CN112129845A - Method for rapidly detecting content of B vitamins in pig intestinal contents/feces - Google Patents

Abstract

The invention discloses a method for rapidly detecting the content of B vitamins in pig intestinal contents/feces, which comprises the following steps: weighing the cecum contents/feces of the pigs as samples, adding methanol/acetonitrile/water solution, and performing homogeneous extraction to obtain a sample solution; carrying out UPLC-MS/MS-MRM detection analysis on the sample solution to finally obtain the content of 8B vitamins in the sample; the 8B vitamins are: vitamin B₁Vitamin B₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, vitamin B₉Vitamin B₁₂. The method utilizes the MRM technology, combines an ultra-high performance liquid chromatography system and a mass spectrometer, is based on the targeted metabonomics of the MRM method, and canAnd the content of various B vitamins in samples such as pig intestinal contents, excrement and the like is rapidly detected.

Description

Method for rapidly detecting content of B vitamins in pig intestinal contents/feces

Technical Field

The invention belongs to the field of animal husbandry and veterinary medicine, and particularly relates to a method for rapidly detecting B vitamins in intestinal contents of pigs.

Background

The B vitamins are the components of various coenzymes and enzymes and participate in important metabolic activities in the body. The intestinal microorganism synthesis is one of the important sources of host B vitamins, the B vitamins are important metabolites of the intestinal microorganisms, have important effects on the aspects of intestinal development, immunologic function, nutrition metabolism and the like, and are widely applied to livestock and poultry breeding as feed additives.

The method can accurately master the dynamic change of the B vitamins in intestinal contents, and has important significance for researching the biological functions of the B vitamins in the intestinal. At present, most of the existing methods such as high performance liquid chromatography (HLPC), high performance capillary electrophoresis (HPLC), microbiological method, fluorescence method and the like are detection methods aiming at the content of single or a few B vitamins and have the problems of long time consumption, complex pretreatment, high detection limit, low specificity and the like.

At present, the methods are mainly used for detecting the B vitamins in food or feed premix with high B vitamin content, and because the content of the B vitamins in animal intestinal contents/feces is extremely low, the methods are difficult to accurately detect the content of the B vitamins in the intestinal contents, so that the research of regulating and controlling the intestinal health by utilizing the dynamic change of the content of the B vitamins is severely limited.

The latest research finds that targeted metabonomics research is carried out by combining an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology with a Multiple Reaction Monitoring (MRM) technology, and the high-efficiency quantitative determination can be carried out on low-content biological samples. The Liangqiang et al establishes a method for determining the contents of folic acid, nicotinic acid, biotin and pantothenic acid in food by using an UPLC-MS/MS-MRM technology, and the Wanren et al establishes a method for analyzing the contents of folic acid and biotin in a compound vitamin tablet by using an UPLC-MS/MS-MRM technology through tests. However, there is still no method for efficiently and quantitatively determining the content of various B vitamins in pig intestinal contents/feces.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for rapidly detecting a plurality of B vitamins in pig intestinal contents, the method utilizes an MRM technology, combines an ultra-high performance liquid chromatography system and a mass spectrometer, is a targeted metabonomics based on the MRM method, and can rapidly detect the content of the plurality of B vitamins in samples such as pig intestinal contents, excrement and the like.

In order to solve the technical problems, the invention provides a method for rapidly detecting the content of B vitamins in pig intestinal contents/feces, which comprises the following steps:

1) sample pretreatment;

weighing 200 +/-10 mg of cecum contents/feces of pigs as samples, adding the samples into 750 +/-50 mu L of methanol/acetonitrile/water solution, and performing homogeneous extraction to obtain a sample solution;

methanol/acetonitrile/water in a volume ratio of 2:2: 1;

