A method for determining calcium beta-hydroxy-beta-methylbutyrate in high protein food products

Abstract

The present application relates to a food detection method, and particularly to a method for determining calcium beta-hydroxy-beta-methylbutyrate in high-protein food. 18 The sample is extracted by methanol water ultrasonic extraction, purified by PXC solid phase extraction column, separated by HPLC, eluted by acetonitrile-0.02 mol / L potassium dihydrogen phosphate solution as the mobile phase at a flow rate of 1.0 mL / min, detected by a UV detector, and quantified by an external standard method. The mass concentration of calcium beta-hydroxy-beta-methylbutyrate in the range of 0.020-2.00 mg / mL has a good linear relationship with the peak area, the correlation coefficient is 0.9993, the quantitative limit of special medical purpose formula food is 0.4 g / 100g, and the detection limit is 0.2 g / 100g; the quantitative limit of sports nutrition food is 1.0 g / 100g, and the detection limit is 0.5 g / 100g. The standard addition recovery is between 92.9% and 104%, and the relative standard deviation is 0.94%-3.93%. The method has simple pretreatment, good purification effect and high specificity, and can meet the content determination of calcium beta-hydroxy-beta-methylbutyrate in special medical purpose formula food and sports nutrition food.

Description

TECHNICAL FIELD

[0001] The present application relates to a food detection method, in particular to a method for determining calcium β-hydroxy-β-methylbutyrate in high-protein food by solid phase extraction purification-high performance liquid chromatography. BACKGROUND

[0002] Calcium β-hydroxy-β-methylbutyrate (CaHMB) is one of the common supplement forms of β-hydroxy-β-methylbutyric acid (HMB), which is a derivative of essential amino acid leucine in the body through its metabolite α-ketoisocaproic acid, and is widely present in citrus fruits, vegetables, legumes, fish and seafood. In addition to being used for protein synthesis, a small amount of leucine in the human body can produce a small amount of β-hydroxy-β-methylbutyric acid after metabolism. Under normal dietary conditions, an adult can generally produce about 200-400 mg of β-hydroxy-β-methylbutyric acid per day. HMB has the effects of promoting muscle growth, enhancing immunity, reducing cholesterol, and preventing cardiovascular diseases.

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

[0004] .

[0005] Calcium β-hydroxy-β-methylbutyrate is officially included in the new resource food (new food raw material) directory, with a specified edible amount of ≤3 grams per day, and a use range of beverages, milk and dairy products, cocoa products, chocolate and chocolate products, candies, baked foods, sports nutrition foods, and special medical use formula foods. Infants, children, pregnant women and lactating women should not consume it, and the label and instructions should indicate the unsuitable population and the edible limit. At present, the CaHMB products sold on the market are mainly concentrated in special medical use formula foods and sports nutrition foods and other foods.

[0006] The current 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, the Chinese invention patent application (publication number: CN106596771A, publication date: April 26, 2017) discloses a method for determining the content of beta-hydroxy-beta-methylbutyric acid in soybean peptide protein powder by high performance liquid chromatography external standard method. In this method, 0.1 mol / L hydrochloric acid solution is used to precipitate proteins, and 0.01 mol / L sodium heptane sulfonate solution-acetonitrile is used as the mobile phase for high performance liquid chromatography external standard method determination. The Chinese invention patent application (publication number: CN110618214A, publication date: December 27, 2019) discloses a method for determining and confirming beta-hydroxy-beta-methylbutyric acid and its calcium salt in liquid beverages. High performance liquid chromatography-tandem mass spectrometry is used to determine and confirm beta-hydroxy-beta-methylbutyric acid in liquid beverages. In this method, 0.1% formic acid methanol solution is used to precipitate proteins.

[0007] The pretreatment methods of these reported documents directly use hydrochloric acid solution or formic acid methanol solution for extraction without purification steps. However, special medical formula foods and sports nutrition foods contain a large amount of amino acids, short peptides and proteins. When these interfering substances in the extract enter the chromatographic column, the separation efficiency is reduced, there are many interfering peaks in the spectrum, and accurate quantification cannot be performed.

