamin precursors on the growth of microorganisms, and overestimate vitamin B12The problem of content.
Drawings
FIG. 1 shows four vitamin B compounds in a dairy product using the present invention12Detection method of (3) detection of 4 vitamin B12The two-dimensional high performance liquid chromatography-mass spectrogram. Wherein, A in figure 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 methylcobalamin; in FIG. 1, E is hydroxocobalamin.
FIG. 2A-FIG. 2C show four vitamins B in the milk product of the present invention12The schematic diagram of the flow path state of the instrument is switched and controlled by a six-way valve adopted in the two-dimensional high performance liquid chromatography-mass spectrum in the detection method; wherein,FIG. 2A is a one-dimensional chromatographic column purification; FIG. 2B shows the valve switching to enrichment Ring, 4 vitamin B12Is enriched into the ring; FIG. 2C shows the switching of the valve to the analytical column, 4 vitamin B species12Separated by an analytical column and detected by mass spectrometry.
FIG. 3A-FIG. 3D show 4 vitamin B12An ion scan; wherein FIG. 3A is 5' -deoxyadenosylcobalamin; FIG. 3B is methylcobalamin; FIG. 3C is hydroxocobalamin; FIG. 3D is cyanocobalamin.
FIG. 4 is a schematic diagram of a comparative example in which one-dimensional liquid size exclusion chromatography is not performed, and two-dimensional liquid reversed-phase chromatography and mass spectrometry are performed after pretreatment. Wherein, A in FIG. 4 is 5' -deoxyadenosylcobalamin; b in FIG. 4 is cyanocobalamin; c in FIG. 4 is methylcobalamin; d in FIG. 4 is hydroxocobalamin.
Detailed Description
The present invention will be described in detail below. The technical features described below are explained based on typical 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 represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, the term "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.
In the present specification, the meaning of "may" 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.
In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, 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 described 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 combined technology, and the dairy product sample to be detected is digested by protease in advance, so that the vitamin B combined with protein in the dairy product sample to be detected12Is released, and the obtained primary extract is subjected to one-dimensional chromatography using size-exclusion column to make macromolecular protein (polypeptide) precede four vitamin B12Is eluted, four vitamin B12Because the molecular weight is small relative to protein (polypeptide), the four vitamin B are eluted later, and the eluent is switched to two-dimensional chromatogram, namely a reversed-phase analytical column according to the elution time, so that the four vitamin B12Is separated and detected by mass spectrometry, wherein the mass spectrometry is specific to four vitamin B12The corresponding charge-to-mass ratio can effectively avoid the interference of inactive cobalamin precursors, improves the accuracy of the method and does not need to adopt cyanide.
Four kinds of vitamin B12And details of inactive cobalamin precursors are given in table 1 below.
Table 1:
Figure BDA0003410862190000081
in the following four vitamin B vitamins in the dairy product of the invention will be addressed12The detection method of (2) is specifically described.
< sample Source and Pre-treatment >
In the detection method of the invention, the sample to be detected is a dairy product.
There is no particular limitation on the "dairy product" or source of the test sample in the present invention, 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), fat free milk, milk powder, whole milk products, butter, buttermilk products, skim milk products, high milk fat products, condensed milk, whipped cream, cheese, ice cream and confectionery products, probiotic beverages or probiotic yoghurt type beverages. Wherein "powdered milk" refers to an artificial dairy product made by evaporating milk to dryness. In some preferred embodiments of the present invention, the sample in the detection method of the present invention is derived from a bovine or ovine milk powder preparation.
Further, due to vitamin B12Is an important index of nutrition of infant formula food (especially formula dairy products). Thus, four vitamin B vitamins in the dairy product of the invention12The detection method is particularly suitable for infant formula dairy products. In some embodiments of the invention, four vitamin B groups12Including hydroxocobalamin, 5' -deoxyadenosylcobalamin, methylcobalamin and cyanocobalamin.