2) and detecting a sample:

centrifuging the sample solution obtained by the pretreatment in the step 1) at a low temperature and a high speed (4 ℃, 14000r/min and 15 min); 500 μ L of supernatant I was passed through an Ostro phospholipid removal plate (to reduce matrix effect interference response and sensitivity), and blown dry with nitrogen (37 ℃ nitrogen); then adding 100 mu L of 0.3% formic acid aqueous solution for redissolution, and centrifuging at high speed and low temperature (4 ℃, 14000r/min and 10 min); carrying out UPLC-MS/MS-MRM detection analysis on the obtained supernatant II;

3) substituting the peak area obtained by detecting the supernatant II into a corresponding B vitamin standard curve (linear equation) to obtain the content of 8B vitamins in the supernatant II, and then converting to obtain the content of 8B vitamins in the sample;

the 8B vitamins are: vitamin B₁Vitamin B₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, vitamin B₉Vitamin B₁₂。

As the method for rapidly detecting the content of the B vitamins in the pig intestinal contents/feces, UPLC-MS/MS-MRM detection and analysis:

the ultra-high performance liquid chromatography comprises the following steps:

separating by using a Waters ACQUITY UPLCI-Class system;

a chromatographic column: waters, HSS T3(2.5 μm,2.1 mm. times.100 mm column); column temperature: 45 ℃; mobile phase: the solution A is 0.3 percent (volume percent) of formic acid aqueous solution, and the solution B is methanol;

the sample injection flow rate is 300 mu L/min, and the sample injection amount is 5 mu L;

the liquid phase gradient was as follows: 0-2.0 min, and 0 for liquid B; 2.0-3.0 min, the liquid B changes from 0 to 50% linearly; the liquid B is linearly changed from 50% to 100% in 3.0-4.5 min; the liquid B is linearly changed from 100% to 0 within 4.5-5.5 min; 5.5-11 min, and 0 for liquid B. The above% is volume%;

the mass spectrometry analysis is as follows:

mass spectrometry was performed in positive ion mode using a 5500QTRAP mass spectrometer (AB SCIEX);

ionization mode: ESI +, ion spray voltage: + 4500V, ion source temperature: 550 ℃, 40.00psi of air curtain gas (CUR), 55.00psi of atomization gas (GS1) and 55.00psi of auxiliary gas (GS2), and detecting the ion pair to be detected by adopting a multi-selection reaction monitoring (MRM) mode, wherein the MRM conditions are as follows:

the improvement of the method for rapidly detecting the content of the B vitamins in the pig intestinal contents/feces provided by the invention comprises the following steps:

mixing vitamin B₁Vitamin B₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, vitamin B₉Vitamin B₁₂The following operations were performed:

preparing a gradient standard working solution; injecting the gradient standard working solution into a gas chromatography-mass spectrometer, selecting an ion monitoring (MRM) mode for measurement, determining the peak position of each B vitamin, recording the peak area of a quantitative ion, and making a standard curve by taking the concentration of each standard solution of the B vitamins as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a linear regression equation.

As a further improvement of the method for rapidly detecting the content of the B vitamins in the pig intestinal contents/feces of the invention:

vitamin B₁The linear equation of (1) is Y-0.238X-8.689;

vitamin B₂The linear equation of (1) is Y ═ 0.0225X-2.172;

the linear equation of nicotinamide is Y ═ 0.0267X-11.060;

the linear equation of nicotinic acid is that Y is 0.0299X + 4.252;

the linear equation for pyridoxine is Y ═ 0.0503X + 0.0981;

the linear equation of pyridoxal is Y ═ 0.0232X-0.0404;

vitamin B₉The linear equation of (A) is Y ═ 0.00800X-0.0328;

vitamin B₁₂The linear equation of (a) is Y-0.00445X-0.0333.

The method of the invention can simultaneously detect a plurality of B Vitamins (VB)₁、VB₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, VB₉、VB₁₂) The method has the advantages of low detection lower limit (0.01-0.1 ng/ml), simple sample pretreatment, rapid detection (1-2 h consumed in the whole process), high flux and the like.

