CN106855545B

CN106855545B - Method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed

Info

Publication number: CN106855545B
Application number: CN201611217764.1A
Authority: CN
Inventors: 朱正鹏; 梁玉树; 何开蓉; 易锡斌; 张遨然; 汪春霞; 王虎; 周丽; 黄李蓉; 梁秋菊
Original assignee: SICHUAN NEW HOPE ANIMAL HUSBANDRY TECHNOLOGY CO LTD; New Hope Liuhe Co Ltd
Current assignee: SICHUAN NEW HOPE ANIMAL HUSBANDRY TECHNOLOGY CO LTD; New Hope Liuhe Co Ltd
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2020-04-28
Anticipated expiration: 2036-12-26
Also published as: CN106855545A

Abstract

The invention discloses a method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed, which comprises the following steps: (1) preparing a multivitamin standard solution comprising at least two or more of the following vitamins: VB₁、VB₃、VB₆、VB₂、VB₇、VB₁₂Vitamin A acetate and vitamin D₃And vitamin E acetate. And detecting the standard solution by adopting HPLC-MS/MS to obtain a standard curve equation. (2) Weighing 5-10g of sample into a volumetric flask, preferably using a 100ml brown volumetric flask, adding the extraction solution, performing ultrasonic extraction for 10-30min, cooling to fix the volume, centrifuging, filtering with a 0.22 μm organic phase filter membrane, and performing analysis and test by adopting the same gradient elution program and mass spectrum detection parameters as those in the step 1. (3) And (4) calculating the content of the multivitamins in the sample according to the measurement results of the step 1 and the step 2. The detection method of the invention realizes one-time continuous detection analysis treatment of vitamin components from various sources in complex compound feed components.

Description

Method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed

Technical Field

The invention relates to a High Performance Liquid Chromatography (HPLC) detection method, in particular to a method for simultaneously detecting multiple vitamins in feed by combining high performance liquid chromatography with mass spectrum-mass spectrum, which comprises the simultaneous detection of fat-soluble vitamins and water-soluble vitamins.

Background

Because the solubility difference between the water-soluble vitamin and the fat-soluble vitamin is large, the methods for simultaneously measuring the water-soluble vitamin and the fat-soluble vitamin have fewer reports.

Semahat Kucukkolbasi et al reported a method for simultaneously determining the content of water-soluble vitamins and fat-soluble vitamins in a multivitamin tablet. The method adopts a trifluoroacetic acid-methanol (TFA-MeOH) system to extract vitamins in a vitamin complex tablet, the TFA-MeOH system is mobile phase gradient elution, the vitamins are separated in a Phenomenex Gemini C18 column, and a Diode Array Detector (DAD) is adopted to detect vitamin B at different wavelengths₁、B₃、B₉、B₁₂、V_CP-aminobenzoic acid (PABA), A, D₃And K, a fluorescence detector (FLD) for the detection of vitamin E and B at different wavelengths₂. The method can detect 11 vitamins simultaneously within 40 min, and is suitable for detecting vitamins in vitamin complex tablet. However, its determination of feed samples with low vitamin content is under further investigation.

Luyan and the like adopt a double-ternary high performance liquid chromatography multi-valve multi-column technology and simultaneously analyze 21 vitamins in a sample. The method adopts water to extract water-soluble vitamins in a sample, and adopts a methanol-dichloromethane system to extract fat-soluble vitamins in the sample. Allowing the water-soluble vitamins to enter an Acclaim PA chromatographic column, and performing gradient elution by using a phosphate buffer solution, an acetonitrile-phosphate buffer solution and methanol-methyl butyl ether as mobile phases; the fat-soluble vitamin enters an Acclaim C18 chromatographic column and is eluted by taking methanol-acetonitrile and methyl butyl ether as mobile phases in a gradient way. The VWD 3400RS uv detector detects vitamins at different wavelengths. The analysis result of the actual sample shows that the method is suitable for detecting vitamin supplement medicines and the like rich in vitamins, but in the beverage, only a few water-soluble vitamins are detected due to the limitation of the solubility of the vitamins, and similarly, a part of low-content vitamins in the chicken feed sample are not detected.