In some embodiments of the invention, before performing two-dimensional high performance liquid chromatography-tandem mass spectrometry, the dairy product sample to be detected needs to be subjected to a pretreatment step, so that the sample is suitable for detection and the detected object is preliminarily enriched.
Specifically, in the sample pretreatment step, protease is used for carrying out enzymolysis reaction on a dairy product sample to be detected, and the supernatant is obtained by centrifugation, so that the primary extraction solution is obtained. Through the sample pretreatment step of the invention, the combined action of the buffer solution and the protease is used to combine the protein in the dairy product sample to be detected with the vitamin B12Is released for subsequent two-dimensional high performance liquid chromatography separation and mass spectrum detection, 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 present invention, the amount of the enzyme may be adjusted according to the amount of the dairy product sample to be tested, for example, 50000-100000U protease is added to 5-10 g dairy product sample to be tested.
The enzymatic reaction is usually carried out in a buffer, which may be selected from sodium acetate buffer. In some preferred embodiments of the invention, the buffer is a sodium acetate buffer. The concentration of the sodium acetate buffer solution is 40-60 mmol/L, preferably 40-55 mmol/L, more preferably 45-55 mmol/L, and further preferably 50 mmol/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, and more preferably 4.0. + -. 0.1. In some embodiments of the present invention, the amount of the buffer solution can be adjusted according to the amount of the dairy product sample to be tested added, for example, 5-10 g of the dairy product sample to be tested is added in every 100mL of the buffer solution. In another embodiment 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 present invention, after the enzymatic reaction is performed in a buffer solution that is less than the final volume (constant volume) of the buffer solution, the buffer solution is added to dilute the reaction solution to the final volume (constant volume). Under the condition of smaller volume, the protease can be more fully contacted with a dairy product sample to be detected so as to exert the enzymolysis effect and improve the detection efficiency. For example, in the case that the final volume of the buffer 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 for the enzymatic reaction, and then the volume may be increased to 100 mL.
The temperature and time of the enzymolysis reaction can be adjusted according to the type of the selected protease, for example, in some embodiments of the present invention, the enzymolysis reaction is performed under 35-39 ℃ for 15-50 min. Preferably, heating in a water bath at 37 ℃ for 15-50 min; more preferably, the heating is carried out in a 37 ℃ water bath for 20-40 min, and even more preferably, the heating is carried out in a 37 ℃ water bath for 30 min. The heating method is not particularly limited, and may be, for example, heating in a water bath.
In some embodiments of the invention, a centrifugation step is further included after the enzymatic reaction. In the inventionIn some specific embodiments, the enzymolysis reaction product is shaken up and then centrifuged, and the centrifugation condition is 7000-15000 r/min for 5-15 min. Preferably, centrifugation conditions are 10000 rpm for 10 min. Centrifuging, collecting supernatant as vitamin B12The primary extract of (1).
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 the two-dimensional high performance liquid chromatography separation step. The adding amount of the stable isotope internal standard substance is that 5-20 ng of the stable isotope internal standard substance is added in every 5-10 g of dairy product sample to be detected. The use of stable isotope internal standard substance can avoid potential matrix effect in the 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 detect vitamin B in a sample12The individual components of (a) are separated. 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 present invention, the primary extract obtained in the sample pretreatment step is chromatographically separated using two-dimensional high performance liquid chromatography to separate the four vitamin B compounds12And separating from the primary extraction liquid to reduce mass spectrum interference. The two-dimensional high performance liquid chromatography comprises a one-dimensional liquid size exclusion chromatography and a two-dimensional liquid reversed phase chromatography, the two-dimensional liquid size exclusion chromatography and the two-dimensional liquid reversed phase chromatography 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 present invention, the one-dimensional chromatography uses a size-array chromatographic column to make the macromolecular protein (polypeptide) in the primary extraction solution precede 4 vitamin B12Is eluted (purified by a liquid phase size exclusion method) with 4 vitamin B12Because the molecular weight is small relative to protein (polypeptide), the protein is eluted later, and the eluent is switched to a two-dimensional liquid phase reverse phase analytical column according to the elution time (for example, an online fraction switching method adopting six-way valve switching) so as to ensure that 4 vitamin B12Is separated.
