An ion chromatography method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids and its application

Abstract

The present invention belongs to the technical field of analytical chemistry, and specifically relates to an ion chromatography method for simultaneously determining the content of lactic acid and a variety of short-chain fatty acids and its application. The method of the present invention includes adding a metal salt to a sample to be tested, filtering, and diluting with a solvent, wherein the metal salt is selected from one or both of zinc sulfate heptahydrate and potassium ferrocyanide. The present invention realizes a detection method for simultaneously analyzing the content of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid in a linear range of 1-500 μg / mL by investigating the pre-treatment process of the sample to be tested, and achieves the effect of simple pre-treatment method, small amount of chemical reagent, accurate qualitative and quantitative analysis and good sensitivity; by selecting the type of mobile phase and its concentration in the chromatographic conditions, the reagent is simple and interference-free, and the accuracy is high, which provides a basis for the determination of lactic acid and short-chain fatty acids in animal metabolism research.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and specifically relates to an ion chromatography method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids and an application thereof. Background Art

[0002] Lactic acid is converted from pyruvate and is a product of anaerobic glycolysis. Lactic acid is continuously produced during normal metabolism and exercise. Generally speaking, when the energy of tissues cannot be met through aerobic respiration, tissues cannot obtain enough oxygen or cannot process oxygen quickly enough, the concentration of lactic acid will rise. The continuous accumulation of lactic acid will cause acidosis in or around muscle cells, which will seriously disrupt multiple mechanisms in muscle cells. Short-chain fatty acids are generally organic acids with a carbon chain of 1-6 carbon atoms, including formic acid, acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, butyric acid, etc. They are the most intensively studied metabolites. Their content in the intestine mainly depends on the composition of the intestinal flora, the content of dietary fiber, the digestion time of food in the intestine, and the metabolic capacity of the host and intestinal flora.

[0003] Chinese invention patent CN102128885A discloses a method for rapid and simultaneous determination of 16 kinds of inorganic anions and organic acids in tobacco. The invention adopts an ion chromatography analysis method, and the elution procedure is: 0.000-8.000min, the concentration of potassium hydroxide is 1mmol / L; 8.000-28.000min, the concentration of potassium hydroxide is 1mmol / L; 28.000-35.000min, the concentration of potassium hydroxide is 30mmol / L; 35.000-40.000min, the concentration of potassium hydroxide is 50mmol / L; 40.000-40.001m in, the concentration of potassium hydroxide is 50mmol / L; at 40.001-50.000min, the concentration of potassium hydroxide is 1mmol / L; when the analysis is finished, the concentration of potassium hydroxide is 1mmol / L; the ion chromatography test conditions are: anion exchange column: IonPacAS11-HC (2mm×50mm) and IonPacAG11-HC (2mm×250mm); column temperature: 30°C; flow rate: 0.30mL / min; injection volume: 25μL; suppressor current: 50mA, but the analysis method is gradient elution, which has the disadvantages of complex process, poor separation effect and long time consumption.

[0004] Another Chinese invention patent CN101566607A discloses a method for measuring organic acid radicals in cigarette paper. The invention is to configure organic acid radical standard liquid samples with different concentrations and fumarate radical standard liquid samples with different concentrations; take 10 μL of each for high performance liquid chromatography analysis, and plot the peak area against the concentration of the organic acid radical to obtain a standard curve; cut the cigarette paper sample to be tested into pieces in a stoppered conical flask, add a sulfuric acid solution with pH=2, and oscillate at 200 rpm for 25 minutes. After the oscillation liquid is filtered through a filter membrane, take 10 μL of the sample treatment liquid to be tested for high performance liquid chromatography analysis; determine the chromatographic peak position of each organic acid radical according to the retention time for qualitative analysis, and read the concentration value of the organic acid radical in the standard curve using the measured peak area. However, the eluent reagent of this analysis method is an organic solvent such as methanol and acetonitrile, and the reagent usage is large and toxic, which is harmful to the health of the operator, and it is difficult to detect the above 7 organic acid radicals at the same time.