The raw material composition of the compound feed is complex, the vitamin source is more, the content is lower, and the compound feed can not be detected by the above two methods. There is a need to develop a new method for simultaneously detecting water-soluble vitamins and fat-soluble vitamins in low vitamin content samples such as feed.

Disclosure of Invention

The invention aims to overcome the defects that the components in the compound feed are complex, the vitamin source is more, the content is lower and the simultaneous and complete detection cannot be realized in one detection in the prior art, and provides a method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in the feed. The method can complete the detection and analysis of multiple vitamins in the compound feed in the process of one-time detection, has the characteristic of high monitoring and analysis efficiency, and can greatly simplify the multiple detection and analysis of the compound feed.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed comprises the following steps:

(1) preparing a multivitamin standard solution according to detection requirements, wherein the prepared vitamin standard solution comprises at least two or more of the following vitamins: VB₁、VB₃、VB₆、VB₂、VB₇、VB₁₂Vitamin A acetate and vitamin D₃Vitamin E acetate (standard solution). Particularly in the case of at least one of each of water-soluble vitamins and fat-soluble vitamins. Preferably, in particular, the water-soluble vitamins: VB₁、VB₃、VB₆、VB₂、VB₇、VB₁₂And a fat-soluble vitamin: vitamin A acetate and vitamin D₃And vitamin E acetate. Namely, a standard solution of at least one of the above water-soluble vitamins and a standard solution of at least one of the above fat-soluble vitamins. According to the actual detection purpose, the preparation condition of the corresponding standard solution is adjusted, and the standard solution containing both water-soluble vitamins and fat-soluble vitamins, or a plurality of different vitamins, is prepared.

And detecting the standard solution by adopting HPLC-MS/MS to obtain a standard curve equation.

Gradient elution is adopted in the detection process. The mobile phase comprises mobile phase A: aqueous methanol phase, and mobile phase B: aqueous formic acid phase.

Wherein the ratio of the mobile phase A: the volume proportion of methanol in the methanol water solution is more than 95 v%; mobile phase B: the formic acid content of the aqueous formic acid solution is less than 1% by weight.

(2) Weighing sample, preferably weighing 5-10g sample, placing in volumetric flask, preferably 100ml brown volumetric flask, adding extraction solution, and ultrasonic extracting for 10-30min, preferably 15-25min, preferably 20 min; cooling, fixing volume, centrifuging, filtering with 0.22 μm organic phase membrane, and analyzing with the same chromatographic parameters (including gradient elution procedure) and mass spectrum detection parameters as in step 1.

The extraction solution is prepared by mixing a first component of the extraction solution and a second component of the extraction solution according to the weight ratio of 25-35: 65-75 volume ratio, and mixing to obtain the invented solution.

The first component of the extraction solution is 0.03-0.07v% ammonia solution (by volume).

The second component of the extraction solution is 0.1-0.3% BHT methanol solution (m/vol, mass volume percent, g/L).

(3) And (4) calculating the content of the multivitamins in the sample according to the detection results of the step 1 and the step 2.

The detection method provided by the invention designs a targeted extraction scheme, fully extracts vitamin components from multiple sources in complex compound feed components, and then combines with a gradient elution solution combination which is adjusted and optimized to realize one-time continuous detection analysis treatment on multiple vitamins, so that the multiple vitamin components in the compound feed can be rapidly and continuously detected and analyzed, the problems of detection efficiency and accuracy of the multiple vitamins in the existing compound feed can be obviously optimized, the detection analysis time consumption is reduced, the detection analysis efficiency is improved, and the problem of complicated work of the existing detection analysis is avoided.