In the present invention, the size exclusion column used in the one-dimensional liquid phase 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. with a pore size of
Figure BDA0003410862190000111
Preferably a size exclusion chromatography column of
Figure BDA0003410862190000112
More preferably
Figure BDA0003410862190000113
Too small pore diameter can not be separated and is easy to block, and too large pore diameter can easily cause vitamin B12And protein/polypeptide efflux, increasing interference. Size exclusion chromatography columns are commercially available, for example, commercially available Agilent Bio SEC-5: 5190 chromatography column 2523, which has an inner diameter of 4.6mm, a length of 300mm, a particle size of 5 μm, and a pore diameter of
Figure BDA0003410862190000114
In some embodiments of the invention, the chromatographic conditions of one-dimensional liquid phase size exclusion chromatography comprise: the mobile phase is a polar solvent aqueous solution with the volume percentage of 5-15%, and from the viewpoint of compatibility with the reversed phase chromatography and elution purification, the polar solvent is preferably acetonitrile. In some preferred embodiments, the mobile phase is 5-10% by volume of aqueous acetonitrile, more preferably 10% by volume of aqueous acetonitrile; the flow rate is 0.4. + -. 0.2mL/min, preferably 0.4. + -. 0.1mL/min, more preferably 0.4 mL/min. The column temperature of the chromatographic column is 40 +/-3 ℃, preferably 40 +/-2 ℃ and 40 +/-1 ℃. The sample injection volume in the one-dimensional liquid phase size exclusion chromatography is 500 +/-100 mu L, preferably 500 +/-50 mu L, more preferably 500 +/-20 mu L and 500 +/-10 mu L.
In some embodiments of the invention, the detection wavelength used in one-dimensional liquid phase size exclusion chromatography is 550 nm. 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 missing junctions, migration time drift.
In the present invention, the two-dimensional liquid reverse phase chromatography may employ any reverse phase chromatography column suitable for separating vitamins, for example, a non-polar chromatography column, which may typically be a C18 reverse phase chromatography column. The reverse phase chromatography column may be obtained commercially, for example as a commercially available Zorbax Plus-C18 RRHD having an internal diameter of 2.1mm, a length of 150mm and a particle size of 1.8 μm.
In some embodiments of the invention, the chromatographic conditions of two-dimensional liquid reverse phase chromatography comprise: the mobile phase comprises an A phase and a B phase, wherein the A phase is a buffer solution phase, so that the separation performance of reverse phase chromatography can be improved on one hand, and the ionization performance in electrospray can be increased on the other hand due to the compatibility with mass spectrum. In some embodiments, phase A can be 7 to 15mmol/L ammonium formate aqueous solution, preferably 9 to 12mmol/L ammonium formate aqueous solution, and more preferably 10mmol/L ammonium formate aqueous solution; the phase B is a polar solvent, preferably the same as the polar solvent in the one-dimensional chromatography, and can be acetonitrile, so that the operation convenience of automatic detection is improved. Gradient elution is adopted in two-dimensional liquid phase reversed phase chromatography, and the flow rate is as follows: 0.4. + -. 0.2mL/min, preferably 0.4. + -. 0.1 mL/min. The column temperature of the chromatographic column is 35 +/-3 ℃, preferably 35 +/-2 ℃ and 35 +/-1 ℃.
In some more specific embodiments of the invention, the gradient elution comprises:
0 min: 80-95% of phase A and 5-20% of phase B;
1 min: 80-95% of phase A and 5-20% of phase B;
10 min: 40-60% of phase A and 40-60% of phase B;
12 min: 40-60% of phase A and 40-60% of phase B;
12.1 min: 5-20% of phase A and 80-95% of phase B;
15 min: 5-20% of phase A and 80-95% of phase B;
16 min: 80-95% of phase A and 5-20% of phase B;
18 min: 80-95% of phase A and 5-20% of phase B.
