A method for determining calcium beta-hydroxy-beta-methylbutyrate in milk and milk products

Abstract

The present application relates to food detection method, especially to a kind of solid phase extraction purification-high performance liquid chromatography method for determining calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products.The sample is extracted by water oscillation, dichloromethane is extracted, and is purified by PXA solid phase extraction column, with acetonitrile-0.02 mol / L potassium dihydrogen phosphate solution as mobile phase, using C 18 Chromatographic column separation, gradient elution is carried out at flow rate 1.0 mL / min, and is detected by ultraviolet detector, and is quantified by external standard method.Calcium beta-hydroxy-beta-methylbutyrate is linear in the concentration range of 0.020~2.00 mg / mL, and the correlation coefficient is 0.9995.The average recovery of standard addition of 3 concentration levels is between 92.4%~103%, and the relative standard deviation is 1.25%~3.77%, the detection limit of the method is 0.05 g / 100g, and the limit of quantification is 0.1 g / 100g.The method has good purification effect, good separation, strong specificity, good repeatability, and is accurate and reliable, and can meet the determination of calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products.

Description

TECHNICAL FIELD

[0001] The present application relates to a food detection method, in particular to a method for determining the content of calcium β-hydroxy-β-methyl butyrate in milk and dairy products by solid phase extraction purification-high performance liquid chromatography. BACKGROUND

[0002] β-hydroxy-β-methyl butyric acid (HMB) is a five-carbon organic acid, which is a derivative of leucine in the body through its metabolite α-ketoisocaproic acid. HMB has the biological functions of reducing muscle damage, promoting protein synthesis, maintaining cell membrane integrity, and enhancing immunity. Due to the instability of HMB, it is usually converted into calcium salt (CaHMB) by synthetic method for storage and use. GB 24154-2015 "General Standard for Sports Nutrition Foods" GB 29922-2022 "General Standard for Special Medical Purpose Formula Foods (Draft)" and the National Health and Family Planning Commission No. 1 Announcement in 2011 stipulate that the daily intake of CaHMB should not exceed 3g. As a global innovative nutrient, HMB has been widely used in various foods such as dairy products, chocolate products, beverages, energy bars, etc.

[0003] The structural formula of calcium β-hydroxy-β-methyl butyrate is as follows:

[0004] .

[0005] At present, the detection methods of CaHMB in CaHMB products mainly include gas chromatography, high performance liquid chromatography, gas chromatography-tandem mass spectrometry, high performance liquid chromatography-mass spectrometry, etc. For example, Chinese invention patent application (publication number: CN106596771A, publication date: 2017-04-26) discloses a method for determining the content of β-hydroxy-β-methyl butyric acid in soybean peptide protein powder by high performance liquid chromatography external standard method. In this method, 0.1mol / L hydrochloric acid solution is used to precipitate proteins, and 0.01mol / L sodium heptanesulfonate solution-acetonitrile is used as the mobile phase for high performance liquid chromatography external standard method determination. Chinese invention patent application (publication number: CN110618214A, publication date: 2019-12-27) discloses a method for determining and confirming β-hydroxy-β-methyl butyric acid and its calcium salt in liquid beverages. High performance liquid chromatography-tandem mass spectrometry is used to determine and confirm β-hydroxy-β-methyl butyric acid in liquid beverages. In this method, 0.1% formic acid methanol solution is used to precipitate proteins.

[0006] The pretreatment methods of these reported literatures directly use hydrochloric acid solution or formic acid methanol solution for extraction without purification step. Due to the complex composition of milk and dairy products and the high content of proteins, the accurate detection of target substances is greatly disturbed, and purification and enrichment must be carried out.

