Method for simultaneously determining beta-hydroxyl beta-methyl butyrate and amino acid in food

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously determining beta-hydroxyl beta-methyl butyrate and amino acid in food. Comprising the following steps: S1, taking an amino acid standard substance and calcium beta-hydroxy-beta-methylbutyrate to prepare a mixed standard working solution; taking an internal standard substance to prepare an internal standard solution; s2, mixing a to-be-detected object with the internal standard solution, and performing ultrasonic and centrifugal treatment to obtain a to-be-detected sample; s3, respectively injecting the mixed standard working solution and the sample to be detected into a two-dimensional liquid chromatography-mass spectrometry system for analysis; the two-dimensional liquid chromatogram comprises a first-dimensional liquid chromatogram and a second-dimensional liquid chromatogram. According to the invention, beta-hydroxy beta-methylbutyrate and amino acid can be accurately detected at the same time, and the detection requirements of substances in sports nutritious food are met; the problems that a traditional method is low in step-by-step detection efficiency, complex in pretreatment and insufficient in anti-interference capacity are solved, and a solution is provided for optimization of a sports nourishment formula.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection. Specifically, it relates to a method for simultaneously determining β-hydroxy-β-methylbutyrate and amino acids in food. Background Art

[0002] Currently, in the field of sports nutrition foods, the synchronous detection of β-hydroxy-β-methylbutyrate (HMB) and amino acid components has become an increasingly urgent need. As a key muscle growth promoter and anti-catabolic agent, HMB plays an important role in the healthy performance of athletes and fitness enthusiasts. The discovery of HMB provides a new way for the human body to enhance muscle mass and endurance. It can promote protein synthesis and reduce muscle breakdown, thus playing an important role in improving sports performance. However, the separation challenge of HMB in food matrices is an issue that cannot be ignored. Due to the high structural similarity between HMB and leucine, when HMB coexists with other amino acids in food, its resolution is often low. This characteristic limits the wide application of HMB in the formulation design of sports nutrition products because a low resolution may result in a low concentration of HMB components in the final product, affecting its due sports nutrition effects. Therefore, solving the separation problem of HMB in food matrices not only helps to improve its value as a sports nutrition food additive but also further promotes its application and development in this field.

[0003] Chinese Patent Application CN113804806A discloses a method for determining amino acids in bird's nest by ultra-high performance liquid chromatography-tandem mass spectrometry, belonging to the technical field of analytical detection. By using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), a quantitative detection method for amino acids in bird's nest is established, and the analysis of the content of various amino acid components in bird's nest can be completed within 12 minutes. The determination method disclosed in this invention is simple, rapid, accurate, sensitive, and has good reproducibility, and is suitable for the rapid detection of amino acid content in bird's nest and other protein-rich foods. However, this invention is not applicable to the method for simultaneously determining other substances such as β-hydroxy-β-methylbutyrate. Summary of the Invention

[0004] The present invention provides a method for simultaneously determining β-hydroxy-β-methylbutyrate and amino acids in food, comprising the following steps:

[0005] S1, taking amino acid standard substances and calcium β-hydroxy-β-methylbutyrate to prepare a mixed standard working solution; taking an internal standard substance to prepare an internal standard solution;

[0006] S2, mixing the analyte with the internal standard solution, and performing ultrasonic and centrifugal treatments to obtain a test sample;

[0007] S3, inject the mixed standard working solution and the sample to be tested into a two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) system for analysis.

[0008] The amino acid standard substances are: L-Alanine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine, γ-Aminobutanoic acid, L-Glutamine, L-Glutamic acid, Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, Theanine, L-Threonine, L-Tyrosine, L-Valine, N-Acetylneuraminic acid.

[0009] The internal standard substances are: L-Phenylalanine-d8, L-Alanine-d4, L-Methionine-13C5,15N, Glycine-d5, L-Glutamic acid-d5, L-Argininehydrochloride-13C6,15N4, L-Lysine-d4 monohydrochloride, L-Tyrosine-d4, L-Leucine-13C6, L-Proline-d3, L-Serine-13C3,15N, L-Aspartic acid-d3, L-Asparagine-d3 hydrate, L-Valine-13C5,15N, L-Isoleucine-d10, L-Histidine-13C6,15N3 hydrochloride monohydrate, L-Threonine-d5, HMB-d6.

[0010] The two-dimensional liquid chromatography includes a first-dimensional liquid chromatography and a second-dimensional liquid chromatography; the mobile phases of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography both include mobile phase A and mobile phase B, and the mobile phase A includes an ammonium acetate aqueous solution and formic acid.

