CN117825562A - Method for measuring content of beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food - Google Patents
Method for measuring content of beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food Download PDFInfo
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- 235000013305 food Nutrition 0.000 title claims abstract description 27
- 235000014268 sports nutrition Nutrition 0.000 title abstract description 15
- 239000011550 stock solution Substances 0.000 claims abstract description 17
- 239000012224 working solution Substances 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- 239000012488 sample solution Substances 0.000 claims abstract 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 29
- WLJUMPWVUPNXMF-UHFFFAOYSA-L calcium;3-hydroxy-3-methylbutanoate Chemical compound [Ca+2].CC(C)(O)CC([O-])=O.CC(C)(O)CC([O-])=O WLJUMPWVUPNXMF-UHFFFAOYSA-L 0.000 claims description 19
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 9
- 235000016709 nutrition Nutrition 0.000 claims description 9
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 6
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 6
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 6
- 239000004246 zinc acetate Substances 0.000 claims description 6
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000003113 dilution method Methods 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
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- 238000002360 preparation method Methods 0.000 claims description 4
- 230000000750 progressive effect Effects 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000010829 isocratic elution Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- JGFBQFKZKSSODQ-UHFFFAOYSA-N Isothiocyanatocyclopropane Chemical compound S=C=NC1CC1 JGFBQFKZKSSODQ-UHFFFAOYSA-N 0.000 claims description 2
- PWLNAUNEAKQYLH-UHFFFAOYSA-N butyric acid octyl ester Natural products CCCCCCCCOC(=O)CCC PWLNAUNEAKQYLH-UHFFFAOYSA-N 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- UUIQMZJEGPQKFD-UHFFFAOYSA-N n-butyric acid methyl ester Natural products CCCC(=O)OC UUIQMZJEGPQKFD-UHFFFAOYSA-N 0.000 claims description 2
- 239000013558 reference substance Substances 0.000 claims description 2
- 230000002441 reversible effect Effects 0.000 claims description 2
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- 239000002245 particle Substances 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 9
- AXFYFNCPONWUHW-UHFFFAOYSA-N 3-hydroxyisovaleric acid Chemical compound CC(C)(O)CC(O)=O AXFYFNCPONWUHW-UHFFFAOYSA-N 0.000 description 6
- YZHOZVWZUXBPMD-UHFFFAOYSA-N calcium;3-hydroxy-3-methylbutanoic acid Chemical compound [Ca].CC(C)(O)CC(O)=O YZHOZVWZUXBPMD-UHFFFAOYSA-N 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
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- 238000000926 separation method Methods 0.000 description 3
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- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
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- 125000001909 leucine group Chemical class [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention discloses a method for measuring the content of beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food, which is a high-performance liquid chromatography. The method comprises the following steps: (1) preparing a standard stock solution and a standard working solution; (2) preparing a sample solution to be tested; (3) high performance liquid chromatography conditions; (4) establishing a standard working curve; (5) analysis of results. The method fills up the detection blank of the method for measuring the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutrition food, is simple, convenient and quick, has good reproducibility, and is suitable for daily detection of the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutrition food.
Description
Technical Field
The invention relates to a method for measuring the content of beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food.
Background
Beta-hydroxy-beta-methylbutyrate (HMB) is a five-carbon organic acid, a derivative of leucine produced in vivo by its metabolite alpha-ketoisohexide. HMB has effects of promoting muscle protein synthesis, inhibiting muscle protein decomposition, maintaining lean body mass, improving exercise capacity, maintaining cell membrane integrity, enhancing immune function, and reducing inflammatory reaction. However, due to the active nature of HMB, HMB is often converted to calcium salt, i.e., calcium beta-hydroxy-beta-methylbutyrate (CaHMB), in commercial processes for storage and use. At present, the food is mainly applied to animal nutrition, sports nutrition and functional food. In GB 24154-2015, food safety national Standard sports nutrient food general rule, caHMB is listed as one of the ingredients recommended for addition in animal nutrition foods, but no reference test method is found. Therefore, the invention provides a method for measuring the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutritional food, which has important practical significance.