[0007] Chinese invention patent application (publication number: CN115236213A, publication date: 2022-10-25) discloses a method for determining calcium beta-hydroxy-beta-methylbutyrate in a sample, which comprises: providing a sample to be tested in liquid form, adding potassium ferrocyanide and zinc acetate, constant volume, ultrasonic, filtration, and the obtained filtrate is used as a liquid chromatography sample for determination of calcium beta-hydroxy-beta-methylbutyrate. Although the method improves the precipitant, there are still interfering substances in the separation process, which will still cause the separation efficiency to decrease after entering the chromatographic column, and there are many interference peaks in the spectrum, which leads to the inability to accurately quantify. SUMMARY

[0008] In order to solve the above technical problems, the purpose of the present application is to provide a method for determining calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products by solid phase extraction purification-high performance liquid chromatography; the method has good purification effect, high accuracy and strong specificity, and can meet the content determination of calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products, and provide strong technical support for the quality control of CaHMB products.

[0009] In order to achieve the above purpose, the following technical solutions are adopted in the present application:

[0010] A method for determining calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products, the method comprising the following steps:

[0011] 1) Sample extraction

[0012] Accurately weigh 1.0 g of sample into a 50 mL centrifuge tube, add 10 mL of water, mix well, add 2 mL of dichloromethane, shake for 4-8 min, centrifuge at 8000-10000 r / min for 4-8 min, transfer the supernatant to a 25 mL volumetric flask, extract the sample residue with water again, combine the two extraction liquids, dilute to the mark with water, shake well, take 2.5 mL of sample solution in a 15 mL centrifuge tube, add 5 mL of water, mix well, and purify;

[0013] 2) Purification

[0014] Take the liquid to be purified through the activated solid phase extraction column, the solid phase extraction column uses a strong basic anion exchange column, sequentially wash with water and 0.005% hydrochloric acid methanol solution, discard the washing liquid; elute with 0.1% hydrochloric acid methanol solution, collect the eluate, remove 1 mL of water from the eluate, mix well, blow with nitrogen at 20-45 ℃ until less than 1.0 mL, dilute to 1 mL with water, vortex mix, filter through a filter membrane, and then sample;

[0015] 3) Chromatographic conditions

[0016] Chromatographic column: C18 Column; column temperature: 35 °C; mobile phase: A phase: 0.05 mol / L potassium dihydrogen phosphate solution, pH 3.0; B phase: acetonitrile, elution gradient 0-17 min, 98% A; 17-17.5 min, 98%-40% A; 17.5-21.0 min, 40% A; 21.0-21.5 min, 40%-98% A; 21.5-35 min, 98% A; the above percentages are volume ratios; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 214 nm;

[0017] 4) Quantitative determination by external standard method

[0018] Precisely take the control solution, inject it into the liquid chromatograph, draw the standard curve according to the concentration and peak area, calculate the regression equation; another sample solution is precisely taken and injected into the liquid chromatograph, the chromatogram is recorded, and the content of β-hydroxy-β-methyl butyric acid calcium in the sample is calculated by the regression equation.

[0019] As preferred, in step 1), 1.0 g of the sample is weighed in a 50 mL centrifuge tube, accurately to 0.01 g, 10 mL of water is added, mixed, 2 mL of dichloromethane is added, shaken for 5 min, centrifuged at 8000 r / min for 5 min, the supernatant is transferred to a 25 mL volumetric flask, the sample residue is extracted once more with 10 mL of water, the two extraction liquids are combined, and the volume is adjusted to the mark with water, shaken well, 2.5 mL of the sample solution is taken in a 15 mL centrifuge tube, 5 mL of water is added, mixed, and purified.

[0020] As preferred, in step 2), the liquid to be purified is passed through the activated solid phase extraction column, and 5 mL of water and 5 mL of 0.005% hydrochloric acid methanol solution are used for elution in sequence, and the eluate is discarded. 6 mL of 0.1% hydrochloric acid methanol solution is used for elution, the eluate is collected, 1 mL of water is added to the eluate, mixed, blown with nitrogen at 40 °C until less than 1.0 mL, adjusted to 1 mL with water, vortexed, filtered through a filter membrane, and then injected.

[0021] As preferred, in step 2), the mixed strong anion exchange column uses Waters Oasis MAX, Dikma ProElut PXA or Anspu CNW Poly-Sery MAX.

[0022] As preferred, in step 2), the mixed strong anion exchange column uses Dikma ProElut PXA column.