[0011] The mobile phase B includes an ammonium acetate aqueous solution, acetonitrile and formic acid.

[0012] The concentration of ammonium acetate in the ammonium acetate aqueous solution is 5 - 20 mmol / L.

[0013] Optionally, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 8 - 15 mmol / L.

[0014] The volume ratio of formic acid in the mobile phase A is 0.01 - 0.5%.

[0015] Optionally, the volume ratio of formic acid in the mobile phase A is 0.05 - 0.15%.

[0016] The volume percentage of acetonitrile in the mobile phase B is 80 - 95%.

[0017] Optionally, the volume percentage of acetonitrile in the mobile phase B is 85 - 95%.

[0018] The column particle type of the first - dimensional liquid chromatography is HSS (high - strength silica particles), and the bonded phase of the chromatographic column is T3 (tri - bonded C 18 , end - capped).

[0019] The column particle type of the second - dimensional liquid chromatography is BEH (ethylene - bridged hybrid particles), and the bonded phase of the chromatographic column is an amide group.

[0020] The gradient elution program of the first - dimensional liquid chromatography includes: 0 - 15 min, the volume ratio of the mobile phase B is 0.5 - 3%; 15 - 20 min, the volume ratio of the mobile phase B is 90 - 98%.

[0021] The flow rate of the gradient elution of the first - dimensional liquid chromatography is: 0.2 - 0.5 mL / min.

[0022] The gradient elution program of the second - dimensional liquid chromatography includes: 0 - 0.6 min, the volume ratio of the mobile phase B is 85 - 95%; 0.6 - 0.7 min (excluding the endpoint values), the volume ratio of the mobile phase B is 5 - 15%; 0.7 - 9.1 min, the volume ratio of the mobile phase B is 95 - 100%; 9.1 - 12 min (excluding the endpoint values), the volume ratio of the mobile phase B is 75 - 85%; 12 - 16 min, the volume ratio of the mobile phase B is 3 - 7%;

[0023] The flow rate of the gradient elution of the second - dimensional liquid chromatography is: 0 - 0.6 min, the flow rate is 0.2 - 0.5 mL / min; 0.7 - 1.6 min (excluding the endpoint values), the flow rate is 1.5 - 2.5 mL / min; 1.6 - 9.1 min, stop the flow; 9.1 - 16 min (excluding the endpoint value 9.1), the flow rate is 0.2 - 0.5 mL / min.

[0024] The capture column stationary phase of the second - dimensional liquid chromatography is C 8 (octyl) reversed - phase stationary phase.

[0025] The mass spectrometry conditions are: the acquisition mode is MSE, the electrospray ionization source (ESI), the monitoring mode is dynamic multiple reaction monitoring (dMRM); the capillary voltage is 3500 - 4500 V, the dwell time is 30 - 50; the collision gas is high - purity N 2 , the pressure is 0.1 - 0.2 MPa; the nebulizing gas and the drying gas are both N 2, the atomizing gas pressure is 35 - 55 psi, the drying gas temperature is 280 - 300 °C, and the drying gas flow rate is 8 - 12 L / min; the sheath gas temperature is 290 - 310 °C, and the sheath gas flow rate is 12 - 16 L / min.

[0026] Aiming at the industry gap in the synchronous detection of β-hydroxy-β-methylbutyrate (HMB) and amino acid components in sports nutrition foods, the present invention constructs an orthogonal separation system of dual chromatographic columns, breaks through the limitation of single chromatographic polarity adaptation, and effectively solves the problem of insufficient resolution when HMB and amino acids coexist in complex food matrices; combined with on-line enrichment and gradient elution techniques, it realizes the automatic purification of high-concentration excipients (such as whey protein, sugars) in sports supplements, avoiding the cumbersome pretreatment such as multiple centrifugations and extractions in traditional methods.