The method can quickly detect the content of various B vitamins in the samples such as pig intestinal contents, excrement and the like, and has application prospect in the detection of other livestock and poultry samples.

The invention utilizes UPLC-MS/MS-MRM technology to carry out targeted metabonomics analysis, and efficiently and quantitatively detects the content of 8B vitamins in the pig cecal contents/feces by optimizing the pretreatment steps and test conditions of the sample.

The method for detecting the content of the nicotinic acid in the feed by using a microbiological method, such as Dongshuang, has the advantages that the average recovery rate is 98.75 percent, the RSD is less than 10 percent, the effective range of the detection result is 25-500 ng, and the detection period is 1-2 days. The method for detecting the niacin has the advantages that the average recovery rate of the detected niacin is 104%, the RSD is less than 5%, the recovery rate and the accuracy are basically consistent with those of a microbiological method, the detection limit is lower (0.01-0.1 ng/mL), and the detection period is shortened to 40 min.

Fan and the like utilize HPLC-FLD technology to detect vitamin B in tea₃And vitaminsB₇The detection limit of the content of (1) is 2.22-2.56 ng/mL, and the detection period is 2 h. Schmidt et al use HPLC to detect vitamin B in milk₆And vitamin B₂The average recovery rate is 91.1-111.2%, RSD is less than or equal to 6.7%, the sample separation time is 6min, the detection period is 45min, and the vitamin B of the invention₆Average recovery rate of 97-108% and RSD<9.6% of vitamin B₂The content average recovery rate is 108 percent and the RSD<17.5％。

The content of folic acid and nicotinic acid in food is respectively detected by UPLC-MS/MS technology, the average recovery rate is 92.1-118.1%, the RSD is 1.49-5.99%, and the detection limit is 0.04-0.40 ng/mL. Method for measuring vitamin B in vitamin complex tablets by using HPLC-MS (high Performance liquid chromatography-Mass Spectrometry) method by Wang and the like₉The average recovery rate of (A) is 99.9-108.7%, the RSD is 1.0%, and the detection limit is 0.33 mug/g. The method of the invention measures vitamin B₉The average recovery was 103% and the RSD was 6.81%.

The currently reported methods for detecting the content of B vitamins mostly detect the content of single or a few B vitamins. The method can detect VB with high flux₁、VB₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, VB₉、VB₁₂The recovery rate of the content of the 8B vitamins is basically consistent with that of a microbial method or a liquid phase method, and the lower limit of detection is lower than that of other methods.

The invention adopts ultra-high performance liquid chromatography, uses 0.3 percent formic acid aqueous solution-methanol as a mobile phase for gradient elution, combines MS for mass spectrometry under a positive ion mode and MRM for ion pair detection, and establishes a high-efficiency and quantitative detection method for VB in pig intestinal contents/feces₁、VB₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, VB₉、VB₁₂And in the methods for detecting the content of 8B vitamins, the analysis time is less than 7min, the whole sample detection process can be completed within 40min, the detection lower limit is low (0.01-0.10 ng/mL), the recovery rate is 88.0-110.0%, and the RSD of a quality control sample is less than 18%. The method for detecting the content of the B vitamins in the pig intestinal contents/feces is favorable for the interaction research of the B vitamins and intestinal microorganisms.

In summary, compared with the prior art, the invention has the following advantages and effects:

the invention utilizes a multi-reaction monitoring technology, combines an ultra-high performance liquid chromatography system and a mass spectrometer, screens parent ions with B vitamins specificity in a targeted manner, selectively performs collision induction on the parent ions, removes interference of other ions, and only acquires mass spectrum signals of selected MS/MS2 ions, thereby realizing more specific, sensitive and accurate analysis on the B vitamins. The method can detect the content of various B vitamins in intestinal contents, can detect a large number of samples simultaneously, and has the advantages of simple and rapid pretreatment, high throughput and the like.