[0023] As preferred, the milk and dairy products are milk, yogurt, cheese sticks or milk powder.

[0024] As preferred, the method is linear well in the concentration range of 0.020-2.00 mg / mL of the calcium beta-hydroxy-beta-methylbutyrate, with a correlation coefficient of 0.9995. The average spiked recovery rate of the three concentration levels is between 92.4% and 103%, the relative standard deviation is 1.25%-3.77%, the detection limit of the method is 0.05 g / 100g, and the quantitative limit is 0.1 g / 100g.

[0025] As preferred, the configuration method of the standard stock solution and the working solution is as follows:

[0026] The calcium beta-hydroxy-beta-methylbutyrate standard stock solution 50.0 mg / mL: 1.25 g of the calcium beta-hydroxy-beta-methylbutyrate standard is accurately weighed to 0.0001 g, dissolved with water, and then diluted to 25 mL. After mixing, it is transferred to a brown glass bottle, sealed and stored at 4 DEG C in the dark, and the storage period is 6 months;

[0027] The calcium beta-hydroxy-beta-methylbutyrate standard intermediate solution 10.0 mg / mL: 2 mL of the calcium beta-hydroxy-beta-methylbutyrate standard stock solution is accurately taken into a 10 mL volumetric flask, diluted to the mark with water, mixed, and prepared into a standard intermediate solution with a mass concentration of 10 mg / mL. It is sealed and stored at 4 DEG C in the dark, and the storage period is 3 months;

[0028] The calcium beta-hydroxy-beta-methylbutyrate standard series working solution: an appropriate amount of the standard intermediate solution is taken, and water is used to prepare a series of working solutions with concentrations of 0.020 mg / mL, 0.050 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, 1.00 mg / mL and 2.00 mg / mL, respectively. It is prepared immediately before use.

[0029] The application adopts the above technical scheme, the sample is extracted by water oscillation, extracted by dichloromethane, purified by a PXA solid phase extraction column, acetonitrile-0.02 mol / L potassium dihydrogen phosphate solution is used as a mobile phase, and C 18 The column is separated, gradient elution is carried out at a flow rate of 1.0 mL / min, an ultraviolet detector is used for detection, and an external standard method is used for quantification. The calcium beta-hydroxy-beta-methylbutyrate is linear well in the concentration range of 0.020-2.00 mg / mL, with a correlation coefficient of 0.9995. The average spiked recovery rate of the three concentration levels is between 92.4% and 103%, the relative standard deviation is 1.25%-3.77%, the detection limit of the method is 0.05 g / 100g, and the quantitative limit is 0.1 g / 100g. The method has good purification effect, good separation, strong specificity, good repeatability, and is accurate and reliable, and can meet the content determination of the calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 6 is a graph of the recovery of CaHMB in different matrices by solid phase extraction column.

[0031] Figure 2 Figure 7 is a graph of the column capacity of the solid phase extraction column.

[0032] Figure 3 Figure 8 is a graph of the effect of different elution volumes on the recovery of CaHMB.

[0033] Figure 4 Figure 9 is a comparison of the purification effect (A) spectrum of the un-purified sample; (B) spectrum of the purified sample.

[0034] Figure 5 Figure 10 is a chromatogram of CaHMB in actual samples (A) formula milk powder sample; (B) modulated milk powder sample; (C) milkshake sample. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0036] 1. Experimental section