[0027] The S3 detection method: Inject the test sample into the 2D-LC-MS system for analysis. This system includes an automatic sampler, liquid chromatographic column 1 (the chromatographic column of the first-dimensional liquid chromatography), liquid chromatographic column 2 (the chromatographic column of the second-dimensional liquid chromatography), liquid chromatographic pump 1, liquid chromatographic pump 2, a trapping column, six-port valve 1, six-port valve 2, and a detector. It mainly includes three processes: HMB transfer, amino acid component separation, and HMB separation; as Figure 1 (A) shows that from 0 to 0.6 min, the 3rd position of six-port valve 1 is connected to the first-dimensional hydrophilic interaction liquid chromatographic column, the 2nd position is connected to the 1st position of six-port valve 2, the 1st position and the 3rd position of six-port valve 2 are connected, the 3rd position leads to the waste liquid, and the output end of liquid chromatographic pump 1 flows through chromatographic column 1 into the waste liquid; as Figure 1 (B) shows that from 0.6 to 1.6 min, control the switching of the six-port valve so that the 5th and 4th positions of six-port valve 1 are connected to the trapping column, the 7th position of six-port valve 2 is connected to the detector, the 6th position of six-port valve 2 is connected to the output end of liquid chromatographic pump 2, and the 8th position is connected to a three-way valve for water replenishment. The HMB in the sample flows through chromatographic column 1 into the trapping column; as Figure 1 (C) shows that from 1.6 to 9.0 min, control the switching of the six-port valve so that the 1st and 8th positions of six-port valve 1 are connected, and the amino acid components in the sample flow through chromatographic column 1 into the detector; as Figure 1 (D) shows that from 9.0 to 18.0 min, control the switching of the six-port valve so that the 7th position of six-port valve 1 and the 5th position of six-port valve 2 are connected to chromatographic column 2, the 6th position of six-port valve 1 and the 4th position of six-port valve 2 are connected, and the HMB in the trapping column flows through chromatographic column 2 into the detector.

[0028] Beneficial effects

[0029] 1. This application can accurately detect β-hydroxy-β-methylbutyrate and amino acids simultaneously, meeting the detection requirements of substances in sports nutrition foods.

[0030] 2. It solves the problems of low efficiency in step-by-step detection, cumbersome pre-treatment and insufficient anti-interference ability of traditional methods, and provides a solution for the optimization of sports nutrition formula.

[0031] 3. By selecting a specific orthogonal separation system of double chromatographic columns, it breaks through the limitation of single chromatographic polarity adaptation and effectively solves the problem of insufficient resolution when HMB and amino acids coexist in complex food matrices.

[0032] 4. The mass spectrometry acquisition mode of the present invention is MSE, the electrospray ionization source (ESI), and the monitoring mode is dynamic multiple reaction monitoring (dMRM), which can improve the sensitivity and specificity of detection.

[0033] 5. The present invention can improve the detection accuracy and precision through a specific elution program. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a working state diagram of the 2D-LC-MS system in Example 1, A: 0 - 0.6 min; B: 0.6 - 6 min; C: 1.6 - 9 min; D: 9 - 18 min.

[0035] Figure 2 It is the extracted ion chromatogram of each compound in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0036] Example 1

[0037] A method for simultaneously determining β-hydroxy-β-methylbutyrate and amino acids in food, comprising the following steps:

[0038] S1, Take amino acid standard substances and calcium β-hydroxy-β-methylbutyrate to prepare a mixed standard working solution; Take the internal standard substance and prepare a 1 μg / mL internal standard solution using 0.1 M hydrochloric acid solution;

[0039] S2, Weigh 0.1 g (accurate to 0.0001 g) of the sample precisely into a 5.0 mL centrifuge tube, add 20 μL of the internal standard solution, add 2.0 mL of 0.1 M hydrochloric acid solution, vortex and mix well, extract ultrasonically for 10 min, centrifuge at 3000 rpm for 15 min, transfer 1.0 mL of the supernatant to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, transfer the supernatant to the inner insert tube of a plastic or glass sample bottle, and discharge the bottom bubbles, and store it in a 4°C refrigerator before injection to obtain the sample to be tested;

[0040] S3, Inject the mixed standard working solution and the sample to be tested into a two-dimensional liquid chromatography - mass spectrometry (2D-LC-MS) system for analysis respectively.

[0041] The quantitative upper limit (LLOQ), quantitative lower limit (LLOD), linear range, regression equation, and correlation coefficient of the mixed standard working solution are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] The amino acid standard substances are: L-Alanine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine, γ-Aminobutanoic acid, L-Glutamine, L-Glutamic acid, Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, Theanine, L-Threonine, L-Tyrosine, L-Valine, N-Acetylneuraminic acid.

[0046] The internal standard substances are: L-Phenylalanine-d8, L-Alanine-d4, L-Methionine-13C5,15N, Glycine-d5, L-Glutamic acid-d5, L-Argininehydrochloride-13C6,15N4, L-Lysine-d4 monohydrochloride, L-Tyrosine-d4, L-Leucine-13C6, L-Proline-d3, L-Serine-13C3,15N, L-Aspartic acid-d3, L-Asparagine-d3 hydrate, L-Valine-13C5,15N, L-Isoleucine-d10, L-Histidine-13C6,15N3 hydrochloride monohydrate, L-Threonine-d5, HMB-d6.