[0005] Since lactic acid and short-chain fatty acids play an important role in maintaining the normal physiological activities of the body, there is an urgent need in the art for a detection method that is simple to operate, sensitive and accurate, environmentally friendly, and can simultaneously determine the content of lactic acid and multiple short-chain fatty acids. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides an ion chromatography method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids comprises adding a metal salt solution to a sample to be tested, filtering, and diluting with a solvent.

[0009] Preferably, the metal salt is selected from one or both of zinc sulfate heptahydrate and potassium ferrocyanide.

[0010] Preferably, the sample to be tested includes at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

[0011] Preferably, before adding the metal salt solution to the sample to be tested, the sample to be tested needs to be added with water and shaken.

[0012] Preferably, the mass volume ratio of the sample to be tested to water is 0.2-0.3 g:2-4 mL.

[0013] Preferably, the shaking speed is 1000-2800 rpm, and the shaking time is 4-6 min.

[0014] Preferably, the concentration of the metal salt solution is 100-150 g / L.

[0015] Preferably, the mass volume ratio of the sample to be tested and the metal salt solution is 0.2-0.3 g:200-400 μL.

[0016] Preferably, the filtering further includes adding water and centrifuging.

[0017] Preferably, the filtering membrane is a 0.22 μm filter membrane.

[0018] Preferably, the volume ratio of the metal salt solution to water is 0.2-0.4:4-6.

[0019] Preferably, the centrifugal speed is 8000-12000 rpm, and the centrifugal time is 8-12 min.

[0020] Preferably, the mass volume ratio of the sample to be tested to the solvent is 0.2-0.3 g:4-6 mL.

[0021] Preferably, the solvent is water.

[0022] Preferably, the method further comprises preparing a standard solution, comprising dissolving the standard solution in water.

[0023] Preferably, the standard comprises at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

[0024] Preferably, the method adopts ion chromatography for detection and analysis, wherein the chromatographic column in the ion chromatography conditions is Metrosep Organic Acids and the regeneration liquid is lithium chloride solution.

[0025] Preferably, the molar concentration of the lithium chloride solution is 90-110 mmol / L.

[0026] Preferably, in the ion chromatography conditions, the mobile phase is aqueous sulfuric acid, the flow rate is 0.5-0.8 mL / min, the column temperature is 20-30° C., and the elution is isocratic.

[0027] Preferably, the molar concentration of the aqueous sulfuric acid solution is 0.4-0.6 mmol / L; the isocratic elution is 0-30 min, with 100% mobile phase.

[0028] Preferably, the short-chain fatty acid comprises at least one of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

[0029] The present invention also provides the use of the above method in detecting and analyzing a product containing at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention provides an ion chromatography method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids. By examining the pretreatment process of the sample to be tested, a detection method for simultaneously analyzing the contents of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid in a linear range of 1-500 μg / mL is realized, achieving the effects of simple pretreatment method, small amount of chemical reagents, accurate qualitative and quantitative analysis and good sensitivity.

[0032] (2) The ion chromatography method of the present invention for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids selects the type and concentration of the regeneration liquid in the chromatographic conditions, and the reagents are simple and non-interfering, with high accuracy.

[0033] (3) The ion chromatography method provided by the present invention for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids adopts isocratic elution, which is simpler to operate than multi-gradient elution and reduces the cost of sample analysis.

[0034] (4) The method of the present invention can be applied to the determination of lactic acid and short-chain fatty acids in samples such as fecal fluid, intestinal contents and rumen fluid, providing a basis for the determination of lactic acid and short-chain fatty acids in animal metabolism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the chromatogram of the standard solution of Example 1.

[0036] Figure 2 This is the chromatogram of lactic acid and short-chain fatty acids in the intestinal contents of pigs in Example 2.

[0037] Figure 3 This is the chromatogram of lactic acid and short-chain fatty acids in the intestinal contents of pigs in Example 2 (partially enlarged view).

[0038] Figure 4 This is the chromatogram of lactic acid and short-chain fatty acids in cat feces in Example 1.

[0039] Figure 5 This is the chromatogram of lactic acid and short-chain fatty acids in cat feces in Example 1 (partial magnification).

[0040] Figure 6 This is the chromatogram of lactic acid and short-chain fatty acids in rumen fluid of Example 1.