[0008] The Chinese invention patent application (publication number: CN115236213A, publication date: October 25, 2022) discloses a method for determining beta-hydroxy-beta-methylbutyric acid calcium in a sample, which comprises: providing a liquid sample to be tested, adding potassium ferrocyanide and zinc acetate, making up the volume, ultrasonicating, filtering, and using the obtained filtrate as a liquid chromatography sample solution for determination of beta-hydroxy-beta-methylbutyric acid calcium. Although this method improves the precipitating agent, there are still interfering substances in the separation process, which enter the chromatographic column and still cause a reduction in separation efficiency, there are many interfering peaks in the spectrum, and accurate quantification cannot be performed. SUMMARY

[0009] In order to solve the above technical problems, the purpose of the present application is to provide a method for determining beta-hydroxy-beta-methylbutyric acid calcium in high-protein foods by solid phase extraction purification-high performance liquid chromatography. This method has simple pretreatment, good purification effect and high specificity, and can meet the content determination of beta-hydroxy-beta-methylbutyric acid calcium in special medical formula foods and sports nutrition foods.

[0010] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0011] A method for determining beta-hydroxy-beta-methylbutyric acid calcium in high-protein food by solid phase extraction purification-high performance liquid chromatography, which comprises the following steps:

[0012] 1) sample extraction

[0013] 1.0 g of sample was precisely weighed in a 100 mL volumetric flask, 60-100 mL of methanol aqueous solution 8:2 (v / v) was added, mixed, ultrasonic extraction was performed for 3-8 min, after cooling to room temperature, methanol aqueous solution 8:2 (v / v) was added to the mark, shaken, and an appropriate amount of sample solution was taken in a centrifugal tube, centrifuged at 10,000-12,000 r / min for 3-8 min, the supernatant was taken and purified.

[0014] 2) purification

[0015] Immediately after activation, 2-4 mL of the purified liquid was removed and loaded onto the column, a mixed strong cation exchange column was used as the purification column, the effluent was discarded, 2-3 mL of the purified liquid was added, the effluent was collected, filtered through a filter membrane, and then injected.

[0016] 3) chromatographic conditions

[0017] Chromatographic column: C 18 column; column temperature: 30°C; mobile phase: A phase: 0.02 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; 21.5-25 min, 98% A; the above percentages are volume ratios; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 208 nm.

[0018] 4) quantitative determination by external standard method

[0019] The control solution was precisely measured and injected into the liquid chromatograph, the standard curve was drawn according to the concentration and peak area, and the regression equation was calculated; another sample solution was precisely measured and injected into the liquid chromatograph, the chromatogram was recorded, and the content of beta-hydroxy-beta-methylbutyric acid calcium in the sample was calculated by the regression equation.

[0020] As preferred, in step 1), 80 mL of methanol aqueous solution 8:2 (v / v) was added, mixed, ultrasonic extraction was performed for 5 min, after cooling to room temperature, methanol aqueous solution 8:2 (v / v) was added to the mark, shaken, an appropriate amount of sample solution was taken in a 15 mL centrifugal tube, centrifuged at 10,000 r / min for 5 min, 5 mL of supernatant was taken and purified.

[0021] As preferred, the mixed-mode strong cation exchange column in step 2) is Waters Oasis MCX, Dikma ProElut PXC or Anpel CNW Poly-Sery MCX.

[0022] As preferred, the mixed-mode strong cation exchange column in step 2) is Dikma ProElut PXC.

[0023] As preferred, the high-protein food is a special medical purpose formula food and a sports nutrition food.

[0024] As preferred, the high-protein food is a milk protein partially hydrolyzed formula food, an amino acid formula food, a whey protein bar and an oat cereal bar.

[0025] As preferred, the method has a good linear relationship between the mass concentration of calcium beta-hydroxy-beta-methylbutyrate in the range of 0.020-2.00 mg / mL and the peak area, the correlation coefficient is 0.9993, the limit of quantification of the special medical purpose formula food is 0.4 g / 100g, the detection limit is 0.2 g / 100g; the limit of quantification of the sports nutrition food is 1.0 g / 100g, the detection limit is 0.5 g / 100g; the average standard addition recovery is between 92.9% and 104%, and the relative standard deviation is 0.94%-3.93%.