[0047] The two-dimensional liquid chromatography includes a first-dimensional liquid chromatography and a second-dimensional liquid chromatography; the mobile phases of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography are both mobile phase A and mobile phase B. Mobile phase A includes an aqueous ammonium acetate solution and formic acid; mobile phase B includes an aqueous ammonium acetate solution, acetonitrile and formic acid; the concentration of ammonium acetate in the aqueous ammonium acetate solution is 10 mmol / L; the volume ratio of formic acid in mobile phase A is 0.1%; the volume ratio of acetonitrile in mobile phase B is 90%.

[0048] The particle type of the chromatographic column of the first-dimensional liquid chromatography is HSS (high-strength silica particles), and the bonded phase of the chromatographic column is T3 (tri-bonded C 18, end-capped), column specification: ACQUITY UPLC HSS T3, 2.1 mm × 100 mm, 1.8 μm; the column particle type of the second-dimensional liquid chromatography is BEH (ethylene-bridged hybrid particles), the bonded phase of the column is an amide group, and the column specification: ACQUITY UPLC BEH Amide, 2.1 mm × 100 mm, 1.7 μm.

[0049] The gradient elution program of the first-dimensional liquid chromatography includes: 0 - 15 min, the volume ratio of mobile phase B is 1%; 15 - 20 min, the volume ratio of mobile phase B is 95%; the flow rate of the gradient elution of the first-dimensional liquid chromatography is: 0.3 mL / min.

[0050] The gradient elution program of the second-dimensional liquid chromatography includes: 0 - 0.6 min, the volume ratio of mobile phase B is 90%; 0.6 - 0.7 min, the volume ratio of mobile phase B is 10%; 0.7 - 9.1 min, the volume ratio of mobile phase B is 100%; 9.1 - 12 min, the volume ratio of mobile phase B is 80%; 12 - 16 min, the volume ratio of mobile phase B is 5%;

[0051] The flow rate of the gradient elution of the second-dimensional liquid chromatography is: 0 - 0.6 min, the flow rate is 0.3 mL / min; 0.7 - 1.6 min, the flow rate is 2 mL / min; 1.7 - 9.1 min, stop flow; 9.1 - 16 min, the flow rate is 0.3 mL / min.

[0052] The capture column fixed phase C of the second-dimensional liquid chromatography 8 (octyl) reversed-phase fixed phase, column specification: Agilent Zorbax SB-C 8 , 4.6 mm × 50 mm, 5 μm.

[0053] The mass spectrometry conditions are as follows: using an Agilent 6495 triple quadrupole tandem mass spectrometry system. The acquisition mode is MSE, the electrospray ionization source (ESI), and the monitoring mode is dynamic multiple reaction monitoring (dMRM). The capillary voltage is 4000 V, and the dwell time is 40. The collision gas is high-purity N 2 , with a pressure of 0.1 MPa. Both the nebulizing gas and the drying gas are N 2 , the nebulizing gas pressure is 45 psi, the drying gas temperature is 290 °C, and the drying gas flow rate is 10 L / min. The sheath gas temperature is 300 °C, the sheath gas flow rate is 14 L / min, and the collision energy is shown in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] The S3 detection method: Inject the test sample into a 2D-LC-MS system for analysis. This system includes an autosampler, liquid chromatography column 1 (the column of the first-dimensional liquid chromatography), liquid chromatography column 2 (the column of the second-dimensional liquid chromatography), liquid chromatography pump 1, liquid chromatography pump 2, a trapping column, six-port valve 1, six-port valve 2, and a detector. It mainly includes three processes: HMB transfer, amino acid component separation, and HMB separation; as Figure 1 (A) shows, from 0 to 0.6 min, the 3rd position of six-port valve 1 is connected to the first-dimensional hydrophilic interaction liquid chromatography column, the 2nd position is connected to the 1st position of six-port valve 2, the 1st position and the 3rd position of six-port valve 2 are connected, the 3rd position leads to the waste liquid, and the output end of liquid chromatography pump 1 flows through column 1 into the waste liquid; as Figure 1 (B) shows, from 0.6 to 1.6 min, control the switching of the six-port valve so that the 5th and 4th positions of six-port valve 1 are connected to the trapping column, the 7th position of six-port valve 2 is connected to the detector, the 6th position of six-port valve 2 is connected to the output end of liquid chromatography pump 2, and the 8th position is connected to a three-way valve for water replenishment. The HMB in the sample flows through column 1 into the trapping column; as Figure 1 (C) shows, from 1.6 to 9.0 min, control the switching of the six-port valve so that the 1st and 8th positions of six-port valve 1 are connected. The amino acid components in the sample flow through column 1 into the detector; as Figure 1 (D) shows, from 9.0 to 18.0 min, control the switching of the six-port valve so that the 7th position of six-port valve 1 and the 5th position of six-port valve 2 are connected to column 2, the 6th position of six-port valve 1 and the 4th position of six-port valve 2 are connected, and the HMB in the trapping column flows through column 2 into the detector.