Further, the extraction solution is: 0.05v% aqueous ammonia solution: 0.2 (g/L)% BHT methanol 25-35: 65-75 volume ratio to prepare the mixed solution. Most preferably, the extraction solution is: 0.05% aqueous ammonia solution: 0.2% BHT methanol 30: 70, and (b) dissolving. For example, the BHT methanol solution was prepared by dissolving 20mg of BHT in 100mL of methanol.

Further, in the gradient elution process, two mobile phases are adopted, which are respectively: mobile phase A: 96-99v% methanol aqueous solution containing 0.01-0.03v% ammonia water, mobile phase B: 0.01-0.03wt% aqueous formic acid solution. In the process of gradient elution, the mobile phase B is used as a main solution for elution, then the mobile phase A is gradually transited to be the main solution for elution, and finally the mobile phase B is recovered to be the main elution solution.

Examples are as follows: the mobile phase A is prepared by uniformly mixing methanol and water according to the volume ratio of 96-99:1-4 to obtain 96-99v% methanol aqueous solution, and then mixing the methanol aqueous solution and ammonia water according to the volume ratio to prepare a mixed solution with the volume ratio of the ammonia water of 0.01-0.03v%, namely the mobile phase A.

Examples are as follows: and (3) preparing a mobile phase B, namely weighing 10-30mg of formic acid, adding the formic acid into 99.97-99.99g of water, and fully shaking up to obtain the mobile phase B, wherein the weight percentage of the formic acid is 0.01-0.03 wt%.

Further, the gradient elution procedure was: mobile phase A: 98v% aqueous methanol containing 0.02v% aqueous ammonia, mobile phase B: 0.02wt% formic acid solution, flow rate of 0.3mL/min, run for 25min, use gradient elution program, gradient conditions: 0-2 min, and 0.5-0.5% of mobile phase A; 2-8.5 min, and 0.5-56% of mobile phase A; 8.5-8.6 min, and 56.0-99.5% of mobile phase A; 8.6-20 min, 99.5-99.5% of mobile phase A; 20-20.01 min, and 99.5-0.5% of mobile phase A.

Further, in the detection and analysis process, the chromatographic column is selected from one of the following: C18-MG II (pH 2-10)3 μm2.0mm I.D.. times 150mm column, C18-MG III (pH 2-10)3 μm2.0mm I.D.. times 150mm column, ADME 3 μm2.1mm I.D.. times 150mm column (pH 2-10) (also known as Capcell PAK ADME 2.1mm I.D. times 150mm, 3 μm), AQ 5 μm2.1mm I.D. times 150mm column, Agilent ZORBIpse XDB-C182.1X 50mm column, Waters ACQUITYUPLC HST 31.8 μm 3.0X 50mm column and Waters ACQUITY UPLC BEH 1.7 μm 2.1X 50mm column. Preferred chromatographic columns are: capcell PAK ADME 2.1mm I.D. times 150mm, 3 μm, ADME 3 μm2.1mm I.D. times 150mm column (pH 2-10), using pH range 2-10.

Further, the mass spectrometer operating parameters are preferably as follows: ESI positive ion scan mode; multiple Reaction Monitoring (MRM) parameters: the temperature of the drying gas is 325 ℃, the flow rate of the drying gas is 11L/min, the pressure of the atomizing gas is 45psi, the temperature of the sheath gas is 300 ℃, the flow rate is 9L/min, the voltage of the capillary tube is 4000V, and the voltage of the nozzle is 500V.

Further, in the mass spectrometer detection and analysis process, the detection parameter conditions of the multivitamins in the MRM monitoring mode are shown in table 2.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the method for simultaneously detecting multiple fat-soluble vitamins and water-soluble vitamins in the feed, disclosed by the invention, a corresponding extraction scheme, chromatographic parameters and mass spectrum parameters are designed according to the complex vitamin source condition in a feed sample and the property characteristics of the multiple vitamins, so that the HPLC-MS/MS can complete the detection and analysis of multiple different vitamins in one detection and analysis process.

