CN115616120A - Method for simultaneously measuring contents of 11 feeding organic acids in organic acidifier - Google Patents
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Abstract
The invention discloses a method for simultaneously measuring the content of 11 feeding organic acids in organic acidifier, which comprises the following steps: preparing standard working solutions of 11 feeding organic acids; (2) Analyzing the standard working solution by using an ion chromatography instrument to obtain an ion chromatogram; (3) respectively drawing a standard curve of 11 feeding organic acids; (4) Pretreating a sample, and analyzing by using an ion chromatograph to obtain an ion chromatogram of the sample solution; (5) And calculating the content of each organic acid by a standard curve according to the chromatographic peak area corresponding to each organic acid in the ion chromatogram of the sample solution. The invention adopts an ion chromatography detection method, adopts three mixed mobile phases of sulfuric acid aqueous solution-acetone-acetonitrile to realize simultaneous determination of covering 11 organic acids through an organic acid column, and can be qualitatively and quantitatively analyzed within 40 minutes. The method has the characteristics of simple operation, wide linear range, good repeatability, high accuracy, wide application range, low detection limit, good stability and the like.
Description
Technical Field
The invention relates to a method for simultaneously measuring the content of organic acids, in particular to a method for simultaneously measuring the content of 11 feeding organic acids in an organic acidifier, belonging to the field of measurement of the content of the feeding organic acids.
Background
Acidity regulators (also known as acidifiers) are a very common class of feed additives in products of the feed industry. The addition of acidifying agent in the daily ration can regulate pH value of gastrointestinal tract, promote digestion and absorption of nutrient substances, inhibit reproduction of harmful microorganisms in intestinal tract, and promote growth of animals. In recent years, since antibiotics for feed are banned in succession in various countries, acidifiers, which are safe animal growth promoting additives, are regarded as one of products capable of effectively replacing antibiotics in feed, and are receiving attention in the livestock industry. The acidifier comprises inorganic acidifier and organic acidifier, wherein the organic acidifier has good health and palatability, has low corrosivity, and is relatively safe in the processing process, so that the acidifier is widely applied.
The organic acid content measuring methods reported in the literature mainly include gas chromatography, liquid chromatography, capillary electrophoresis, liquid chromatography tandem mass spectrometry, ion chromatography and the like. When the gas chromatography is used for measuring high-boiling organic acid, derivatization is generally needed, and the operation is complicated; the application of liquid chromatography in organic acid determination is reported more, but the requirement on mobile phase preparation in the determination process is higher, the sensitivity of an ultraviolet detector is low, and the separation effect of a common liquid chromatography column is not good; the capillary electrophoresis method is to be improved in reproducibility compared with other analysis methods; the liquid chromatography tandem mass spectrometry has high instrument and equipment requirements, low detection linear range and is not suitable for the determination of high-content organic acidifier. The ion chromatography analysis of organic acid has the advantages of simple and convenient sample pretreatment, no need of derivatization, high sensitivity and the like. At present, organic acidifiers applied in the market mostly contain several kinds of organic acids, and the content of different organic acids is greatly different, so far, a method for accurately and quickly simultaneously determining the content of 11 feeding organic acids in the organic acidifiers is lacked.
Disclosure of Invention
The invention mainly aims to provide a method for simultaneously measuring the content of 11 feeding organic acids in an organic acidifier;
the above purpose of the invention is realized by the following technical scheme:
a method for simultaneously measuring the contents of 11 feeding organic acids in organic acidifiers comprises the following steps: (1) Preparing standard working solutions of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid; (2) Analyzing the standard working solution by adopting an ion chromatograph to obtain an ion chromatogram of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid; (3) Respectively drawing standard curves of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid according to the chromatographic peak area of each organic acid in the ion chromatogram to the concentration of each organic acid in the standard working solution; (4) Pretreating a non-coated or coated organic acidifier sample to be measured, and analyzing by using an ion chromatograph to obtain a sample solution ion chromatogram; (5) And calculating the content of each organic acid by a standard curve according to the chromatographic peak area corresponding to each organic acid in the ion chromatogram of the sample solution.
As a preferred embodiment of the present invention, the method for preparing the standard working solution of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid in step (1) comprises: weighing each organic acid standard substance in a volumetric flask, adding water or ethanol to a constant volume to a scale, and preparing an organic acid standard solution mother solution; accurately transferring the organic acid standard solution mother liquor with the corresponding volume into the same 100 mL volumetric flask, and metering the volume by using a mobile phase to prepare a mixed standard solution.
As a preferred embodiment of the present invention, the chromatographic conditions for the ion chromatography analysis in step (2) or step (4) comprise:
the mobile phase is as follows: consists of 0.5 mmol/L sulfuric acid water solution, acetone and acetonitrile, wherein,
0.5 mmol/L sulfuric acid aqueous solution by volume ratio: acetone: acetonitrile = 84:6:10.
ion chromatographic column: an Organic acid column, wherein the Organic acid column is preferably Metrosep Organic Acids 250/7.8;
flow rate: 0.5 mL/min;
sample introduction volume: 20. mu L;
column temperature: 30 o C；
A detector: suppressor-conductivity detector.