In some preferred embodiments of the invention, the gradient elution comprises:
0 min: 90% of phase A and 10% of phase B;
1 min: 90% of phase A and 10% of phase B;
10 min: 50% of phase A and 50% of phase B;
12 min: 50% of phase A and 50% of phase B;
12.1 min: 10% of phase A and 90% of phase B;
15 min: 10% of phase A and 90% of phase B;
16 min: 90% of phase A and 10% of phase B;
18 min: 90% of phase A and 10% of phase B.
< Mass Spectrometry detection >
In some embodiments of the invention, the four vitamin B components after two-dimensional high performance liquid chromatography are separated12And (5) carrying out mass spectrum detection.
The conditions for mass spectrometric detection include: with charged spray ion source, positive ion scanning mode, capillary temperature: 300 +/-50 ℃; capillary voltage: +3.5 + -0.5 KV.
Mass spectrometric detection the collection of quantitative ion pairs is as follows:
mother ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
mother ion m/z 672.80 of methylcobalamin, quantitative ion m/z 147.1;
mother ion m/z 673.79 of hydroxycobalamin, quantitative ion m/z 147.1;
the parent ion m/z 790.34 of 5' -deoxyadenosylcobalamin, and the quantitative ion m/z 147.1.
The present invention recognizes that vitamin B will be tested in a quantitative assay12The quantitative ions of all the components are determined to be 147.1m/z, so that the interference of interference peaks is avoided more easily, especially the interference of inactive cobalamin can be avoided, and the precision of quantitative analysis is improved effectively.
< construction of Standard Curve >
The basis of quantitative analysis comparison is determined through establishing a standard curve, and the detection limit and the quantification limit of a detection system or a detection method can be determined at the same time.
According to the inventionFour vitamins B in dairy products12The detection method also comprises the step of constructing a standard curve, and specifically, four vitamin B are respectively adopted12Preparing a series of standard substance working solutions with different concentrations, and performing the two-dimensional high performance liquid chromatography separation step and the mass spectrometry detection step according to the respective target compounds (namely, four vitamin B compounds) of the detected standard substance working solutions12) And (4) plotting the peak area of the parent ion chromatographic peak corresponding to the concentration to obtain a standard curve regression equation.
In addition, when the standard curve is created, 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, respectively obtaining four vitamin B in the dairy product sample to be detected12To calculate the four vitamin B12The content of (b) in the dairy sample to be tested.
The present invention provides vitamin B12The detection method of each component can be used for detecting the end commodity of the dairy product, and can also be used as an online detection monitoring method for dairy product production through automatic setting.
Examples
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
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)3CN, chromatographically pure).
1.1.3 methanol (CH)3OH, chromatographically pure).
1.1.4 formic acid (HCOOH, chromatographically pure).
1.1.5 acetic acid (CH)3COOH, chromatographic purity).
1.1.6 sodium acetate (CH)3COONa)。
1.1.7 ammonium Formate (HCOONH)4Chromatographically pure).
1.1.8 pepsin (activity, 38U/mg).
1.1.9 cyanocobalamin-dimethylbenzimidazole-13C7Isotope internal standard: the purity is more than or equal to 95 percent and is 1 mu g/mL.
1.2 preparation of reagents
1.2.1 sodium acetate buffer (50 mmol/L): 4.1g of anhydrous sodium acetate (1.1.6) are weighed out, dissolved in 950mL of water and adjusted to pH 4.0. + -. 0.1 with acetic acid (1.1.5) and made up to 1000mL with water.
1.2.2 acetonitrile-water solution (10%, volume ratio): accurately measure 100mL acetonitrile (1.1.2), dissolve with water and make up to 1000 mL.