In conclusion, the method for rapidly detecting multiple B vitamins in intestinal contents is formed by using an ultra-high performance liquid chromatography system and a mass spectrometer and combining the MRM technology, has the advantages of high efficiency, low detection lower limit and the like, and can be applied to detection of B vitamins in feces, serum, tissue samples and the like.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a XIC profile of the standard mixture.

In fig. 1:

VB₁vitamin B₁ vitamin B₁(ii) a Niacin acid is nicotinic acid; nicotinamide, Nicotinamide; pyridoxal is Pyridoxal; VB₁₂Vitamin B₁₂ vitamin B₁₂(ii) a Pyridoxine is Pyridoxine; VB₉Vitamin B₉vitamin B₉；VB₂Vitamin B₂ vitamin B₂。

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Examples 1,

1. Reagent and apparatus

Formic acid (Sigma-Aldrich,94318, USA), methanol (Fisher Chemical, A452-4, USA), acetonitrile (Sigma-Aldrich,34851-4L, USA), isopropanol (Fisher Chemical, A461-4, USA), vitamin B standard (all from Sigma-Aldrich, USA), and water for testing was ultrapure water.

5500QTRAP mass spectrometer (AB SCIEX, USA), ultra high performance liquid chromatography system (Waters ACQUITY UPLC I-Class, USA), chromatography column (HSST 32.5 μm,2.1mm x 100mm column, USA), low temperature high speed centrifuge (Eppendorf,5430R, USA), high speed homogenizer (T25, IKA, Germany), vortex shaker (MS3 IKA, Germany).

2. Conditions of the apparatus

2.1) ultra high performance liquid chromatography conditions

The samples were isolated using a Waters ACQUITY UPLCI-Class system. A chromatographic column: waters, HSS T3(2.5 μm,2.1 mm. times.100 mm column); column temperature: 45 ℃; mobile phase: the solution A was 0.3% (by volume) formic acid aqueous solution, and the solution B was methanol. The sample was placed in an autosampler at 8 ℃ at a flow rate of 300. mu.L/min and a sample volume of 5. mu.L.

The relevant liquid phase gradients are as follows: 0-2.0 min, and 0 for liquid B; 2.0-3.0 min, the liquid B changes from 0 to 50% linearly; the liquid B is linearly changed from 50% to 100% in 3.0-4.5 min; the liquid B is linearly changed from 100% to 0 within 4.5-5.5 min; 5.5-11 min, and 0 for liquid B. The above% is volume%.

Note: "liquid B is 50%", i.e., 50% of liquid B, 50% of liquid A, and the rest in analogy.

2.2) conditions for Mass Spectrometry

Mass spectrometry was performed in positive ion mode using a 5500QTRAP mass spectrometer (AB SCIEX). Ionization mode: ESI +, ion spray voltage: + 4500V, ion source temperature: the ion pair to be detected is detected by adopting a multi-selection reaction monitoring (MRM) mode at 550 ℃, 40.00psi of air curtain gas (CUR), 55.00psi of atomization gas (GS1) and 55.00psi of auxiliary gas (GS2), and the MRM conditions are shown in table 1.

TABLE 1 MRM conditions

3. Preparation of standard curve

3.1) preparation of stock solutions of standards

And (4) taking the standard substance, and diluting the standard substance with an aqueous solution to obtain a series of standard working solutions with gradient contents. The method comprises the following specific steps:

vitamin B₁Vitamin B₂Pyridoxal, pyridoxine (collectively vitamin B)₆) Vitamin B₉The standard products are as follows: preparing standard working solutions with contents of 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.00, 25.00 and 100.00ng/mL in sequence;

nicotinamide and nicotinic acid (collectively vitamin B)₃) The standard products are as follows: preparing standard working solutions with the contents of 5.0, 12.5, 25.0, 50.0, 125.0, 250.0, 500.0, 1250.0 and 5000.0 ng/mL in sequence;

vitamin B₁₂And (3) standard substance: preparing standard working solution with contents of 0.50, 1.25, 2.50, 5.00, 12.50, 25.00, 50.00, 125.00 and 500.00ng/mL in sequence.