Disclosure of Invention
The invention aims to provide a method for measuring the content of beta-hydroxy-beta-methylbutyric acid calcium in sports nutrition food, which is used for making up and solving the defect that no method for detecting the content of beta-hydroxy-beta-methylbutyric acid calcium in sports nutrition food exists at present, so that the method can accurately analyze the content of beta-hydroxy-beta-methylbutyric acid calcium in sports nutrition food.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the method is a method for detecting the content of beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food, and is mainly characterized by comprising the following steps of:
(1) Standard stock solution and standard working solution are prepared, wherein the standard stock solution is 1.05mg/mL standard stock solution, and the standard working solution is a series of standard working solutions with the concentration of 21.0 mug/mL, 52.5 mug/mL, 105 mug/mL, 210 mug/mL, 525 mug/mL and 1050 mug/mL prepared from the standard stock solution of beta-hydroxy-beta-methyl calcium butyrate by adopting a progressive dilution method and using 0.1 mol/L hydrochloric acid solution.
(2) Preparing a sample to be tested: placing a sample to be detected in a 25mL colorimetric tube, adding 20ml of 0.1 mol/L hydrochloric acid solution, mixing uniformly by vortex, placing in an ultrasonic oscillator for ultrasonic treatment for 30min, placing to room temperature, adding 1ml of 100g/L potassium ferrocyanide solution and 1ml of 200g/L zinc acetate solution, fixing the volume to a scale mark by using 0.1 mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5min at 4500 r/min, taking a proper amount of supernatant, passing through a 0.45 mu m water phase filter membrane, and waiting for liquid chromatography measurement;
(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a reverse C18 column; mobile phase: the phase A is 0.1% phosphoric acid aqueous solution, and the phase B is methanol; isocratic elution: 0.1% phosphoric acid aqueous solution (a)/methanol (B) =90/10; run time: 20min; column temperature: 30 ℃; sample injection amount: 20. Mu.L; flow rate: 1.0 mL/min; the detection wavelength is 210nm;
(4) Drawing a standard curve: injecting a series of standard working solutions of beta-hydroxy-beta-calcium methylbutyrate into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), qualifying with retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by each concentration and the concentration;
(5) Analysis of results: and (3) injecting the sample filtrate obtained in the step (2) into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), measuring the peak area of the beta-hydroxy-beta-methyl calcium butyrate in the sample, qualitatively determining the retention time, quantifying according to the standard curve manufactured in the step (4), and calculating the content of the beta-hydroxy-beta-methyl calcium butyrate in the sample to be measured.
Compared with the prior art, the invention has the following outstanding advantages:
1. the invention fills the blank in the field of detecting the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutrition food, and needs to establish a new technology to meet the detection requirement of the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutrition food.
2. According to the invention, a C18 column is selected as a stationary phase, when common mobile phase is acetonitrile water or methanol water, peak shape, separation degree and symmetry of beta-hydroxy-beta-methyl calcium butyrate are poor, in order to solve the problem, 0.1% phosphoric acid is selected as a mobile phase aqueous solution through continuous search, meanwhile, the composition and proportion of the mobile phase aqueous solution and an organic phase are continuously optimized, and finally chromatographic conditions suitable for content separation of the beta-hydroxy-beta-methyl calcium butyrate are obtained through research, so that target substances and impurities can be completely separated, and meanwhile, optimal peak shape, separation degree and symmetry are obtained.
3. The analysis instrument used in the invention is a high-efficiency chromatograph equipped with a photodiode array detector, is a general instrument which is easy to purchase in the market, and is easier to popularize and apply compared with instruments with high manufacturing cost such as a liquid chromatography-mass spectrometer and the like.
The invention establishes a high-efficiency chromatographic method for measuring the content of the beta-hydroxy-beta-methylbutyric acid calcium in the sports nutrition food. The method is simple and quick, has good reproducibility, is suitable for quantitative detection of the content of the beta-hydroxy-beta-methyl calcium butyrate in the sports nutritional food, and can provide technical support for related production enterprises, supervision departments and import and export detection departments.