1.2.3 aqueous ammonium formate solution (10 mmol/L): 0.63g ammonium formate (1.1.7) was weighed out, dissolved in 950mL of water, adjusted to pH 4.0. + -. 0.1 with formic acid (1.1.4) and made up to 1000mL with water.
1.3 Standard substance
The standard substances used were obtained from Sigma-Aldrich and the details are given in Table 2.
Table 2: information on standard substance used in examples of the present invention
Figure BDA0003410862190000151
1.4 preparation of Standard solution
1.4.1 Standard stock solutions (1 mg/mL): respectively weighing 10mg (accurate to 0.01mg) of 4 VB12 standard substances, dissolving with water, transferring to a 10mL volumetric flask, adding 5mL of methanol (1.1.3), adding water to a constant volume to a scale, subpackaging 0.5mL of the solution in each part, transferring to a 1mL jaw sample bottle, sealing, and storing at-20 ℃ in a dark place for later use.
1.4.2 Standard working solution (1. mu.g/mL): accurately sucking 10 μ L (1.4.1) of 4 kinds of VB12 standard stock solutions into a 10mL volumetric flask, fixing the volume to the scale with water, and storing at 4 ℃ in a dark place.
1.4.3 isotope internal standard working solution: 0.5mL (1. mu.g/mL) of VB12 isotope internal standard solution was taken and diluted with water to 10mL, giving a 50ng/mL concentration. Stored at 4 ℃ in the dark.
1.4.4 Standard series 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 the standard working solution (1.4.2) were accurately pipetted, and 200. mu.L of the isotope internal standard solution (1.4.3) was added simultaneously, and the volume was adjusted to 10mL with 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 an internal isotope standard concentration of 1 ng/mL. This standard series of solutions was prepared prior to use.
1.5 materials
1.5.1 microporous Filter Membrane: aqueous phase, 0.22 μm.
1.5.2 size exclusion chromatography column (one-dimensional chromatography): 4.6X 300mm,5 μm,
Figure BDA0003410862190000161
Agilent Bio SEC-5：5190-2523。
1.5.3 reverse phase chromatography column (two-dimensional chromatography): zorbax Plus-C18 RRHD, 2.1X 150mm, 1.8 μm.
Instrument and equipment
2.1 two-dimensional high performance liquid chromatography-high resolution mass spectrometer: an electrospray ion source.
2.2 balance: the sensory was 0.01g and 0.1 mg.
2.3, centrifuge: the rotating speed is not less than 10000 r/min.
2.4 quantitative pipettor: 10 mu L to 1 mL.
2.5 centrifuge tube: 15 mL.
2.6 shaking table with constant temperature water bath.
Third, sample pretreatment
Accurately weighing 5-10 g (accurately to 0.01g) of a sample in a 150mL brown conical flask, sequentially adding 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), fully mixing to fully dissolve the sample, and carrying out water bath at 37 ℃ for 30 min. 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), shaken well and centrifuged at 10000 rpm for 10min, and 5mL of supernatant was aspirated for use.
Four, liquid chromatogram-tandem mass spectrum reference condition
Fig. 2A to 2C are schematic diagrams of a connection system of a device for combining liquid chromatography with mass spectrometry according to an embodiment of the present invention.
4.1 liquid chromatography reference conditions
4.1.1 reference conditions for one-dimensional chromatography
A chromatographic column: 4.6X 300mm,5 μm,
Figure BDA0003410862190000171
or the equivalent.
Mobile phase: 10% aqueous acetonitrile (1.2.2).
Flow rate: 0.4 mL/min.
Detection wavelength: 550 nm.
Column temperature of the chromatographic column: 40 ℃;
sample introduction volume: 500 mu L of the solution;
the switching control schematic diagram of the six-way valve is shown in fig. 2A to fig. 2C, and the switching time is set according to the detection condition of the ultraviolet detector.
4.1.2 two-dimensional chromatography (gradient Pump) chromatography reference conditions
A chromatographic column: 2.1X 150mm, 1.8 μm.