As a preferred embodiment of the invention, the method for pretreating the non-coated organic acidifier sample to be tested in the step (4) comprises the following steps: adding a mobile phase into a non-coated organic acidifier sample, performing ultrasonic extraction, centrifuging, and filtering supernate with a microporous filter membrane to obtain the product;
as a preferred embodiment of the invention, the method for pretreating the coating organic acidifier sample to be tested in the step (4) comprises the following steps: adding petroleum ether into the coated organic acidifier sample, performing ultrasonic treatment, adding ultrapure water until the sample is completely dissolved, separating in a separating funnel, collecting the solution at the lower layer, centrifuging, and filtering the supernatant with a microporous filter membrane to obtain the organic acidifier.
As a preferred embodiment of the present invention, the method for calculating the content of each organic acid by a calibration curve from the chromatographic peak area corresponding to each organic acid in the chromatogram of the sample solution in step (5) comprises:
and (3) quantifying by adopting a standard curve external standard method, wherein the calculation formula is as follows:
in the formula:
Xcontent of organic acids in the sample, unit: mg/g;
c-the concentration of the organic acid determined in the sample solution, mg/L;
V-volume of extract, mL;
m- sample weighing Mass, g
K-dilution factor.
The invention adopts an ion chromatography detection method, adopts three mixed mobile phases of sulfuric acid aqueous solution-acetone-acetonitrile, and realizes the determination of 11 organic acids which are allowed to be used in feed additive variety catalog for the first time through an organic acid column, and the 11 organic acids can be qualitatively and quantitatively analyzed within 40 minutes. The linear range of the method for simultaneously measuring the 11 organic acids is 5-1000 mg/L; the detection limits for the 11 organic acids are as follows: 0.011 mg/L citric acid, 0.012 mg/L tartaric acid, 0.012 mg/L malic acid, 0.011 mg/L fumaric acid, 0.027 mg/L lactic acid, 0.010 mg/L formic acid, 0.016 mg/L acetic acid, 0.023 mg/L propionic acid, 0.032 mg/L butyric acid, 0.107 mg/L sorbic acid, 0.098 mg/L benzoic acid; the recovery rate of each organic acid component is between 92.9 and 112.2 percent, and the relative standard deviation is between 0.2 and 2.4 percent; the method has the characteristics of simplicity in operation, wide linear range, good repeatability, high accuracy, wide application range, low detection limit, good stability and the like, and has an application prospect in synchronous determination of the organic acidifier.
Drawings
FIG. 1 is a chromatogram of 11 feeding organic acids; from left to right, citric acid, tartaric acid, malic acid, fumaric acid, lactic acid, formic acid, acetic acid, propionic acid, butyric acid, sorbic acid and benzoic acid.
FIG. 2 is a standard curve for citric acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 3 is a standard curve for tartaric acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 4 is a standard curve of malic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 5 is a standard curve for fumaric acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 6 is a standard curve of lactic acid at concentrations of 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in this order.
FIG. 7 is a standard curve for formic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 8 is a standard curve for acetic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 9 is a standard curve for propionic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 10 is a standard curve of butyric acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 11 is a standard curve for sorbic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 12 is a standard curve for benzoic acid; the concentration is 5 mg/L, 10mg/L, 50mg/L, 100mg/L, 200mg/L, 400mg/L, 800mg/L and 1000 mg/L in sequence.
FIG. 13 is a chromatogram of an assay of a sample of organic acidulant; 11.18 Fumaric acid at min, formic acid at 13.92 min, propionic acid at 15.76 min, miscellaneous peak at 16.78min, sorbic acid at 30.74 min, and benzoic acid at 36.34 min.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Experimental example 1 establishment of method for simultaneously measuring contents of 11 feeding organic acids in organic acidifier
Test method 1
1.1 preparation of Mobile phase solution
Preparing a mobile phase: 0.5 mmol/L aqueous sulfuric acid-acetone-acetonitrile = 84:6:10
1.1.1 Preparation of 0.1 mol/L sulfuric acid aqueous solution: adding 25 mL of primary water into a 100 mL beaker, slowly adding 2.72 mL of concentrated sulfuric acid, transferring the primary water into a 500 mL volumetric flask after the temperature is recovered to the room temperature, and fixing the volume of the primary water to the scale.
1.1.2 Preparing 0.5 mmol/L sulfuric acid aqueous solution: accurately transferring 5.00 mL of 0.5 mol/L sulfuric acid aqueous solution into a 1000 mL volumetric flask, and metering the primary water to the scale.
1.1.3 preparation of mobile phase: in a 1L solvent bottle, 60 mL of acetone, 100 mL of acetonitrile and 840 mL of 0.5 mmol/L sulfuric acid aqueous solution are added, and mixed by ultrasound.