Drawings
FIG. 1 is a liquid chromatographic separation of standard solution of calcium beta-hydroxy-beta-methylbutyrate in the sports nutritional food of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings of the specification of the specific embodiments. The present embodiment is implemented on the premise of the present technology, and a detailed embodiment and a specific operation procedure are now given, but the scope of the present invention is not limited to the following examples.
Examples
1. Reagents and materials
Unless otherwise indicated, all reagents were analytically pure and water was the secondary water specified in GB/T6682.
1.1 acetonitrile: chromatographic purity.
1.2 standard: beta-hydroxy-beta-methylbutyrate calcium CAS:135236-72-5, 98.1% pure, supplied by Fuzhou Yongle Biotechnology Co., ltd.
1.3 preparation of solution: 0.1 mol/L hydrochloric acid solution: 8.3 parts of mL parts of hydrochloric acid (36% -38%) are taken, carefully poured into a proper amount of water and diluted to 1000mL parts of water, and evenly mixed. 100g/L potassium ferrocyanide solution: 100g of potassium ferrocyanide was weighed and the volume was set to 1000mL with water. 200g/L zinc acetate solution: 200g of zinc acetate was weighed out and the volume was set to 1000mL with water. 0.1% phosphoric acid aqueous solution: 1.00mL of phosphoric acid was measured, and the volume was fixed with water to 1000mL. Preparation of standard stock solution: 0.05348g of beta-hydroxy-beta-methylbutyrate calcium reference substance is accurately weighed, dissolved by 0.1 mol/L hydrochloric acid solution and fixed to a volume of 50mL, and standard stock solution with a concentration of 1.05mg/mL is prepared. Preparing a standard working solution: standard stock solution of beta-hydroxy-beta-methyl calcium butyrate is prepared into serial standard working solutions with the concentration of 21.0 mug/mL, 52.5 mug/mL, 105 mug/mL, 210 mug/mL, 525 mug/mL and 1050 mug/mL by adopting a progressive dilution method and using 0.1 mol/L hydrochloric acid solution. All standard stock solutions and standard working solutions were kept in a refrigerator at a temperature of 4 ℃.
2. Apparatus and device
2.1 high performance liquid chromatograph: a photodiode array detector is provided.
2.2 analytical balance (precision 0.1mg and 0.01 mg).
2.3 vortex oscillator
2.4 ultrasonic generator: the power is greater than 180W.
2.5 high speed centrifuge
2.6 aqueous phase filtration membrane: 0.45 μm.
3. Method of
3.1 conditions for high Performance liquid chromatography
a) Chromatographic column: c18 column (250 mm X4.6 mm,5 μm);
b) Mobile phase: 0.1% phosphoric acid aqueous solution (a)/methanol (B) =90/10 isocratic elution;
c) Run time: 20min;
d) Column temperature: 30 ℃;
e) Sample injection volume: 20. Mu.L;
f) Flow rate: 1.0 mL/min;
e) Detection wavelength: 210nm;
3.2 Standard Curve drawing
The standard working solution (1.3) of the beta-hydroxy-beta-methylbutyrate calcium is taken and measured according to the chromatographic condition of 3.1. And (3) carrying out linear regression on the mass concentration (X, mug/mL) corresponding to the peak area (Y) of the analyte to draw a standard curve, thereby obtaining a linear regression equation.
4. Sample testing procedure
4.1 sample pretreatment
Weighing a sample to be measured of 0.2g to 0.1mg, adding 20ml of 0.1 mol/L hydrochloric acid solution into a 25mL colorimetric tube, mixing uniformly by vortex, placing into an ultrasonic oscillator, performing ultrasonic treatment for 30min, placing into room temperature, adding 1ml of 100g/L potassium ferrocyanide solution and 1ml of 200g/L zinc acetate solution, fixing the volume to a scale mark by using 0.1 mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5min at 4500 r/min, taking a proper amount of supernatant, and performing liquid chromatography measurement by using a 0.45 mu m water phase filter membrane;
4.2 sample testing
The prepared sample filtrate was chromatographed under the same chromatographic conditions, and quantified as retention time, by external standard method.