2. In the detection method, the existing vitamin extraction scheme of the feed raw materials is particularly optimized and designed, various vitamin components with complex sources in the feed can be fully extracted by utilizing different extraction schemes, and the detection and analysis result on an HPLC-MS/MS instrument is accurate and reliable.

3. In the detection method, the designed mobile phase and gradient elution program realize high-efficiency separation according to the characteristics of a plurality of fat-soluble and water-soluble vitamins for detection and analysis, and provide reliable basic conditions for subsequent qualitative and quantitative work of mass spectrometer detection and analysis.

Description of the drawings:

FIG. 1 is MRM map of 9 vitamins in broiler feed

Fig. 2 is a graph showing the results obtained using 0.02mol/L ammonium acetate (pH 4.0): 0.2% (g/L) BHT +0.05 v% ammonia methanol solution ═ 50: 50 as mobile phase.

FIG. 3 is a graph showing the results of using 0.05% by volume aqueous ammonia: 0.2% (m/vol) BHT methanol 30: 70 as mobile phase.

Detailed Description

The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.

1 Instrument, reagent and sample Material preparation

(1) Agilent 1260-.

(2) Model AB-135 electronic analytical balance (Metlerlitoduo Shanghai Co., Ltd.).

(3) LD5-2B centrifuge (Beijing King Li centrifuge Co., Ltd.).

(4) KQ-500DE model digital control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan).

(5) Vortex mixer MS3 (IKA, germany).

(6) Acetonitrile, methanol (chromatographic purity, merck company.

(7) Formic acid, ammonium acetate, ammonia (analytical purity, Tianjin Kemi Euro Chemicals Co., Ltd.).

(8) Sodium hydroxide, sodium chloride (analytically pure, Shanghai national drug group).

(9) Thiamine (C)₁₂H₁₇CLN₄Hcl, molecular weight: 337.3) (content 99.5%. Sigma company).

(10) Riboflavin (C)₁₇H₂₀N₄O₆Molecular weight: 376) (content 99.5%. Sigma Co.).

(11) Nicotinic acid (C)₆H₅NO₂Molecular weight: 123) (content 99.5%. Sigma Co.).

(12) Pyridoxine (C)₈H₁₁NO₃HCL, molecular weight: 205.6) (content 99.5%. Sigma company).

(13) Biotin (C)₁₀H₁₆N₂O₃S, molecular weight: 244.23) (content 99.5%. Sigma company).

(14) Cobalamin (C)₆₃H₈₈CoN₁₄O₁₄P, molecular weight: 1355.37) (content 99.5%. Sigma company).

(15) Vitamin A acetate (C)₂₂H₃₂O₂Molecular weight: 328.5).

(16) Vitamin E acetate (C)₃₁H₅₂O₃Molecular weight: 472.74).

(17) Vitamin D₃(C₂₇H₄₄O, molecular weight: 384.63).

Samples of fish feed, broiler feed, piglet feed, layer feed, suckling pig feed and boar feed were provided by the Sichuan new hope animal technology Co.

2 preparation of extraction solution and nine mixed vitamin standard solutions

2.1 preparation of the extraction solution

Extracting solution: 0.05% aqueous ammonia solution: 0.2% (m/vol) BHT methanol 30: mixing at 70 vol% to obtain extractive solution

2.2 preparation of Standard solutions of nine Mixed vitamins

Stock solution of water-soluble vitamin mix standard substance: accurately weighing 5mg of VB₁、VB₃、VB₆；50mg VB₂、VB₇、VB₁₂Standard substance in a 500mL brown volumetric flask, 0.01mol ammonium acetate (pH 4.0) is used for preparing a mixed standard substance stock solution (with VB) with the mass concentration of 10.0mg/L₁Meter).