[0037] 1.1 Instruments, materials and reagents

[0038] The high-performance liquid chromatograph (U3000 equipped with a DAD detector) was purchased from Thermo Fisher Scientific, USA; the electronic balance (AE260) was purchased from Mettler Toledo, Switzerland; the vortex mixer (WH-861) was purchased from Huailida Experimental Equipment Co., Ltd., Taicang; the ultrasonic cleaner (P300H) was purchased from Elmasonic, Germany; the high-speed refrigerated centrifuge (Multifuge X1R) was purchased from Thermo, USA; the nitrogen blowing instrument (N-EVAP 111) was purchased from Tokyo Rika, Japan; the ultrapure water instrument (Synergy 185) was purchased from Millipore, USA; the microporous filtration membrane (0.22 µm, organic phase) was purchased from Shanghai Dikema Technology Development Co., Ltd.; the Oasis MAX solid phase extraction column (6 mL, 500 mg) was purchased from Waters Technology (Shanghai) Co., Ltd., the ProElut PXA solid phase extraction column (6 mL, 500 mg) was purchased from Shanghai Dikema Technology Development Co., Ltd., and the Poly-Sery MAX solid phase extraction column (6 mL, 500 mg) was purchased from Shanghai Anpu Experimental Technology Co., Ltd., and was activated with 5 mL of methanol and 5 mL of water in sequence before use. TM 111) purchased from Tokyo Rika, Japan; the ultrapure water instrument (Synergy 185) was purchased from Millipore, USA; the microporous filtration membrane (0.22 µm, organic phase) was purchased from Shanghai Dikema Technology Development Co., Ltd.; the Oasis MAX solid phase extraction column (6 mL, 500 mg) was purchased from Waters Technology (Shanghai) Co., Ltd., the ProElut PXA solid phase extraction column (6 mL, 500 mg) was purchased from Shanghai Dikema Technology Development Co., Ltd., and the Poly-Sery MAX solid phase extraction column (6 mL, 500 mg) was purchased from Shanghai Anpu Experimental Technology Co., Ltd., and was activated with 5 mL of methanol and 5 mL of water in sequence before use.

[0039] Acetonitrile, methanol, dichloromethane were chromatographically pure, purchased from Merck (Darmstadt, Germany); hydrochloric acid, phosphoric acid were analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd.; potassium dihydrogen phosphate was analytically pure, purchased from Shantou Xilong Scientific Co., Ltd.

[0040] 1.2 Preparation of standard stock solution and working solution

[0041] 1.2.1 β-hydroxy-β-methylbutyric acid calcium standard stock solution (50.0 mg / mL): 1.25 g (accurate to 0.0001 g) of β-hydroxy-β-methylbutyric acid calcium standard was accurately weighed, dissolved in water, and then diluted to 25 mL. After mixing, it was transferred to a brown glass bottle and stored at 4 ℃ in the dark. The storage period was 6 months.

[0042] 1.2.2 β-hydroxy-β-methylbutyric acid calcium standard intermediate solution (10.0 mg / mL): 2 mL of β-hydroxy-β-methylbutyric acid calcium standard stock solution was accurately pipetted into a 10 mL volumetric flask, and then diluted to the mark with water. After mixing, a standard intermediate solution with a mass concentration of 10 mg / mL was prepared. It was stored at 4 ℃ in the dark. The storage period was 3 months.

[0043] 1.2.3 β-hydroxy-β-methylbutyric acid calcium standard series working solution: an appropriate amount of standard intermediate solution was pipetted, and water was added to prepare a series of working solutions with concentrations of 0.020 mg / mL, 0.050 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, 1.00 mg / mL, and 2.00 mg / mL. It was prepared immediately before use.

[0044] 1.3 Sample pretreatment

[0045] 1.3.1 Sample extraction

[0046] 1.0 g (accurate to 0.01 g) of sample was weighed into a 50 mL centrifuge tube, 10 mL of water was added, mixed, 2 mL of dichloromethane was added, oscillated for 5 min, and centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a 25 mL volumetric flask, and the sample residue was extracted with 10 mL of water again. The two extraction solutions were combined, diluted to the mark with water, shaken well, and 2.5 mL of sample solution was taken into a 15 mL centrifuge tube. 5 mL of water was added, mixed well, and purified.

[0047] 1.3.2 Purification

[0048] Take the liquid to be purified through the activated solid phase extraction column, sequentially rinse with 5 mL water, 5 mL 0.005% hydrochloric acid methanol solution, and discard the rinsing liquid. Elute with 6 mL 0.1% hydrochloric acid methanol solution, collect the eluent, take 1 mL water and add to the eluent, mix well, blow with nitrogen at 40 ℃ until less than 1.0 mL, and then dilute to 1 mL with water, mix well, filter through a filter membrane, and then sample.