3.2) preparation of Standard Curve

And (3) putting 150 mu L of the standard substance working solution into a 1.5mL centrifuge tube, and mixing 600 mu L of methanol/acetonitrile (volume ratio is 1:1) solution in a vortex manner to prepare a standard curve solution, and establishing a standard curve.

The method comprises the following specific steps:

an extracted ion chromatogram (XIC) of a B-group vitamin standard substance mixture is obtained by using an ultra-high performance liquid chromatography tandem mass spectrometer and is shown in figure 1, and it can be seen from the figure that the chromatographic separation of each B-group vitamin is better, the peak shapes are sharp and symmetrical, and mass spectrum quantification can be carried out on each metabolite.

Injecting the gradient standard working solution into a gas chromatography-mass spectrometer, selecting an ion monitoring (MRM) mode for measurement, determining the peak position of each B vitamin, recording the peak area of a quantitative ion, and making a standard curve equation by taking the concentration of each standard solution of the B vitamins as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a linear regression equation. The content of each substance to be detected has good linearity in a linear range, and the correlation coefficient r is more than 0.995, thereby meeting the detection requirement. The linear regression equation, correlation coefficient and linear range of the method are shown in table 2.

Table 2, linear regression equation, correlation coefficient and linear range of 8B vitamins

2.3 recovery and precision

Vitamin B₁Vitamin B₂Pyridoxine, pyridoxal, vitamin B₉Respectively adding 0.4, 2.0, 20.0ng/mL 3 content standard substance solutions for recovery test, respectively adding 80, 400, 4000 ng/mL 3 content standard substance solutions for recovery test, and vitamin B₁₂The recovery rate test was carried out by adding 3 standard solutions of 1.6, 8.0, 80.0 ng/mL. And mixing all samples in equal amount to prepare a quality control sample, and inspecting the stability of the detection process by using the quality control sample. As can be seen from Table 3, the average recovery rate thereof>89%, RSD value<18 percent, which shows that the test data is stable and reliable.

TABLE 3 recovery and precision

Example 2

1. Sample collection and processing

1.1) sample Collection

In the experimental base of the animal science research institute of Guangdong province academy of agricultural sciences, 35-day-old piglets are selected to collect the fecal sample and the cecal content, 63-day-old piglets are selected to collect the cecal content sample and the fecal sample, and the specific information of the samples is shown in Table 4. In the experiment, all piglets weaned at 21-day age are fed with the feed in a pig farm.

TABLE 4 sample basic information

1.2) pretreatment of samples

Weighing caecum content/feces sample about 200mg, 750 μ L methanol/acetonitrile/water (volume ratio 2:2:1) solution, extracting for 30S, and mixing for 2min by vortex.

2. Sample detection

Centrifuging the sample solution obtained by the pretreatment for 15min at 4 ℃ by using an Eppendorf-5430R low-temperature high-speed centrifuge at 14000R/min; taking 500 mu L of supernatant, passing the supernatant through an Ostro phospholipid removal plate to reduce matrix effect interference response and sensitivity, and drying the supernatant with nitrogen at 37 ℃; adding 100 μ L of 0.3% formic acid water solution for redissolution, centrifuging at 14000r/min at 4 deg.C for 10 min; the supernatant was taken to a sample vial and analyzed according to the chromatographic conditions described above in example 1.

Description of the drawings: and 1 quality control sample can be arranged in the sample queue at intervals of a certain number of test samples and is used for detecting and evaluating the stability and the repeatability of the system. For example: every 4 test samples at intervals set up 1 matter accuse sample, and this matter accuse sample is prepared for all sample equivalent mixing, and each determinand RSD% is less than 30% in the matter accuse sample, and it is normal to believe system stability and repeatability, and the experimental data is reliable and stable.