[0026] As preferred, the configuration method of the standard stock solution and the working solution is as follows:

[0027] The calcium beta-hydroxy-beta-methylbutyrate standard stock solution 50.0 mg / mL: accurately weigh 1.25 g, accurate to 0.0001 g, calcium beta-hydroxy-beta-methylbutyrate standard, dissolve in water, and then dilute to 25 mL, mix well, and then transfer to a brown glass bottle, store at 4 ℃ in the dark, and seal, the storage period is 6 months;

[0028] The calcium beta-hydroxy-beta-methylbutyrate standard intermediate solution 10.0 mg / mL: accurately take 2 mL of the calcium beta-hydroxy-beta-methylbutyrate 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 ℃ in the dark, and seal, the storage period is 3 months;

[0029] The calcium beta-hydroxy-beta-methylbutyrate standard series working solution: respectively take an appropriate amount of standard intermediate solution, and 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 with water, and prepare them immediately before use.

[0030] The present application adopts the above technical scheme. After the sample is extracted by methanol water ultrasonic extraction and purified by a PXC solid phase extraction column, C 18 The column was separated, gradient elution was performed with acetonitrile-0.02 mol / L potassium dihydrogen phosphate solution as the mobile phase at a flow rate of 1.0 mL / min, UV detector detection, and external standard method quantification. The mass concentration of β-hydroxy-β-methyl butyric acid calcium in the range of 0.020~2.00 mg / mL showed a good linear relationship with the peak area, the correlation coefficient was 0.9993, the quantitative limit of the special medical use formula food was 0.4 g / 100g, and the detection limit was 0.2 g / 100g; the quantitative limit of the sports nutrition food was 1.0 g / 100g, and the detection limit was 0.5 g / 100g. The average standard addition recovery was between 92.9% and 104%, and the relative standard deviation was 0.94%~3.93%. The method has simple pretreatment, good purification effect and high specificity, and can meet the content determination of β-hydroxy-β-methyl butyric acid calcium in special medical use formula food and sports nutrition food. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Recovery data of β-hydroxy-β-methyl butyric acid calcium in different matrices by solid phase extraction column (n=3).

[0032] Figure 2 Effect of different elution volumes on recovery.

[0033] Figure 3 Chromatogram of β-hydroxy-β-methyl butyric acid calcium in special medical use formula food (A) and sports nutrition food (B) samples.

[0034] Figure 4 Comparison chromatogram of A without purification and B with purification of milk protein partially hydrolyzed formula food. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only 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 labor are within the protection scope of the present application.

[0036] 1 Experimental part

[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 TM 111) was purchased from Tokyo Denshi, Japan; the ultrapure water instrument (Synergy185) 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 MCX solid-phase extraction column (6 mL, 500 mg) was purchased from Waters Technology (Shanghai) Co., Ltd., the ProElut PXC solid-phase extraction column (6 mL, 500 mg) was purchased from Shanghai Dikema Technology Development Co., Ltd., and the Poly-Sery MCX solid-phase extraction column (6 mL, 500 mg) was purchased from Shanghai Anpu Experimental Technology Co., Ltd. and activated with 5 mL of methanol and 5 mL of water in sequence before use.

[0039] Acetonitrile, methanol, and dichloromethane were chromatographically pure and purchased from Merck (Darmstadt, Germany); hydrochloric acid and phosphoric acid were analytically pure and purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; potassium dihydrogen phosphate was analytically pure and purchased from Shantou Xilong Scientific Co., Ltd., Guangdong; and ammonia water was analytically pure and purchased from Zhejiang Hangzhou Gaojing Fine Chemical 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 with water, and then made up 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, made up to the mark with water, and mixed to prepare a standard intermediate solution with a mass concentration of 10 mg / mL. 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 taken, and water was 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, and was prepared immediately before use.

[0044] 1.3 Sample pretreatment

[0045] 1.3.1 Sample extraction

[0046] 1.0 g (accurate to 0.0001 g) of the sample was weighed into a 100 mL volumetric flask, 80 mL of methanol aqueous solution 8:2 (v / v) was added, mixed, ultrasonic extraction was performed for 5 min, after cooling to room temperature, the sample was diluted to the mark with methanol aqueous solution 8:2 (v / v), shaken well, an appropriate amount of sample solution was taken into a 15 mL centrifuge tube, centrifuged at 10 000 r / min for 5 min, 5 mL of supernatant was taken and purified.

[0047] 1.3.2 Purification

[0048] Immediately after activation, 3 mL of the purified liquid was removed and loaded onto the column, the effluent was discarded, 2 mL of the purified liquid was added, the effluent was collected, and after filtration through a filter membrane, the sample was injected.