Mobile phase: phase A, 10mmol/L aqueous ammonium formate solution (1.2.3); phase B, acetonitrile (1.1.2)
Flow rate: 0.4 mL/min.
Column temperature of the chromatographic column: 35 ℃ is carried out.
Gradient elution: see table 3.
Table 3: gradient elution conditions
Serial number Time (min) A(％) B(％) 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 conditions (ESI +)
4 kinds of vitamin B12The mass spectrum reference conditions of (a) are shown in table 4. The instrument conditions were: scanning mode: a positive ion; capillary temperature: 300 ℃; capillary voltage: +3.5 KV.
Table 4: 4 kinds of vitamin B12Reference condition of mass spectrum of
Serial number Name of Compound Ionized forms 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, 4 kinds of vitamin B12The ion scan is shown in fig. 3A to 3D.
4.3 qualitative determination
The retention time of the target compound chromatographic peak in the sample should be within + -2.5% of the retention time of the corresponding standard chromatographic peak.
The mass spectrometric qualitative ion of each compound should appear, comprising at least one parent ion and two daughter ions, and the relative abundance ratio of the two daughter ions of the target compound in the sample for the same compound in the same test batch should not deviate more than the range specified in table 5 compared to a standard solution 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, sample introduction and detection are carried out on a standard series solution (1.4.4) from low concentration to high concentration, peak areas of parent ion chromatographic peaks of all target compounds are plotted corresponding to the concentration, a standard curve regression equation is obtained, and the linear correlation coefficient of the standard curve regression equation is greater than 0.99.
4.5 measurement of sample solution
Sampling the solution to be tested obtained in the step of sample pretreatment, and checking a standard curve to obtain 4 vitamin B in the test solution12According to the test data processing, the content of the substance to be tested in the sample is calculated. The response value of the substance to be tested in the test solution should be within the linear range of the standard curve, and the substance should be re-measured after being diluted properly when exceeding the linear range.
Fifth, processing test data
4 vitamin B species in the sample12In micrograms per kilogram (μ g/kg), calculated according to the following formula:
Figure BDA0003410862190000191
in the formula:
x-the amount of the component to be measured in the sample in micrograms per kilogram (. mu.g/kg).
C-concentration of the component to be tested in the standard solution in nanograms per milliliter (ng/mL).
Ci-determining the concentration of the component to be determined in nanograms per milliliter (ng/mL) in the liquid.
A-peak area of the component to be measured in the determination liquid.
AsiPeak area of internal standard substance in standard solution.
V-volume volumetric volume in milliliters (mL).
CsiConcentration of internal standard substance in standard solution in nanograms per milliliter (ng/mL).
Ai-determining the peak area of the internal standard substance in the solution.
As-standardPeak area of the component to be measured in the solution.
m is the sample weight in grams (g).
The calculation result retains two significant digits.
Sixth, degree of precision
The absolute difference between the results of two independent tests obtained under reproducible conditions is not more than 10% of the arithmetic mean.
Example 1
The actual measurement result according to the invention is as follows:
Figure BDA0003410862190000201
wherein, the actual test content is the background value plus the theoretical addition, and the error range meets the relevant requirements of the attached table F1 in GB/T27404- & 2008 & lt & gt laboratory quality control Specification food physicochemical detection Standard & gt. Therefore, the actual measurement result of the method meets the standard, and the four vitamin B can be accurately detected12。
Comparative example 1
In this comparative example, the sample was not subjected to one-dimensional chromatography, but was subjected to two-dimensional chromatography and mass spectrometry directly after pretreatment (the same conditions as in the example), and the detection results are shown in fig. 4. As shown in the mass spectrogram of FIG. 4, the interference of the detection result is large, and four vitamin B can not be distinguished12。
In conclusion, the dairy product provided by the invention contains four vitamin B12Under the condition of keeping out of the sun, under the combined action of sodium acetate buffer (pH 4.0) and pepsin, 4 vitamin B are extracted from the sample12. Purifying the extractive solution by one-dimensional liquid size exclusion method, separating the purified sample solution by two-dimensional liquid phase reversed phase separation by on-line flow fraction switching method, and determining 4 vitamin B by mass spectrometry12The content of (a). The invention is especially suitable for 4 vitamin B in infant formula milk powder12The measurement of (1). The invention relates to 4 vitamin B in infant formula milk powder12The detection limit of (2) and the quantification limit of (5) are respectively 0.2. mu.g/Kg and 0.5. mu.g/Kg.