3. As a result:

b vitamins in a sample solution to be detected and the peak area thereof are determined according to a detection method: and (3) substituting peak areas obtained by detecting the supernatant into corresponding standard substance curves to obtain the content of 8B vitamins in the supernatant, and then converting:

to obtainThe content of 8B vitamins in the sample.

3.1) 24 fecal samples of 35-day-old piglets and 24 fecal samples of 63-day-old piglets were subjected to the method for measuring the content of B-group vitamins, and the detection results of the samples are shown in Table 5.

As can be seen from Table 5, the content of nicotinic acid in the feces of the piglets is the highest, the average value is 19619.99 ng/g, the average value measured by the content of nicotinic acid in the feces of the piglets at the age of 35 days is 20315.60 ng/g, the maximum value is 58093.78 ng/g, and the minimum value is 6268.05 ng/g; the measured average value of the content of the nicotinic acid in the feces of 63-day-old piglets is 18924.38 ng/g, the maximum value is 55388.18 ng/g, and the minimum value is 8222.01 ng/g. Vitamin B in piglet manure₁₂The content is the lowest, the average value is 18.37ng/g, and vitamin B is contained in the excrement of the piglets aged 35 days₁₂The average value of the content measurement was 10.92ng/g, the maximum value was 17.06ng/g, and the minimum value was 8.95 ng/g. Vitamin B in excrement of 63-day-old piglet₁₂The average value of the content measurement is 24.46ng/g, the maximum value is 82.59ng/g, and the minimum value is 9.17 ng/g. With the increase of the age of the day, the vitamin B in the B vitamins in the excrement of the piglets is removed₁₂The externally measured contents all decreased to different degrees.

TABLE 5 stool sample detection results

3.2) determination of B-group vitamin content in pig cecal content

The vitamin B content was measured by the method using 18 cecal content samples of 35-day-old piglets, 24 cecal content samples of 63-day-old piglets, and the detection results of the samples are shown in Table 6. As can be seen from Table 6, the content of nicotinic acid in the cecal intestine of the piglets is highest, the average value is 15058.25 ng/g, the average value measured by the content of nicotinic acid in the cecal intestine of the piglets aged 35 days is 12095.51 ng/g, the maximum value is 27791.28 ng/g, and the minimum value is 7158.77 ng/g; the content of nicotinic acid in the cecal contents of 63-day-old piglets is measured to be 17280.31 ng/g on the average, 43840.81 ng/g on the maximum and 3608.92 ng/g on the minimum. Vitamin B in piglet cecum contents₁₂Lowest content, average valueIs 13.40ng/g, vitamin B in the cecal contents of piglets aged 35 days₁₂The average value of the content measurement is 12.55ng/g, the maximum value is 30.11ng/g, and the minimum value is 9.40 ng/g; vitamin B in caecum contents of 63-day-old piglets₁₂The average value of the content measurement is 14.00ng/g, the maximum value is 38.12ng/g, and the minimum value is 9.30 ng/g. With the increase of the age of the day, vitamin B is removed from B vitamins in the caecum contents of the piglets₁Vitamin B₆The measured contents are increased in different degrees; vitamin B in piglet excrement₁Vitamin B₆The content decreases with increasing age.

TABLE 6 detection of cecal content

Comparative example 1, ESI + of mass spectrometer positive ion working mode in chromatography-mass spectrometry is changed into ESI-; the rest are equal, and a corresponding standard curve is obtained; the rest is equivalent to the invention.

The results of the recovery test and the precision test conducted in the same manner as in the comparative example are shown in Table 7 below.