[0049] 1.4 Chromatographic conditions

[0050] Chromatographic column: C 18 (4.6 x 250 mm, 5.0 μm); column temperature: 30 °C; mobile phase: A phase: 0.02 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; 21.5-25 min, 98% A; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 208 nm.

[0051] 2 Results and discussion

[0052] 2.1 Optimization of extraction reagent

[0053] Special medical use formula food and sports nutrition food substrate contains high content of protein, in order to ensure that the sample matrix can be fully extracted, and ensure that the protein precipitate is completely, reduce the interference peak of spectrum, the complete protein precipitation has important role. The method of protein precipitation includes: organic solvent precipitation method, heavy metal salt precipitation method, acid precipitation method and the like. The present application optimizes five kinds of protein precipitation schemes. A: methanol aqueous solution 8:2 (v / v); B: acetonitrile aqueous solution 8:2 (v / v); C: lead acetate solution; D: potassium ferrocyanide and zinc acetate solution; E: trichloroacetic acid solution. The representative sample of Tangchen Beijian plant protein powder is selected for protein precipitation by the above four methods, and the machine is determined, and the recovery rate of the added recovery is used to determine which method is the best. The results show that the acetonitrile aqueous solution 8:2 (v / v) is better than the methanol aqueous solution 8:2 (v / v) in the effect of precipitating protein, but the target recovery rate is low (28%, 94%); the lead acetate solution, the potassium ferrocyanide and the zinc acetate solution are used to precipitate the protein, and the effect of precipitating the protein is good, but there is an interference peak in the peak position of the target, which affects the accurate quantification of the result, and causes the recovery rate to be high (108% and 125%). And the acid precipitation protein method (recovery rate 101%) can meet the requirements of the recovery rate in the sample standard addition recovery experiment, and there is a higher interference peak near the target peak. Therefore, the methanol protein precipitation method is finally adopted.

[0054] The extraction efficiency of different volume ratios of methanol aqueous solution (2:8, 4:6, 6:4, 8:2, 10:0) (v / v) is further investigated, and the results show that the methanol aqueous solution (2:8 and 4:6) (v / v) is used to extract the sample, 5mL of the extraction solution is passed through the column, and the solution almost does not flow out; the methanol aqueous solution 6:4 (v / v) is used to extract the sample, and all the extraction solution can flow out slowly, but the time is too long; the methanol aqueous solution (8:2, 10:0) (v / v) is used to extract the sample, and all the extraction solution can flow out smoothly. When the methanol aqueous solution 10:0 (v / v) is used for extraction, it is found that the recovery rate of the target is very high, but the actual positive sample recovery rate is low; and when the methanol aqueous solution 8:2 (v / v) is used for extraction, the recovery rate of the blank sample addition recovery and the actual positive sample meets the requirements, therefore, the methanol aqueous solution 8:2 (v / v) is finally selected as the sample extraction solution.

[0055] 2.2 Extraction time optimization

[0056] In order to ensure that the calcium β-hydroxy-β-methylbutyrate in the sample can be completely extracted out, the influence of different extraction times on the recovery rate is investigated. The representative matrix amino acid formula milk powder is selected in a 100 mL volumetric flask, 20 mL of water is added and mixed, an appropriate amount of methanol is added and mixed, and then ultrasonic extraction is carried out for 5 min, 10 min, 15 min and 20 min, respectively. After cooling to room temperature, the volume is adjusted to the mark with methanol, shaken well, and then an appropriate amount of sample solution is taken into a 15 mL centrifugal tube, centrifuged at 10 000 r / min for 5 min, 3 mL of supernatant is taken, passed through a cation exchange column, and the effluent is discarded. Then 2 mL of supernatant is taken and passed through the column, and the effluent is collected. After membrane filtration, the sample is detected. The results show that with the increase of extraction time, the recovery rates of the target substances are 94.0%, 94.7%, 93.2% and 93.1% respectively, the recovery rates meet the requirements, and there is no obvious increase, and the impurity peaks on the chromatogram also have no obvious difference. Therefore, the extraction time of 5 min is finally selected.