[0049] 1.4 Chromatographic conditions

[0050] Chromatographic column: C 18 (4.6x250 mm, 5.0 μm); column temperature: 35 ℃; mobile phase: A phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 3.0); B phase: acetonitrile, elution gradient 0-17 min, 98% A; 17-17.5 min, 98%-40% A; 17.5-21.0 min, 40% A; 21.0-21.5 min, 40%-98% A; 21.5-35 min, 98% A; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 214 nm.

[0051] 2 Results and discussion

[0052] 2.1 Optimization of extraction solvent

[0053] The components of milk and dairy products are complex, and the protein content is high, which greatly interferes with the accurate detection of target substances, so purification and enrichment must be performed. Commonly used extraction reagents for precipitating proteins, degreasing, and removing other impurities are organic reagents such as dichloromethane, n-hexane, and petroleum ether. In the experiment, representative matrix milk powder was selected to compare the purification effects of dichloromethane, n-hexane, and petroleum ether extraction reagents on target substances. It was found in the experiment that the protein, fat, and other impurities in the extraction solution were extracted into the lower layer of the solution, and the water phase was in the upper layer, which was more conducive to the removal of the supernatant for the next experimental operation; n-hexane and petroleum ether had poor extraction effect and smaller density than water, and the water phase was in the lower layer when n-hexane or petroleum ether was added to the aqueous solution, which was not convenient for removing the lower layer of the purified liquid. Therefore, dichloromethane was finally selected as the liquid-liquid extraction reagent.

[0054] 2.2 Optimization of solid phase extraction column

[0055] Calcium β-hydroxy-β-methylbutyrate contains a carboxyl group, so it can retain target compounds through ion exchange principle. In the experiment, three different brands of mixed strong anion exchange columns, Waters Oasis MAX, Dikma ProElutPXA, and Anpul CNW Poly-Sery MAX, were compared for their purification effects on four representative food matrices (milk, yogurt, cheese stick, and milk powder).

[0056] From Figure 1 It can be seen that there is no significant difference in the recovery rate and purification effect of the three brands of anion exchange columns in four typical substrates of milk, yogurt, cheese sticks and milk powder. Compared with the other two, the solution flow rate is easier to control when using Dikma ProElut PXA column, so the Dikma ProElut PXA column is finally used as the solid phase extraction column for sample pretreatment.

[0057] 2.3 Column capacity investigation

[0058] The experiment uses a pure standard solution of CaHMB to pass through the column, and investigates the influence of the sample loading amount of 0.1, 1, 5, 10, 25 and 50 mg on the recovery rate. 1 mL of CaHMB standard solution is taken and passed through the PXA purification column, and then the purification procedure in section 1.3.2 is operated. From Figure 2 It can be seen that when the column loading amount is less than 1 mg, the recovery rate is more than 100%, when the column loading amount reaches 5 mg, the recovery rate is only 71.2%, and when the column loading amount continues to increase, the recovery rate gradually decreases. Therefore, the maximum column loading amount of the target analyte of the present application needs to be controlled to be less than 1 mg.

[0059] 2.4 Elution liquid optimization

[0060] The elution liquid is an important factor affecting the recovery rate and purification effect of the target compound. The present application investigates the influence of methanol, 0.005% hydrochloric acid methanol solution and 0.01% hydrochloric acid methanol solution on the recovery rate and purification effect. The representative substrate milk powder is selected, the sample extraction is carried out according to section 1.3.1, 2.5 mL of the extraction solution is taken and passed through the column for purification, and then 5 mL of water is used for elution, and then methanol, 0.005% hydrochloric acid methanol solution and 0.01% hydrochloric acid methanol solution are used for elution, respectively, 0.1% hydrochloric acid methanol solution is used for elution, the eluate is collected, 1 mL of water is added to the eluate, mixed, concentrated to 1 mL under nitrogen blowing at 40°C water bath, and then passed through the membrane and analyzed and detected by the instrument. The recovery rate results are 103%, 100% and 94%, respectively, and when methanol is used for elution, an interfering peak appears at the peak of the target compound. With the increase of the proportion of hydrochloric acid, the recovery rate gradually decreases, so 0.005% hydrochloric acid methanol solution is finally selected as the elution solvent.