TABLE 7 recovery and precision in ESI-mode

As can be seen from the data in Table 7, in the mass spectrum ESI-mode, the recovery rate of the detection method is obviously lower than that in the ESI + mode, and the RSD value is obviously higher than that in the ESI + mode, so that the mass spectrum ESI + mode is selected for detection.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims (4)

1. The method for rapidly detecting the content of B vitamins in pig intestinal contents/feces is characterized by comprising the following steps:

1) sample pretreatment;

weighing 200 +/-10 mg of cecum contents/feces of pigs as samples, adding 750 +/-50 mu L of methanol/acetonitrile/water solution, and performing homogeneous extraction to obtain a sample solution;

methanol/acetonitrile/water in a volume ratio of 2:2: 1;

2) and detecting a sample:

centrifuging the sample solution obtained by the pretreatment in the step 1) at a low temperature and a high speed; taking 500 mu L of supernatant I, passing through an Ostro phospholipid removing plate, and drying by nitrogen; then adding 100 mu L of 0.3% formic acid aqueous solution for redissolution, and centrifuging at low temperature and high speed; detecting and analyzing the obtained supernatant II;

3) substituting the peak area obtained by detecting the supernatant II into a corresponding B vitamin standard curve to obtain the content of 8B vitamins in the supernatant II, and then converting to obtain the content of 8B vitamins in the sample;

the 8B vitamins are: vitamin B₁Vitamin B₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, vitamin B₉Vitamin B₁₂。

2. The method for rapidly detecting the content of B vitamins in pig intestinal contents/feces according to claim 1, characterized in that the detection analysis comprises:

the ultra-high performance liquid chromatography comprises the following steps:

separating by using a Waters ACQUITY UPLCI-Class system;

a chromatographic column: waters, HSS T3; column temperature: 45 ℃; mobile phase: the solution A is 0.3 percent (volume percent) of formic acid aqueous solution, and the solution B is methanol;

the sample injection flow rate is 300 mu L/min, and the sample injection amount is 5 mu L;

the liquid phase gradient was as follows: 0-2.0 min, and 0 for liquid B; 2.0-3.0 min, the liquid B changes from 0 to 50% linearly; the liquid B is linearly changed from 50% to 100% in 3.0-4.5 min; the liquid B is linearly changed from 100% to 0 within 4.5-5.5 min; 5.5-11 min, wherein the liquid B is 0; the above% is volume%;

the mass spectrometry analysis is as follows:

performing mass spectrometry by using a 5500QTRAP mass spectrometer in a positive ion mode;

ionization mode: ESI +, ion spray voltage: + 4500V, ion source temperature: 550 ℃, 40.00psi of air curtain gas, 55.00psi of atomization gas and 55.00psi of auxiliary gas, and adopts a multi-selection reaction monitoring mode to detect the ion pair to be detected, wherein the MRM conditions are as follows:

3. the method for rapidly detecting the content of B vitamins in pig intestinal contents/feces according to claim 2, which is characterized in that:

mixing vitamin B₁Vitamin B₂Nicotinic acid, nicotinamide, pyridoxal, pyridoxine, vitamin B₉Vitamin B₁₂The following operations were performed:

preparing a gradient standard working solution; injecting the gradient standard working solution into a gas chromatography-mass spectrometer, selecting an ion monitoring (MRM) mode for measurement, determining the peak position of each B vitamin, recording the peak area of a quantitative ion, and making a standard curve by taking the concentration of each standard solution of the B vitamins as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to obtain a linear regression equation.

4. The method for rapidly detecting the content of B vitamins in pig intestinal contents/feces according to claim 3, wherein the method comprises the following steps:

vitamin B₁The linear equation of (1) is Y-0.238X-8.689;

vitamin B₂The linear equation of (1) is Y ═ 0.0225X-2.172;

the linear equation of nicotinamide is Y ═ 0.0267X-11.060;

the linear equation of nicotinic acid is that Y is 0.0299X + 4.252;

the linear equation for pyridoxine is Y ═ 0.0503X + 0.0981;

the linear equation of pyridoxal is Y ═ 0.0232X-0.0404;

vitamin B₉The linear equation of (A) is Y ═ 0.00800X-0.0328;

vitamin B₁₂The linear equation of (a) is Y-0.00445X-0.0333.

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