[0057] 2.3 Optimization of solid phase extraction column

[0058] CaHMB contains a carboxyl group, which does not undergo ion exchange on a cation exchange column. Amino acids and other interfering substances contain amino groups, which are adsorbed on the cation exchange column by ion exchange. Therefore, according to the properties of the target compound and the interfering substances, a mixed strong cation exchange column is used to adsorb impurities to achieve the purpose of purification. The purification effects of three different brands of strong cation exchange columns, Waters Oasis MCX, Dikma ProElut PXC and Anpul CNW Poly-Sery MCX, on four representative food matrices (partially hydrolyzed formula with milk protein, amino acid formula, whey protein bar and oat cereal bar) are compared. 1 g of the above samples is weighed into a 50 mL centrifugal tube, and ultrasonic extraction is carried out with methanol water solution 8:2 (v / v). An appropriate amount of extract is centrifuged at 10 000 rpm for 5 min, 5 mL of supernatant is taken for solid phase extraction purification, 3 mL of purified liquid is removed and passed through the column, the effluent is discarded, 2 mL of purified liquid is added, the effluent is collected, and after membrane filtration, the sample is analyzed by instrument. The experimental results are as follows: Figure 1 .

[0059] As can be seen from Figure 1 , the recovery rates of the three brands of cation exchange columns in the four typical matrices of partially hydrolyzed formula with milk protein, amino acid formula, whey protein bar and oat cereal bar all meet the requirements, and there are no interfering peaks in the chromatogram. Compared with Waters, the DIKMA has a faster solution flow rate and higher column efficiency when passing through the column. Therefore, the DIKMA PXC column is finally used as the solid phase extraction column for sample pretreatment.

[0060] 2.4 Optimization of elution volume

[0061] Four representative food matrices (partially hydrolyzed formula, amino acid formula, whey protein bar and oat cereal bar) were selected and mixed with 20 mL water in a 100 mL volumetric flask, then mixed with an appropriate amount of methanol, ultrasonic extraction for 5 min, cooled to room temperature, and then diluted to the mark with methanol, shaken well, and then an appropriate amount of sample solution was taken into a 15 mL centrifuge tube and centrifuged at 10 000 r / min for 5 min, and 8 mL of supernatant was passed through a cation exchange column, and each 1 mL was subjected to a receiving test, and the test results are shown in Table 1. Figure 3 As shown in Table 1, the results show that from the beginning of the elution volume of 3 mL, as the elution volume increases, there is no obvious difference in recovery rate, considering the operability of the experiment, and reducing the impurities eluted, the method of discarding the first 3 mL of sample effluent and receiving the last 2 mL for testing is finally determined.

[0062] 2.5 Methodology

[0063] 2.5.1 Linear range and limit of quantification

[0064] Select 0.020 mg / mL, 0.050 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL of calcium β-hydroxy-β-methylbutyrate standard working solution in section "1.2.3", and determine according to the optimized chromatographic conditions, with the corresponding peak area as the vertical coordinate (Y) Y ), and the mass concentration of the standard solution as the horizontal coordinate (X X ), draw a standard curve and perform linear regression. By adding standard solution to the special medical use formula food and sports nutrition food blank sample without calcium β-hydroxy-β-methylbutyrate, and determining according to the optimized method, the limit of quantification (LOQ) of the method was calculated by signal-to-noise ratio (S / N) S / N ) = 10, and the LOQ of the special medical use formula food was 0.4 g / 100g, and the LOQ of the sports nutrition food was 1.0 g / 100g. Within the linear range of 0.020 ~ 2.00 mg / mL, the linearity was good, and the correlation coefficient (r r 2 ) was greater than 0.9993.

[0065] 2.5.2 Recovery rate and precision

[0066] Method The method uses special medical purpose formula food and sports nutrition food as sample matrix, and uses standard addition method to test the recovery rate and precision of the added sample. The blank special medical purpose formula food sample is added with 0.40, 2.50, 10.00 g / 100g of target substance, and the blank sports nutrition food sample is added with 1.00, 2.00, 10.00 g / 100g of target substance. The 3 concentration levels are determined in parallel for 6 times according to the optimized pretreatment detection method at each addition level. The sample detection parallelity is good, the average standard addition recovery rate is 92.9%~104%, and the relative standard deviation is 0.94%~3.93%. 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 β-hydroxy-β-methylbutyric acid calcium in special medical purpose formula food and sports nutrition food, and can be used for daily analysis and detection.