5. Representation of analysis results
The content of the beta-hydroxy-beta-methylbutyrate calcium in the sample is calculated according to the following formula (1):
wherein:
x-the content of beta-hydroxy-beta-methylbutyrate calcium in the sample is in grams per kilogram (g/kg);
-calculating the concentration (μg/mL) of calcium beta-hydroxy-beta-methylbutyrate in the obtained samples from the standard curve; v-the volume of the sample is determined in milliliters (mL);
m-sample mass in grams (g).
The arithmetic mean of the results of two independent assays obtained under repetitive conditions is indicative of the result retaining three significant digits.
6. Methodology investigation, which includes linearity, detection limit, quantitative limit, recovery, precision.
6.1 Linearity, detection limit, quantitative limit: a series of mixed standard working solutions were prepared with a concentration of calcium beta-hydroxy-beta-methylbutyrate of between 21.0. Mu.g/mL and 1050. Mu.g/mL, and the concentrations were linearly regressed by peak area, and the linear equation and correlation coefficients are shown in Table 1. The result shows that the beta-hydroxy-beta-methyl calcium butyrate has good linear relation within 21.0-1050 mug/mL, the correlation coefficient r is 0.9999, and the method can completely meet the detection requirement of the beta-hydroxy-beta-methyl calcium butyrate in sports nutrition food. The detection limit and the quantitative limit of the method are shown in Table 2.
TABLE 1 regression equation for beta-hydroxy-beta-methylbutyrate calcium, correlation coefficient, linear range
Peak area; x: mass concentration, μg/mL.
TABLE 2 detection limit and quantitative limit of method for beta-hydroxy-beta-methylbutyrate calcium
6.2 Recovery rate and precision: under the optimized detection condition, a blank matrix protein powder sample is taken for standard adding recovery rate test, standard adding levels are respectively 0.01 mg/kg, 0.02 mg/kg and 0.10 mg/kg, each level is repeatedly analyzed for 6 times, and the results are shown in Table 3. As shown in Table 3, the standard recovery rate of the method is 98.4% -99.2%, the relative standard deviation RSD is 0.9% -2.1%, and the result shows that the method is suitable for daily analysis and detection of the content of the beta-hydroxy-beta-methylbutyric acid calcium in the sports nutritional food.
Table 3 protein powder sample labelling recovery experimental results (n=6)
6.3 stability: taking three mass concentration beta-hydroxy-beta-methyl calcium butyrate standard solutions of 21.0 mug/mL, 105 mug/mL and 1050 mug/mL in the standard curve range, placing the standard solutions for 2 h, 5h, 8 h, 18 h and 2-6 days at normal temperature, and examining the daily and daytime stability of the beta-hydroxy-beta-methyl calcium butyrate standard solutions. Experimental results show that the precision of the daily stability of the beta-hydroxy-beta-methylbutyrate standard solution with the low, medium and high mass concentration is 0.1% -1.3%, 0.2% -0.4% and 0.03% -0.1% respectively, and the precision of the daily stability is 0.8% -2.4%, 0.2% -0.8% and 0.04% -0.1% respectively. Therefore, the beta-hydroxy-beta-methylbutyrate calcium has better stability in the day and in the daytime, and is favorable for the experiment.