[0061] 2.5 Elution liquid optimization

[0062] The representative matrix milk powder was selected in a 50 mL centrifuge tube, and extraction was performed according to the extraction procedure in Section 1.3.1 above. 2.5 mL of the extract was taken in a 15 mL centrifuge tube, 5 mL of water was added, and after mixing, solid phase extraction was performed. First, 5 mL of water and 0.005% methanolic hydrochloric acid solution were used for elution, and then 0.05% methanolic hydrochloric acid solution, 0.1% methanolic hydrochloric acid solution, 0.5% methanolic hydrochloric acid solution and 0.1% formic acid methanol solution were used as eluent for elution, respectively. The eluent was collected, 1 mL of water was added to the eluent, mixed, and concentrated to 1 mL under nitrogen blowing at 40°C water bath. After membrane filtration, the machine was analyzed and detected. The experimental recovery rate results were 2.9%, 92.4%, 89.7% and 91.4% in turn, indicating that when 0.05% methanolic hydrochloric acid solution was used for elution, the recovery rate was very low; when 0.5% methanolic hydrochloric acid solution was used for elution, there were interfering peaks near the target affecting quantification; when 0.1% formic acid methanol was used for elution, there was a very high formic acid peak in the chromatogram affecting quantification; when 0.1% methanolic hydrochloric acid solution was used for elution, the recovery rate was the highest, and there was no interfering peak in the chromatogram, so the present application finally selected 0.1% methanolic hydrochloric acid solution as the eluent.

[0063] 2.6 Elution volume optimization

[0064] In order to ensure that the target compound can be completely eluted when passing through the purification column, the influence of different volumes of eluent on the recovery rate of the target compound was investigated. The representative matrix milk powder was selected in a 50 mL centrifuge tube, and extraction was performed according to the extraction procedure in Section 1.3.1 above. 2.5 mL of the extract was taken in a 15 mL centrifuge tube, 5 mL of water was added, and after mixing, solid phase extraction was performed. 5 mL of water and 0.005% methanolic hydrochloric acid solution were used for elution, and then 3, 4, 5, 6, 7 and 8 mL of 0.1% methanolic hydrochloric acid solution were used for elution, respectively. The eluent was collected, 1 mL of water was added to the eluent, mixed, and concentrated to 1 mL under nitrogen blowing at 40°C water bath. After membrane filtration, the machine was analyzed and detected. The recovery rate results are shown in Table 2.6. Figure 3 The results show that when the elution volume is 6 mL, the recovery rate reaches 99.0%, and when the elution volume is increased to 7 mL and 8 mL, the recovery rates are 100.4% and 99.5% respectively, which basically remain unchanged. Therefore, in order to avoid eluting interfering impurities, the present application selects 6 mL as the eluent volume.

[0065] 2.7 Methodology investigation

[0066] 2.7.1 Linear range and limit of quantification

[0067] The standard working solution diluted step by step was injected, and determination was performed according to the optimized chromatographic conditions. The corresponding peak area was taken as the vertical coordinate (Y), Y , and the mass concentration of the standard solution was taken as the horizontal coordinate (X). X), standard curve was drawn, linear regression calculation was carried out, and the standard solution of calcium beta-hydroxy-beta-methylbutyrate in the range of 0.020-2.00 mg / mL showed good linear relationship, and the correlation coefficient (r) was 0.9995. The milk, yogurt, cheese and milk powder without target substance were used as the background, and the detection limit and the quantitative limit of the method were investigated by the standard addition method. The detection limit (LOD) of calcium beta-hydroxy-beta-methylbutyrate was 0.05 g / 100g, and the quantitative limit (LOQ) was 0.10 g / 100g.