Fat-soluble vitamin standard substance stock solution and working intermediate solution: accurately weighing 100mg of vitamin A acetate, vitamin E acetate and vitamin D₃The standard substance is prepared into a standard substance stock solution with the mass concentration of 1000.0mg/L by using methanol (added with 0.2 percent of 2, 6-di-tert-butyl-4-methylphenol (BHT)). Respectively sucking 1mL of each standard substance working intermediate solution, and preparing vitamin A acetate and vitamin D with concentration by using the extractive solution prepared in the above 2.1₃The concentration of the mixed standard substance working solution is 1mg/L, and the concentration of the vitamin E acetate is 20 mug/L.

Working intermediate liquid of mixed vitamin standard substances: accurately transferring 1mL of water-soluble vitamin mixed standard substance stock solution, 1mL of vitamin A acetate, vitamin E acetate and vitamin D₃The working intermediate solution of the standard substance is prepared into a solution with the following concentration by using 70 percent methanol water solution of 0.2 percent BHT for constant volume in a 100mL brown volumetric flask: the concentration of water-soluble vitamin is 100 mug/L (calculated by VB 1) and the concentration of fat-soluble vitamin is 20 mug/L (calculated by VE).

Mixed standard work series: 2 mL, 4 mL, 10 mL and 20mL of the mixed standard substance working solution were placed in a 100mL brown volumetric flask, and diluted to a standard series with concentrations of 0.4, 0.8, 2, 4 and 20. mu.g/L in terms of VE (vitamin E acetate).

3 sample treatment

Weighing 5-10g sample into a 100ml brown volumetric flask, adding the extraction solution (prepared in the above 2.1) for ultrasonic extraction for 20min, cooling to desired volume, centrifuging, filtering with 0.22 μm organic phase filter membrane, and packaging.

4 chromatographic and mass spectrometric conditions

4.1 chromatographic conditions

A chromatographic column: capcell PAK ADME (2.1 mmI.D. times 150mm, 3 μm); mobile phase A: 98% aqueous methanol solution containing 0.02% aqueous ammonia, mobile phase B: 0.02% formic acid in water at a flow rate of 0.3mL/min was run for 25min using a gradient elution procedure with the gradient conditions as shown in Table 1.

TABLE 1 mobile phase elution gradient

4.2 Mass Spectrometry conditions

ESI positive ion scan mode: multiple Reaction Monitoring (MRM) parameters: the temperature of the drying gas is 325 ℃, the flow rate of the drying gas is 11L/min, the pressure of the atomizing gas is 45psi, the temperature of the sheath gas is 300 ℃, the flow rate is 9L/min, the capillary voltage is 4000V, the nozzle voltage is 500V, and other mass spectrum optimization conditions are shown in Table 2.

TABLE 2 Instrument optimization conditions for 9 vitamins in MRM monitoring mode

5 results and discussion

5.1 Effect of different extraction solvents on the results of detection of 9 Mixed vitamins

In order to analyze samples by a simple and quick method, the following two schemes of taking a composite solution as an extracting agent are compared:

the first scheme is as follows: 0.02mol/L ammonium acetate (pH 4.0): 0.2% (m/vol) BHT +0.05 v% ammonia in methanol 50: 50.

scheme II: 0.02mol/L ammonium acetate (pH 4.0): 0.2% (m/vol) BHT + 0.05% ammonia methanol solution ═ 30: 70.

the results are shown in fig. 2 and fig. 3, respectively, where fig. 2 is a graph showing the results obtained with 0.02mol/L ammonium acetate (pH 4.0): 0.2% (g/L) BHT + 0.05% ammonia methanol solution ═ 50: 50 as mobile phase (scheme one). FIG. 3 is a graph showing the results obtained using a 0.05% aqueous ammonia solution: 0.2% (m/vol) BHT methanol 30: 70 as mobile phase (scheme two).

It can be seen that in both cases the fat-soluble vitamins are better separated. However, when the pH or salt content of the extraction solution is high and the ratio of ammonium acetate contained in the ion is large, the peak shape of the vitamin in the HPLC separation of the vitamin is adversely affected to a large extent. Because the salt proportion is larger in the first scheme, the water-soluble vitamin has fat head peaks, and the obtained peak shape is not good. Especially when salt components exist, the problems of broadening of peak patterns, poor separation degree and the like are caused; when the pH is less than 7, the fat-soluble vitamin A peak pattern is not good.