[0041] Figure 7 This is the chromatogram of lactic acid and short-chain fatty acids in the rumen fluid of Example 1 (partially enlarged view).

[0042] Figure 8 This is the chromatogram of the standard solution of Comparative Example 1.

[0043] Fig. 9 This is the chromatogram of the test solution of Comparative Example 2.

[0044] Fig.10 This is the chromatogram of the test solution of Comparative Example 2 (partially enlarged view).

[0045] Fig.11 This is the standard curve of lactic acid.

[0046] Fig.12 This is the standard curve of formic acid.

[0047] Fig.13 This is the standard curve of acetic acid.

[0048] Fig.14 This is the standard curve of propionic acid.

[0049] Fig.15 This is the standard curve of butyric acid.

[0050] Fig.16 This is the standard curve of isobutyric acid.

[0051] Fig.17 This is the standard curve of valeric acid.

[0052] Fig.18 This is the standard curve of isovaleric acid.

[0053] The unclear parts of the drawings in the specification do not affect the understanding of the technical solutions of the present invention by those skilled in the art. DETAILED DESCRIPTION

[0054] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which lactic acid, with a specification of 5 mL and a purity of >98%, is purchased from Panphy Chemical Company; formic acid, with a specification of 50 mL and a purity of >98%, is purchased from Dima Technology Company; acetic acid, with a specification of 500 mL and a purity of >98%, is purchased from West Asia Reagent Company; propionic acid, with a specification of 500 mL and a purity of >98%, is purchased from West Asia Reagent Company; butyric acid, with a specification of 500 mL and a purity of >98%, is purchased from West Asia Reagent Company; isobutyric acid, with a specification of 1 g and a purity of >98%, is purchased from Aladdin Biochemical Technology Co., Ltd.; valeric acid, with a specification of 5 mL and a purity of >98%, is purchased from Aladdin Biochemical Technology Co., Ltd.; isovaleric acid, with a specification of 5 mL and a purity of >98%, is purchased from Aladdin Biochemical Technology Co., Ltd.

[0055] The instruments used in the present invention are: an ion chromatograph of Metrohm 940 Professional IC Vario purchased from Swiss Metrohm Co., Ltd.; an IC Conductivity Detector purchased from Swiss Metrohm Co., Ltd.; and an oscillator of XH-D purchased from Hanno Instruments.

[0056] Preparation of standard solution:

[0057] Lactic acid and 7 short-chain fatty acid stock solutions: Accurately weigh 0.1 g of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid standards into 8 10 mL volumetric flasks, make up to volume with ultrapure water, mix well, and prepare lactic acid and 7 short-chain fatty acid stock solutions with mass concentrations of 10 mg / mL, respectively.

[0058] Mixed intermediate solution: Pipette 10 mL from the lactic acid and 7 short-chain fatty acid stock solutions into a 100 mL volumetric flask, dilute to volume with water, shake well, and prepare a 1000 μg / mL mixed intermediate solution.

[0059] Standard solution: Take 0.1, 0.2, 0.5, 1, 5, 10 and 50 mL of the mixed intermediate solution in a 100 mL volumetric flask, dilute to the mark with ultrapure water, mix well, and prepare standard solutions with mass concentrations of 1, 2, 5, 10, 50, 100 and 500 μg / mL.

[0060] Example 1

[0061] An ion chromatography method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids comprises the following steps:

[0062] 1. Samples: pig intestinal contents, cat feces and cattle rumen fluid.

[0063] 2. Preparation of test solution: Accurately weigh homogenized samples into graduated centrifuge tubes (including 0.25 g of intestinal contents and cat feces, and 0.5 mL of rumen fluid), add about 3 mL of water to soak the samples, shake and extract at 2000 rpm for 5 min, first add 200 μL of 144 g / L ZnSO4·7H2O solution, shake well, then add 100 μL of 106 g / L K4Fe(CN)6·3H2O solution and shake well, add water to make up to 5 mL, centrifuge at 10000 r / min for 10 min, pass the supernatant through a 0.22 μm microporous filter membrane, take 1 mL of filtrate into a 10 mL small test tube, add 4 mL of water, shake well, arrange on the sample injection plate, and wait for instrument analysis.