[0058] As Figure 2 shown, the separation degree of the compound is relatively high.

[0059] Performance test method

[0060] Methodology verification: Test the accuracy and precision of HMB and different amino acids at different concentrations (0.5 ng / mL, 5 ng / mL, and 50 ng / mL). Among them, Table 3 corresponds to n (number of repetitions) = 5, and Table 4 corresponds to n (number of repetitions) = 6.

[0061] Performance test data

[0062] Table 3

[0063]

[0064]

[0065] Table 4

[0066]

[0067]

[0068]

Claims

1. A method for simultaneously determining β-hydroxy β-methylbutyrate and amino acids in food, characterized in that: The following steps are involved: S1, prepare a mixed standard working solution with amino acid standard substances and calcium β-hydroxy-β-methylbutyrate; prepare an internal standard solution with an internal standard substance; S2, mixing the analyte with the internal standard solution, performing ultrasonic and centrifugal treatment to obtain a sample to be tested; S3, injecting the mixed standard working solution and the sample to be tested into a two-dimensional liquid chromatography-mass spectrometry system for analysis respectively; the two-dimensional liquid chromatography includes a first-dimensional liquid chromatography and a second-dimensional liquid chromatography.

2. The method for simultaneously determining β-hydroxyβ-methylbutyrate and amino acids in food according to claim 1, characterized in that: The mobile phases of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography both include mobile phase A and mobile phase B, and the mobile phase A includes an aqueous ammonium acetate solution and formic acid.

3. The method for simultaneously determining β-hydroxyβ-methylbutyrate and amino acids in food according to claim 2, characterized in that: The concentration of ammonium acetate in the ammonium acetate aqueous solution is 5-20 mmol / L.

4. The method for simultaneously determining β-hydroxyβ-methylbutyrate and amino acids in food according to claim 3, characterized in that: The volume proportion of formic acid in the mobile phase A is 0.01-0.5%.

5. The method for simultaneously determining β-hydroxyβ-methylbutyrate and amino acids in food according to claim 4, characterized in that: The volume proportion of acetonitrile in the mobile phase B is 80-95%.

6. The method for simultaneously determining β-hydroxyβ-methylbutyrate and amino acids in food according to claim 1 or 4, characterized in that: The particle type of the chromatographic column of the first-dimensional liquid chromatography is HSS, and the bonded phase of the chromatographic column is T3.

7. The method for simultaneously determining β-hydroxy β-methylbutyrate and amino acids in food according to claim 1, characterized in that: The particle type of the chromatographic column of the second-dimensional liquid chromatography is BEH, and the bonded phase of the chromatographic column is an amide group.

8. The method for simultaneously determining β-hydroxy β-methylbutyrate and amino acids in food according to claim 2, characterized in that: The gradient elution program of the first-dimensional liquid chromatography includes: 0-15min, the volume proportion of mobile phase B is 0.5-3%; 15-20min, the volume proportion of mobile phase B is 90-98%.

9. The method for simultaneously determining β-hydroxy β-methylbutyrate and amino acids in food according to claim 2, characterized in that: The gradient elution program of the second dimension liquid chromatography includes: 0-0.6min, the volume ratio of mobile phase B is 85-95%; 0.6-0.7min, the volume ratio of mobile phase B is 5-15%; 0.7-9.1min, the volume proportion of mobile phase B is 95-100%; 9.1-12min, the volume proportion of mobile phase B is 75-85%; 12-16min, the volume proportion of mobile phase B is 3-7%.

10. The method for simultaneously determining β-hydroxy β-methylbutyrate and amino acids in food according to claim 1, characterized in that: The detection parameters of the mass spectrometer are: the acquisition mode is MSE, the electrospray ion source, and the monitoring mode is dynamic multiple reaction monitoring; the capillary voltage is 3500-4500V, and the residence time is 30-50; the collision gas is high-purity N2, and the pressure is 0.1-0.2MPa; the nebulizing gas and the drying gas are both N2, the nebulizing gas pressure is 35-55psi, the drying gas temperature is 280-300℃, and the drying gas flow rate is 8-12L / min; the sheath gas temperature is 290-310℃, and the sheath gas flow rate is 12-16L / min.

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