Therefore, 0.05% ammonia solution was used: 0.2% (m/vol) BHT methanol 30: about 70 percent of the flow is processed relative to the sample, so that the separation of water-soluble vitamins and the separation of fat-soluble vitamins can be satisfied. In particular, it is preferable to use 0.05% ammonia solution: 0.2% (m/vol) BHT methanol 30: 70, the solution is used as a sample extracting solution, and the response value of each detection component reaches the optimum.

5.2 Effect of different chromatographic columns on the results of detection of 9 Mixed vitamins

The same analytical test work was performed with different columns, respectively, which tried to use the following 7:

C18-MG II (pH 2-10)3 μm2.0mmI.D. times 150mm column,

C18-MG III (pH 2-10)3 μm2.0mmI.D. times 150mm column,

ADME (pH 2-10)3 μm2.1mmI.D. x 150mm column,

AQ 5 μm2.1mm I.D. times 150mm column,

Agilent ZORBAX Eclipse XDB-C182.1X 50mm column,

Waters ACQUITY UPLC HSST 31.8 μm 3.0X 50mm column

And Waters ACQUITY UPLC BEH 1.7 μm 2.1X 50mm column.

And comparing and analyzing the test results, and finding that the separation effects of different chromatographic columns have certain difference. Wherein the AQ 5 μm2.1mmI.D. x 150mm column is used for separating and retaining time control of 6 water soluble vitamins and 3 fat soluble vitamins. Therefore, the protocol of the present invention is preferably chromatographed using a AQ 5 μm2.1mm i.d. × 150mm column.

5.3 influence of different mobile phases and elution gradients on detection results of 9 mixed vitamins

Since water-soluble vitamins are relatively stable under acidic conditions, while fat-soluble vitamin a is inactive under acidic conditions, mobile phase a: 98v% aqueous methanol containing 0.02v% aqueous ammonia, mobile phase B: 0.02wt% formic acid aqueous solution, gradient elution is carried out, thus not only satisfying the separation of water-soluble vitamins, but also satisfying the separation of fat-soluble vitamins.

5.4 Effect of different Mass Spectrometry conditions on the results of detection of 9 Mixed vitamins

The test was carried out using a solution of certain concentration in 0.05% ammonia: 0.2% (m/vol, g/L) BHT methanol 30: 70, respectively carrying out parent ion MS2 SCAN on a single vitamin standard working solution prepared by the solution in a positive ion mode, easily finding that the [ M + H ] + molecular ion peak intensity of each target object is high, and the temperature of an ion source is not obviously changed at 260-350 ℃, so that the set temperature of an instrument is 325 ℃ as the temperature of the ion source, then carrying out MS2 SIM optimization framer, then carrying out Productlon and optimizing Collision Energy, and obtaining qualitative ions and quantitative ions with characteristics. Then continuing to: flow rate, atomizing gas pressure, sheath gas temperature, sheath gas flow rate, capillary voltage, nozzle voltage, etc.

5.5 Linear Range, quantitation Limit, spiked recovery and relative Standard deviation

(1) Linear range

Preparing a mixed standard series working solution with the same matrix as the sample to be detected, enabling the concentration of the mixed standard series working solution to be respectively 0.4, 0.8, 2, 4 and 20 mu g/L in terms of vitamin E acetate), making a standard curve according to the peak area of the selected quantitative ion peak to the mass concentration, and showing linear equations and correlation coefficients of 6 water-soluble vitamins and 3 fat-soluble vitamins in a table 3. The results showed that the linear correlation coefficient (R) was in the range of 0.4 to 20. mu.g/L (in terms of vitamin E acetate) for 6 water-soluble vitamins and 3 fat-soluble vitamins²) The linear correlation is 0.9982-0.9999, which shows good linear correlation.