[0064] 3. Ion chromatography detection conditions: Chromatographic column: Metrosep Organic Acids-250 / 7.8 (250×7.8mm, 9μm); Detector: Conductivity detector (IC Conductivity Detector); Mobile phase: 0.5mmol / L H2SO4 solution; Regeneration solution: 100mmol / L LiCl solution; Gradient program: isocratic elution, 0-30min, 100% mobile phase; Flow rate: 0.6mL / min; Column temperature: 30℃; Injection volume: 20.0μL.

[0065] The standard solution and the test solution were injected into the ion chromatograph respectively, and the chromatograms were obtained under the same chromatographic conditions. The chromatogram of the standard solution is shown in Figure 1 The chromatogram of lactic acid and short-chain fatty acids in pig intestinal contents is shown in Figure 2 and Figure 3 , the chromatogram of lactic acid and short-chain fatty acids in cat feces is shown in Figure 4 and Figure 5 The chromatograms and partial magnifications of lactic acid and short-chain fatty acids in rumen fluid are shown in Figure 6 and Figure 7 .

[0066] Comparative Example 1

[0067] The same as Example 1, the only difference is that the flow rate of the mobile phase is 0.8 mL / min.

[0068] The standard solution was injected into the ion chromatograph to obtain a chromatogram. The chromatogram of the standard solution is shown in Figure 8 It can be seen that as the column pressure increases, the chromatographic peak separation becomes worse.

[0069] Comparative Example 2

[0070] The same as Example 1, the only difference is that during the preparation of the pig intestinal content test solution, the amount of metal salt ZnSO4·7H2O solution added is 400 μL, and the amount of K4Fe(CN)6·3H2O solution added is 200 μL.

[0071] The test sample solution was injected into the ion chromatograph to obtain a chromatogram. The chromatogram of the test solution and the partial enlarged image are shown in Fig. 9 and Fig.10 ,It can be seen that the chromatogram baseline increases, affecting the accurate quantification of organic acids and resulting in a decrease in recovery.

[0072] Methodology Experiment

[0073] (1) Linear range

[0074] Accurately measure a certain volume of the above-mentioned lactic acid and 7 short-chain fatty acid stock solutions and place them in corresponding volumetric flasks, and dilute them step by step with purified water solution to prepare standard solutions with mass concentrations of 1, 2, 5, 10, 50, 100 and 500 μg / mL.

[0075] According to the ion chromatography conditions of Example 1, samples were injected and tested in order from low to high concentrations, and linear regression analysis was performed with the peak area of ​​each component as the ordinate and the mass concentration of each component as the abscissa to obtain the regression equation of the standard curve.

[0076] The standard curves for lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid are shown in Figure 11-Figure 18 .

[0077] The linear relationship equation between the concentration and peak area of ​​lactic acid in the concentration range of 1-500 ppm is: A = 0.0006Q + 0.0327, R 2 =0.9998, good linear relationship;

[0078] The linear relationship equation between formic acid concentration and peak area in the concentration range of 1-500ppm is A=0.0028Q-0.0084, R 2 =0.9999, good linear relationship;

[0079] The linear relationship equation between the concentration and peak area of ​​acetic acid in the concentration range of 1-500ppm is A=0.0017Q-0.0031, R 2 =0.9999, good linear relationship;

[0080] The linear relationship equation between the concentration and peak area of ​​propionic acid in the concentration range of 1-500ppm is A=0.0011Q-0.0551, R 2 =0.9999, good linear relationship;

[0081] The linear relationship equation between the concentration and peak area of ​​butyric acid in the concentration range of 1-500ppm is A=0.0010Q-0.0671, R 2 =0.9997, good linear relationship;

[0082] The linear relationship equation between the concentration and peak area of ​​isobutyric acid in the concentration range of 1-500ppm is A=0.0009Q-0.0636, R 2 =0.9995, good linear relationship;

[0083] The linear relationship equation between the concentration and peak area of ​​valeric acid in the concentration range of 1-500ppm is A=0.0008Q-0.0673, R 2 =0.9996, good linear relationship;

[0084] The linear relationship equation between the concentration and peak area of ​​isovaleric acid in the concentration range of 1-500 ppm is A=0.0008Q-0.0197, R 2 =0.9997, good linear relationship.