[0068] 2.7.2 Recovery and precision

[0069] The milk, yogurt, cheese and milk powder without the measured substance were selected for the standard addition recovery test. Three content levels of 0.10, 6.00 and 10.00 g / 100g were added respectively, and the determination was carried out according to the method. Each level was determined for 6 times, the average standard addition recovery rate was 92.4%-103%, and the relative standard deviation was 1.25%-3.77%. The recovery rate and precision test results are shown in Table 1. The recovery rate and precision meet the requirements of SANTE / 12682 / 2019, can meet the analysis requirements of calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products, and can be used for daily analysis and detection.

[0070] Table 1 Recovery rate of calcium beta-hydroxy-beta-methylbutyrate in different food matrices (n=6)

[0071]

[0072] 2.8 Comparison of purification effect

[0073] The representative sample of milk powder was selected, and two portions of the sample were weighed. One portion of the milk powder sample was extracted according to the steps in section 1.3.1, and the extract was analyzed by machine after membrane filtration, as shown in Figure 4 A, the baseline of the spectrum is very high, there are many interference peaks, and accurate quantitative analysis cannot be carried out; one portion of the milk powder sample was extracted according to the steps in sections 1.3.1 and 1.3.2, and the purified liquid was analyzed by machine after membrane filtration, as shown in Figure 4 B, the baseline of the spectrum is smooth, there is no interference peak near the target peak, the purification effect is good, and the result can be accurately determined.

[0074] 2.9 Determination of actual samples

[0075] Ten kinds of commercially available milk powder and milk shake added with CaHMB were selected, and the detection was carried out according to the method. The determination results are shown in Table 2, and the chromatogram of calcium beta-hydroxy-beta-methylbutyrate in the actual sample is shown in Figure 5The results show that the content of calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products measured by the method is basically consistent with the label value, and the proportion of the measured value to the label value is in the range of 99.3% to 109%. According to the recommended dosage in the product, the 10 products meet the requirement that the daily intake of CaHMB does not exceed 3g / day as required in the announcement.

[0076] Table 2 Determination results of calcium beta-hydroxy-beta-methylbutyrate in actual samples

[0077] Sample name Measured value (g / 100g) Label value (g / 100g) Measured / label value (%) Formula milk powder 1 0.620 0.6 103 Formula milk powder 2 5.96 6.0 99.3 Formula milk powder 3 0.598 0.6 99.7 Preparation milk powder 1 5.83 5.8 100 Preparation milk powder 2 5.82 5.75 101 Preparation milk powder 3 1.12 1.1 102 Preparation milk powder 4 2.95 2.8 105 Smoothie 1 6.61 6.56 101 Smoothie 2 8.66 8.5 102 Smoothie 3 2.18 2.0 109

[0078] 3. Conclusion

[0079] The present application first establishes a method for detecting calcium beta-hydroxy-beta-methylbutyrate in milk and dairy products based on solid phase extraction purification-high performance liquid chromatography. The average recovery rate of the method is in the range of 92.4% to 103%, and the relative standard deviation is 1.25% to 3.77%. Compared with the existing reference method, the present method increases the solid phase extraction purification step, has good purification effect and high specificity, and can meet the daily detection requirements of calcium beta-hydroxy-beta-methylbutyrate in laboratory samples. It can be popularized and applied to the quality screening and supervision detection of milk and dairy products.