[0067] Table 1 Recovery rate experiment of β-hydroxy-β-methylbutyric acid calcium in different food matrix (n=6)

[0068]

[0069] 2.6 Determination of actual samples

[0070] Ten kinds of commercially available special medical purpose formula food and sports nutrition food added with CaHMB were selected, and 6 samples of each were accurately weighed, about 1.0 g (accurate to 0.0001 g), and the sample solution was prepared according to the pretreatment steps in section 1.3. The content of CaHMB in each sample was determined under the chromatographic conditions in section 1.4. The results are shown in Table 2. The chromatogram of β-hydroxy-β-methylbutyric acid calcium in the sample is shown in Figure 3 . The results show that the content of β-hydroxy-β-methylbutyric acid calcium in the special medical purpose formula food and sports nutrition food 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%~119%. 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 in the announcement.

[0071] Table 2 Determination results of β-hydroxy-β-methylbutyric acid calcium in special medical purpose formula food and sports nutrition food

[0072] Sample name Measured value (g / 100g) Label value (g / 100g) Measured value / label value (%) Special medical use formula food 1 0.590 0.5 118 Special medical use formula food 2 5.47 5.4 101 Special medical use formula food 3 0.596 0.6 99.3 Sports nutrition food 1 5.60 5.77 97.1 Sports nutrition food 2 6.20 5.69 109 Sports nutrition food 3 1.11 1.0 111 Sports nutrition food 4 3.10 2.6 119 Sports nutrition food 5 68.1 65.8 103 Sports nutrition food 6 96.6 84.5 114 Sports nutrition food 7 21.8 20.0 109

[0073] 3. Conclusion

[0074] A method for determination of β-hydroxy-β-methylbutyric acid calcium in special medical use formula food and sports nutrition food by solid phase extraction purification-high performance liquid chromatography was established. The average recovery of the method was 92.9%~104%, and the relative standard deviation was 0.94%~3.93%. Compared with other reported methods, the method has the advantages of simple pretreatment, good purification effect and high specificity, and can realize the simultaneous purification and extraction of samples, which can meet the daily detection requirements of β-hydroxy-β-methylbutyric acid calcium in laboratory samples.

Claims

1. A method for determining calcium β-hydroxy-β-methylbutyrate in high-protein foods by solid-phase extraction purification-high performance liquid chromatography, wherein the high-protein foods are special medical purpose formula foods and sports nutrition foods; special medical purpose formula foods and sports nutrition foods are amino acid formula foods, complete nutritional formula foods, partially hydrolyzed infant milk protein formula foods, Red Bull, oat cereal bars, or whey protein bars; characterized in that, The method includes the following steps: 1) Sample extraction Accurately weigh 1.0 g of sample into a 100 mL volumetric flask, add 60-100 mL of methanol-water solution (8:2 v / v) and mix well. Extract by sonication for 3-8 min. After cooling to room temperature, dilute to the mark with methanol-water solution (8:2 v / v), shake well, take an appropriate amount of sample solution into a centrifuge tube, centrifuge at 10000-12000 r / min for 3-8 min, take the supernatant and wait for purification. 2) Purification Immediately after activation, transfer 2-4 mL of the solution to be purified onto the column. The purification column is a mixed strong cation exchange column. Discard the eluent, add another 2-3 mL of the solution to be purified, collect the eluent, filter it through a filter membrane, and then inject it into the column. 3) Chromatographic conditions Column: C 18 column; Column temperature: 30°C; Mobile phase: Phase A: 0.02 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; 21.5-25 min, 98% A; the percentages above are volume ratios; flow rate: 1 mL / min; injection volume: 10 μL; detection wavelength: 208 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, Add 80 mL of methanol-water solution (8:2 v / v) to step 1) and mix well. Extract by sonication for 5 min. After cooling to room temperature, bring the volume up to the mark with methanol-water solution (8:2 v / v), shake well, take an appropriate amount of sample solution into a 15 mL centrifuge tube, centrifuge at 10000 r / min for 5 min, take 5 mL of supernatant and wait for purification.

3. The method according to claim 1, characterized in that, In step 2), the mixed-type strong cation exchange column is Waters Oasis MCX, Dikma ProElut PXC, or Anpu CNW Poly-Sery MCX.

4. The method according to claim 1, characterized in that, In step 2), the mixed-type strong cation exchange column used is the Dikma ProElut PXC.

5. The method according to claim 1, characterized in that, The method described above shows a good linear relationship between the peak area and the concentration of calcium β-hydroxy-β-methylbutyrate in the range of 0.020~2.00 mg / mL, with a correlation coefficient of 0.9993.

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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