[0085] (2) Limit of detection and limit of quantification

[0086] 20 μL of the above standard solution was accurately measured, and the sample was analyzed according to the ion chromatography conditions of Example 1. The quantitative limit was determined by the signal-to-noise ratio S / N ≥ 10, and the detection limit was determined by S / N ≥ 3. Each group of parallel experiments was repeated 5 times. The detection limit and quantitative limit results of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid are shown in Table 1.

[0087] Table 1 Detection limits and quantification limits of lactic acid and seven short-chain fatty acids

[0088]

[0089] From the results in Table 1, it can be seen that the detection limit of this method for lactic acid and seven short-chain fatty acids is in the range of 0.015-0.15 g / kg, and the quantification limit is in the range of 0.067-0.500 g / kg.

[0090] (3) Recovery and precision

[0091] Free lactic acid and short-chain fatty acids naturally exist in cat feces and pig intestinal contents. Different concentrations of lactic acid and short-chain fatty acids were added to samples with known lactic acid and short-chain fatty acid contents, and the test solution was prepared and determined according to the ion chromatography method of Example 1. The test solution was injected into the ion chromatograph, and the chromatographic peak area was obtained under the chromatographic conditions of Example 1. The concentration of the target in the test solution was obtained according to the above standard curve. Each group of parallel experiments was repeated 5 times, and the relative standard deviation and recovery rate were calculated. The experimental results are shown in Table 2-3.

[0092] It can be seen from Tables 2 and 3 that the recovery rates of different concentrations of lactic acid and short-chain fatty acids added to pig intestinal contents and cat feces are between 72.11-116.99%, and the relative standard deviation is <10%, which can meet the detection requirements.

[0093] Table 2 Recovery results of lactic acid and seven short-chain fatty acids added to pig intestinal contents

[0094]

[0095]

[0096] Note: nd means not detected.

[0097] Table 3 Recovery results of cat feces supplemented with lactic acid and 7 short-chain fatty acids

[0098]

[0099] Note: nd means not detected.

[0100] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously determining the contents of lactic acid and multiple short-chain fatty acids, characterized in that: The method comprises adding two metal salt solutions to a sample to be tested, filtering, and diluting with a solvent to obtain the solution; The two metal salt solutions are respectively ZnSO4·7H2O solution and K4Fe(CN)6·3H2O solution; The mass ratio of the sample to be tested and ZnSO4·7H2O is 0.2-0.3:2.88×10 -2 ; The mass ratio of the sample to be tested and K4Fe(CN)6·3H2O is 0.2-0.3:1.06×10 -2 ; The method adopts ion chromatography for detection and analysis, wherein the ion chromatography conditions are: the chromatographic column is Metrosep Organic Acids; the regeneration liquid is a lithium chloride solution with a molar concentration of 90-110 mmol / L; the mobile phase is a sulfuric acid aqueous solution with a molar concentration of 0.4-0.6 mmol / L, the flow rate is 0.6 mL / min, the column temperature is 20-30°C, and the elution is isocratic; the isocratic elution is 0-30 min, and the mobile phase is 100%.

2. The method according to claim 1, characterized in that The sample to be tested includes at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid. Before adding the metal salt solution to the sample to be tested, water is added to the sample to be tested and shaken. The mass volume ratio of the sample to be tested to water is 0.2-0.3g:2-4mL, the shaking speed is 1000-2800rpm, and the shaking time is 4-6min.

3. The method according to claim 1, characterized in that The filtration also includes adding water and centrifuging. The filtration membrane is a 0.22 μm filter membrane. The volume ratio of the metal salt solution to water is 0.2-0.4:4-6. The centrifugal speed is 8000-12000 rpm and the centrifugal time is 8-12 min.

4. The method according to claim 3, characterized in that The mass volume ratio of the sample to be tested to the solvent is 0.2-0.3 g:4-6 mL, and the solvent is water.

5. The method according to claim 1, characterized in that The method further comprises the preparation of a standard solution, which comprises dissolving the standard solution in water, wherein the standard comprises at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

6. Use of the method according to any one of claims 1 to 5 in detecting and analyzing a product containing at least one of lactic acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid or isovaleric acid.

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