TABLE 3 Standard curves and correlation coefficients for nine vitamins

(2) Detection limit and quantification limit

And (3) adding a target compound into the blank extracting solution according to the step of processing the sample in the step 3, and performing on-machine analysis after the processing to obtain the SNR (signal to noise ratio) with the concentration of 0.4 mu g/L in terms of vitamin E acetate. The detection limit is determined by 3 times of signal-to-noise ratio, the lower limit of quantification is determined by 10 times of signal-to-noise ratio, and the detection limit and the quantification limit of 6 water-soluble vitamins and 3 fat-soluble vitamins are obtained by calculation and are shown in table 4.

(3) Recovery rate of added standard

Adding target vitamin standard substances with different concentrations into a solvent blank, processing the sample according to the step 3, detecting the concentration of the sample on a computer, and calculating the standard addition recovery rate, wherein the standard addition amount, the recovery rate and the relative deviation are shown in table 4 (n is 6).

TABLE 4 recovery rate with standard addition, relative deviation, detection limit and quantitation limit (. mu.g/kg)

6 determination of actual samples

The method is used for detecting samples of fish feed, broiler feed, piglet feed, laying hen feed, piglet feed and boar feed, and VB can be detected in several feed samples₁、VB₂、VB₃、VB₆、VB₇、VB₁₂Wherein VB₁The content of (b) is 3731-8939 mu g/kg and VB₂The content of the vitamin B is 8860-14880 mu g/kg and VB₃The content of (b) is 6744-14720 mu g/kg and VB₆The content of (b) is 2293-7136 mu g/kg and VB₇The content is 356-457 mu g/kg and VB₁₂The content of the vitamin D is 339-485 mug/kg₃The content of (b) is 3731-18939 μ g/kg, the content of vitamin E acetate is 18576-38674 μ g/kg, and the content of vitamin A acetate isThe content is 12579-105648 mug/kg. The method proved to be feasible. The method can meet the requirement of detecting 9 vitamins simultaneously in the compound feed.

TABLE 5 results of the method of the invention for testing a variety of different samples

7 small knot

The test establishes an analytical method for simultaneously detecting 3 fat-soluble vitamins and 6 water-soluble vitamins in compound feeds such as fish feed, broiler feed, piglet feed, layer feed, piglet feed, breeding feed and the like by HPLC-MSMS, and totally 9 vitamins, and the method is used for detecting 9 vitamins, wherein the standardized recovery rate is 82.7-102.6%, the relative standard deviation (n is 6) is 3.9-9.1%, the detection limit of each fat-soluble vitamin is 0.11-30.00 mu g/kg, and the lower limit of quantification is 0.38-99.80 mu g/kg. The method is rapid, simple to operate, energy-saving, environment-friendly and accurate in result, meets the requirements of relevant laws and regulations, and meets the daily detection requirements.

Remarking:

the content of each vitamin is calculated by mass fraction, if converted into International Unit (IU), the conversion coefficient is as follows:

1 international unit va (iu) ═ 0.344 μ g vitamin a acetate;

1 international unit ve (iu) ═ 1mg vitamin E acetate;

1 International Unit VD₃(IU) ═ 0.025 μ g but cholecalciferol.

BHT: 2, 6-di-tert-butyl-4-methylphenol, butyl hydrolyzed, commonly known as antioxidant 264.