Finally, it is noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (5)
1. A method for determining the content of calcium beta-hydroxy-beta-methylbutyrate in sports nutritional food, the method comprising:
(1) Preparing standard stock solution and standard working solution, wherein the standard stock solution is 1.05mg/mL standard stock solution, and the standard working solution is a series of standard working solutions with the concentration of 21.0 mug/mL, 52.5 mug/mL, 105 mug/mL, 210 mug/mL, 525 mug/mL and 1050 mug/mL prepared from the standard stock solution of beta-hydroxy-beta-methyl calcium butyrate by adopting a progressive dilution method and using 0.1 mol/L hydrochloric acid solution;
(2) Preparing a sample to be tested: placing a sample to be detected in a 25mL colorimetric tube, adding 20mL of 0.1 mol/L hydrochloric acid solution, mixing uniformly by vortex, placing in an ultrasonic oscillator for ultrasonic treatment for 30min, placing to room temperature, adding 1mL of 100g/L potassium ferrocyanide solution and 1mL of 200g/L zinc acetate solution, fixing the volume to a scale by using 0.1 mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5min at 4500 r/min, taking a proper amount of supernatant, and passing through a 0.45 mu m water-phase filter membrane for liquid chromatography determination;
(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a reverse C18 column; mobile phase: the phase A is 0.1% phosphoric acid aqueous solution, and the phase B is methanol; isocratic elution: 0.1% phosphoric acid aqueous solution (a)/methanol (B) =90/10; run time: 20min; column temperature: 30 ℃; sample injection amount: 20. Mu.L; flow rate: 1.0 mL/min; the detection wavelength is 210nm;
(4) Drawing a standard curve: injecting a series of standard working solutions of beta-hydroxy-beta-calcium methylbutyrate into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), qualifying with retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by each concentration and the concentration;
(5) Analysis of results: and (3) injecting the sample filtrate obtained in the step (2) into a high performance liquid chromatograph, detecting under the chromatographic condition of the step (3), measuring the peak area of the beta-hydroxy-beta-methyl calcium butyrate in the sample, qualitatively determining the retention time, quantifying according to the standard curve manufactured in the step (4), and calculating the content of the beta-hydroxy-beta-methyl calcium butyrate in the sample to be measured.
2. The method for determining the content of calcium beta-hydroxy-beta-methylbutyrate in a sports nutritional food according to claim 1, wherein the standard stock solution in (1) is formulated by the specific steps of: 8.3 g mL of hydrochloric acid (36% -38%) is taken, carefully injected into a proper amount of water and diluted to 1000mL by water, evenly mixed to prepare 0.1 mol/L hydrochloric acid solution, 0.05348g of beta-hydroxy-beta-methyl calcium butyrate reference substance is accurately weighed, and the standard stock solution with the concentration of 1.05mg/mL is prepared by dissolving and fixing the volume to 50mL by using the 0.1 mol/L hydrochloric acid solution; (1) The preparation of the standard working solution comprises the following specific steps: standard stock solutions of beta-hydroxy-beta-methyl calcium butyrate are prepared into serial standard working solutions with the concentration of 21.0 mug/mL, 52.5 mug/mL, 105 mug/mL, 210 mug/mL, 525 mug/mL and 1050 mug/mL by adopting a progressive dilution method through 0.1 mol/L hydrochloric acid solution, and all the standard stock solutions and the standard working solutions are stored at the temperature of 4 ℃ of a refrigerator.
3. The method for measuring the content of beta-hydroxy-beta-methylbutyrate calcium in a sports nutritional food according to claim 1, wherein the preparation of the sample solution to be measured in (2) comprises the following specific steps: weighing 0.2g of a sample to be measured, accurately measuring to 0.1mg, adding 20ml of 0.1 mol/L hydrochloric acid solution into a 25mL colorimetric tube, mixing uniformly by vortex, placing into an ultrasonic oscillator for ultrasonic treatment for 30min, placing to room temperature, adding 1ml of 100g/L potassium ferrocyanide solution and 1ml of 200g/L zinc acetate solution, fixing the volume to a scale by using 0.1 mol/L hydrochloric acid solution, shaking uniformly, centrifuging for 5min at 4500 r/min, taking a proper amount of supernatant, and passing through a 0.45 mu m water phase filter membrane for liquid chromatography measurement.
4. The method for measuring the content of calcium beta-hydroxy-beta-methylbutyrate in a sports nutritional food according to claim 1, wherein the reversed-phase C18 chromatographic column in (3) has an inner diameter of 250mm x 4.6mm and a particle size of 5 μm.
5. The method for measuring the content of calcium beta-hydroxy-beta-methylbutyrate in a sports nutritional food according to claim 1, wherein the standard curve coefficient in (4) is not less than 0.999.
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