Claims

1. A method for determining calcium β-hydroxy-β-methylbutyrate in milk and dairy products, wherein the dairy products are yogurt, milk powder, cheese, or milkshakes, characterized in that, The method includes the following steps: 1) Sample extraction Accurately weigh 1.0 g of sample into a 50 mL centrifuge tube, add 10 mL of water, mix well, add 2 mL of dichloromethane, shake for 4-8 min, centrifuge at 8000-10000 r / min for 4-8 min, transfer the supernatant to a 25 mL volumetric flask, extract the sample residue with water again, combine the two extracts, dilute to the mark with water, shake well, take 2.5 mL of sample solution into a 15 mL centrifuge tube, add 5 mL of water, mix well, and wait for purification; 2) Purification The solution to be purified was passed through an activated solid-phase extraction column and eluted sequentially with water and 0.005% hydrochloric acid-methanol solution, discarding the eluent. Elution was then performed with 0.1% hydrochloric acid-methanol solution, and the eluent was collected. 1 mL of water was added to the eluent, mixed, and purged with nitrogen at 20-45 °C until the volume was less than 1.0 mL. The volume was then adjusted to 1 mL with water, vortexed, and filtered through a membrane before injection. Solid-phase extraction columns such as Waters Oasis MAX, Dikma ProElut PXA, or Anpu CNW Poly-Sery MAX were used. 3) Chromatographic conditions Column: C 18 column; Column temperature: 35 °C; Mobile phase: Phase A: 0.05 mol / L potassium dihydrogen phosphate solution, pH 3.0; Phase B: acetonitrile, elution gradient 0-17 min, 98% A; 17-17.5 min, 98%-40% A; 17.5-21.0 min, 40% A; 21.0-21.5 min, 40%-98% A; 21.5-35 min, 98% A; the percentages above are volume ratios; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 214 nm; 4) Quantitative determination using external standard method Accurately measure the reference solution and inject it into the liquid chromatograph. Plot a standard curve based on concentration versus peak area and calculate the regression equation. Separately, accurately measure the sample solution and inject it into the liquid chromatograph. Record the chromatogram and use the regression equation to calculate the content of calcium β-hydroxy-β-methylbutyrate in the sample.

2. The method according to claim 1, characterized in that, In step 1), weigh 1.0 g of the sample into a 50 mL centrifuge tube, accurate to 0.01 g, add 10 mL of water, mix well, add 2 mL of dichloromethane, shake for 5 min, centrifuge at 8000 r / min for 5 min, transfer the supernatant to a 25 mL volumetric flask, extract the sample residue again with 10 mL of water, combine the two extracts, dilute to the mark with water, shake well, take 2.5 mL of the sample solution into a 15 mL centrifuge tube, add 5 mL of water, mix well, and wait for purification.

3. The method according to claim 1, characterized in that, In step 2), the solution to be purified is passed through the activated solid-phase extraction column and rinsed sequentially with 5 mL of water and 5 mL of 0.005% hydrochloric acid methanol solution, and the rinsing solution is discarded. The solution is then eluted with 6 mL of 0.1% hydrochloric acid methanol solution, and the eluent is collected. 1 mL of water is added to the eluent, mixed, and the volume is reduced to less than 1.0 mL by nitrogen at 40 °C. The volume is then adjusted to 1 mL with water, vortexed, and filtered through a filter membrane before injection.

4. The method according to claim 1, characterized in that, In step 2), the solid-phase extraction column used is a Dikma ProElutPXA column.

5. The method according to claim 1, characterized in that, The method showed good linearity for calcium β-hydroxy-β-methylbutyrate in the concentration range of 0.020~2.00 mg / mL, with a correlation coefficient of 0.9995.

6. The method according to claim 1, characterized in that, The preparation methods for standard stock solutions and working solutions are as follows: 50.0 mg / mL stock solution of calcium β-hydroxy-β-methylbutyrate standard: Accurately weigh 1.25 g (to a minimum of 0.0001 g) of calcium β-hydroxy-β-methylbutyrate standard, dissolve it in water, dilute to 25 mL, mix well, transfer to a brown glass bottle, and store in a sealed container at 4°C protected from light for 6 months. 10.0 mg / mL β-hydroxy-β-methylbutyrate calcium standard intermediate solution: Accurately pipette 2 mL of β-hydroxy-β-methylbutyrate calcium standard stock solution into a 10 mL volumetric flask, dilute to the mark with water, mix well, and prepare a standard intermediate solution with a mass concentration of 10 mg / mL. Store at 4 ℃ protected from light and sealed for 3 months. β-Hydroxy-β-methylbutyrate calcium standard working solutions: Take appropriate amounts of the standard intermediate solution and prepare a series of working solutions with water, with concentrations of 0.020 mg / mL, 0.050 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, 1.00 mg / mL and 2.00 mg / mL respectively. Prepare fresh solutions immediately before use.

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