Claims

1. A method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed comprises the following steps:

(1) preparing a multivitamin standard solution comprising:

water-soluble vitamins: VB₁、VB₃、VB₆、VB₂、VB₇、VB₁₂In at leastIn a first aspect of the present invention,

and

fat-soluble vitamins: vitamin A acetate and vitamin D₃At least one of vitamin E acetate;

detecting the standard solution by adopting HPLC-MS/MS to obtain a standard curve equation;

in the HPLC detection process, gradient elution is adopted, and mobile phases comprise a mobile phase A: aqueous methanol, and mobile phase B: formic acid aqueous solution;

wherein, the mobile phase A: the volume ratio of methanol in the methanol water solution is more than 95 percent, and the ratio of the mobile phase B: the content of formic acid in the formic acid aqueous solution is less than 1 wt%;

gradient elution procedure: the flow rate is 0.3mL/min, and the operation is carried out for 25 min;

gradient conditions: 0-2 min, mobile phase A0.5 → 0.5%; 2-8.5 min, mobile phase A0.5 → 56%; 8.5-8.6 min, mobile phase A56.0 → 99.5%; 8.6-20 min, and 99.5 → 99.5% of mobile phase A; 20-20.01 min, mobile phase A99.5 → 0.5%;

the chromatographic column is selected from one of the following components:

C18-MG II, pH 2-10, 3 μm2.0mmI.D. times 150mm column,

C18-MG III, pH 2-10, 3 μm2.0mmI.D. times 150mm column,

ADME, pH 2-10, 3 μm2.1mmI.D. x 150mm column,

AQ 5 μm2.1mm I.D. times 150mm column,

Agilent ZORBAX Eclipse XDB-C18, 2.1X 50mm column,

Waters ACQUITY UPLC HSS T3, 1.8 μm 3.0X 50mm column,

Capcell PAK ADME, 3 μm 2.1 mm I.D×150 mm，

And Waters ACQUITY UPLC BEH, 1.7 μm 2.1X 50mm column;

the MS working parameters are as follows: ESI positive ion scan mode; multiple reaction monitoring parameters: the temperature of the drying gas is 325 ℃, the flow rate of the drying gas is 11L/min, the pressure of the atomizing gas is 45psi, the temperature of the sheath gas is 300 ℃, the flow rate is 9L/min, the voltage of a capillary tube is 4000V, and the voltage of a nozzle is 500V;

(2) weighing a sample into a volumetric flask, adding an extraction solution, carrying out ultrasonic extraction for 10-30min, cooling to fix the volume, centrifuging, filtering and loading on a machine by using a 0.22 mu m organic phase filter membrane, and carrying out analysis and test by adopting the same chromatographic parameters and mass spectrum detection parameters as those in the step (1);

the extraction solution is prepared by mixing a first component of the extraction solution and a second component of the extraction solution according to the weight ratio of 25-35: 65-75 volume ratio to prepare solution;

the first component of the extraction solution is 0.03-0.07v% ammonia solution;

the second component of the extraction solution is 0.1-0.3% g/L of BHT methanol solution;

(3) and (3) calculating the contents of the fat-soluble vitamins and the water-soluble vitamins in the sample according to the detection results of the step (1) and the step (2).

2. The method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed according to claim 1, wherein the extraction solution is: 0.05% aqueous ammonia solution: 0.2% BHT methanol = 25-35: 65-75 volume ratio to prepare the mixed solution.

3. The method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed according to claim 2, wherein the extraction solution is: 0.05v% aqueous ammonia solution: 0.2% g/L BHT methanol = 30: 70, and (b) dissolving.

4. The method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed according to claim 1, wherein in the gradient elution process, the mobile phase A is 96-99v% methanol aqueous solution containing 0.01-0.03v% ammonia water, and the mobile phase B is 0.01-0.03wt% formic acid aqueous solution.

5. The method for simultaneously detecting a fat-soluble vitamin and a water-soluble vitamin in a feed as claimed in claim 4, wherein said mobile phase A is a 98v% aqueous methanol solution containing 0.02v% aqueous ammonia, and said mobile phase B is a 0.02wt% aqueous formic acid solution.

6. The method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed according to claim 1, wherein the chromatographic column comprises: capcell PAK ADME 2.1mm I.D. times 150mm, 3 μm.

7. The method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed according to claim 1, wherein in the step (2), the extraction solution is added for ultrasonic extraction for 15-25 min.