The above description of exemplary embodiments has been presented only to illustrate the technical solution of the 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 skilled in the art. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to thereby enable others skilled in the art to understand, implement 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 method for detecting the four vitamin B12 in the dairy product can be applied to detection and identification of the content of the four vitamin B12 in the dairy product, especially the dairy product for infants.

Claims (10)

1. Four vitamins B in dairy product12The detection method of (2), characterized in that the detection method comprises:
sample pretreatment: carrying out enzymolysis reaction on a dairy product sample to be detected by adopting protease to obtain an initial extraction solution;
two-dimensional liquid chromatography separation: performing chromatographic separation by using two-dimensional high performance liquid chromatography to obtain four vitamin B12Separating from the primary extract; the two-dimensional high performance liquid chromatography comprises one-dimensional liquid size exclusion chromatography and two-dimensional liquid reversed phase chromatography;
and (3) mass spectrum detection: separating four vitamin B after two-dimensional high performance liquid chromatography12And (5) carrying out mass spectrum detection.
2. The detection method according to claim 1, wherein the protease is one or more selected from the group consisting of pepsin, trypsin, cathepsin, papain and subtilisin.
3. According to claim1 or 2, wherein the four vitamin B compounds are12The quantitative ions are all 147.1 m/z; and/or the four vitamin B12Is hydroxycobalamin, 5' -deoxyadenosylcobalamin, methylcobalamin and cyanocobalamin.
4. The detection method according to any one of claims 1 to 3, wherein the conditions of the one-dimensional liquid phase size exclusion chromatography are as follows: the mobile phase is an aqueous solution of 5-15% by volume of a polar solvent.
5. The detection method according to any one of claims 1 to 4, wherein the conditions of the two-dimensional liquid reverse phase chromatography are as follows: 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 claim 5, wherein the gradient elution in volume percentage comprises:
0 min: 80-95% of phase A and 5-20% of phase B;
1 min: 80-95% of phase A and 5-20% of phase B;
10 min: 40-60% of phase A and 40-60% of phase B;
12 min: 40-60% of phase A and 40-60% of phase B;
12.1 min: 5-20% of phase A and 80-95% of phase B;
15 min: 5-20% of phase A and 80-95% of phase B;
16 min: 80-95% of phase A and 5-20% of phase B;
18 min: 80-95% of phase A and 5-20% of phase B.
7. The detection method according to any one of claims 1 to 6, wherein the conditions for mass spectrometry detection comprise: charged spray ion source and positive ion scanning mode are adopted.
8. The detection method according to claim 7, wherein the mass spectrometric detection is performed by collecting quantitative ion pairs as follows:
mother ion m/z 678.29 of cyanocobalamin, quantitative ion m/z 147.1;
mother ion m/z 672.80 of methylcobalamin, quantitative ion m/z 147.1;
mother ion m/z 673.79 of hydroxycobalamin, quantitative ion m/z 147.1;
the parent ion m/z 790.34 of 5' -deoxyadenosylcobalamin, and the quantitative ion m/z 147.1.
9. The detection method according to any one of claims 1 to 8, further comprising a step of constructing a standard curve, which comprises: respectively adopting four vitamins B12Making a standard curve via the two-dimensional liquid chromatography and mass spectrometry detection.
10. The detection method according to any one of claims 1 to 9, further comprising a step of adding a stable isotope internal standard substance before the step of performing the two-dimensional liquid chromatography separation.
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