2 preparation of Standard solution
Standards of 11 organic acids: accurately weighing the organic acid standard substance in a volumetric flask, adding water or ethanol to a constant volume to a scale, and preparing the organic acid standard solution mother liquor. Accurately transferring the organic acid standard solution mother liquor with the corresponding volume into the same 100 mL volumetric flask, and metering the volume by using a mobile phase to prepare a mixed standard solution.
3 sample pretreatment method
3.1 Pretreatment of a non-coated acidifier sample:
accurately weighing 0.5-2 g (precision of 0.1 mg) of sample in 250 mL conical flask, adding 100 mL of mobile phase, performing ultrasonic extraction for 30 min, centrifuging at 5000 r/min for 10 min, and filtering the supernatant with 0.22 μm microporous membrane.
3.2 Pretreatment of a coated acidifier sample:
accurately weighing 0.5 g-2 g of sample in a 250 mL conical flask, adding 50 mL of petroleum ether, performing ultrasonic treatment at 40-60 ℃ for 10 min, adding 50 mL of ultrapure water, and fully shaking until the sample is completely dissolved. Transferring to a 250 mL separating funnel, collecting the lower layer solution, centrifuging at 5000 r/min for 10 min, and filtering the supernatant with 0.22 μm microporous membrane.
4 ion chromatography conditions
Mobile phase: 0.5 mmol/L aqueous sulfuric acid-acetone-acetonitrile = 84:6:10.
ion chromatographic column: metrosep Organic Acids 250/7.8.
Flow rate: 0.5 mL/min.
Sample introduction volume: 20. μ L.
Column temperature: 30 And oC.
A detector: suppressor-conductivity detector.
5 quantitative analysis
And (3) measuring the standard working solution according to the reference conditions of the instrument, drawing a standard curve by taking the concentration (mg/L) as an abscissa and the area of a response chromatographic peak ((mu S/cm) x min) as an ordinate, and calculating the content of each organic acid by using the corresponding peak area in the chromatogram of the sample solution through the standard curve.
The content of the target organic acid in the sample solution is quantified by adopting a standard curve external standard method, and the calculation formula is as follows:
in the formula:
Xcontent of organic acids in the sample, unit: mg/g;
c-concentration of the determined organic acid in the sample solution, mg/L;
V-volume of extract, mL;
m- weighing a sample, and g;
k-dilution factor.
The result of the calculation retains three significant digits.
6 test results
Fig. 1 is an ion chromatogram of 11 feed organic acids, wherein citric acid, tartaric acid, malic acid, fumaric acid, lactic acid, formic acid, acetic acid, propionic acid, butyric acid, sorbic acid and benzoic acid are sequentially arranged from left to right.
And (2) performing ion chromatography measurement on the standard working solution according to the chromatographic conditions to obtain an ion chromatogram, and drawing standard curves of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid respectively according to the response chromatographic peak area of each organic acid in the ion chromatogram to the concentration of each organic acid in the standard working solution by taking the concentration (mg/L) as a horizontal coordinate and the response chromatographic peak area ((mu S/cm) x min) as a vertical coordinate, wherein the drawn standard curves of the citric acid, the tartaric acid, the malic acid, the fumaric acid, the lactic acid, the formic acid, the acetic acid, the propionic acid, the butyric acid, the sorbic acid and the benzoic acid are respectively shown in figures 2-12.
Test example 2 Linear and detection Limit test of a method for the Simultaneous determination of the content of 11 organic acids for feed in an organic acidifying agent according to the invention
Test method 1
Preparing a series of mixed standard solutions containing 11 organic acid components, sequentially injecting samples under an optimized test chromatographic condition, repeating the sample injection for 2 times, establishing a standard curve by taking a peak area as a vertical coordinate (y) and a mass concentration as a horizontal coordinate (x), and determining coefficients (R2) of the standard curves are all larger than 0.9997 in respective linear ranges. The 3-fold signal-to-noise ratio (S/N) was taken as the detection limit of the method.
2 results of the test
The results of the linear equation, the linear correlation coefficient, the linear range, the detection limit, and the like of the 11 organic acids are shown in table 1.
TABLE 1 Linear equation, linear correlation coefficient, linear Range, and detection Limit results for the 11 organic acids
Test results show that the linear range of the method for simultaneously measuring the 11 organic acids is 5-1000 mg/L;
the detection limit of the method for simultaneously measuring the 11 feeding organic acids is as follows: 0.011 mg/L citric acid, 0.012 mg/L tartaric acid, 0.012 mg/L malic acid, 0.011 mg/L fumaric acid, 0.027 mg/L lactic acid, 0.010 mg/L formic acid, 0.016 mg/L acetic acid, 0.023 mg/L propionic acid, 0.032 mg/L butyric acid, 0.107 mg/L sorbic acid, and 0.098 mg/L benzoic acid.
Experimental example 3 recovery rate and precision test of simultaneous determination method of contents of 11 feeding organic acids in organic acidifier according to the present invention
Test method 1
With the mixed organic acid acidifying agent product as a representative sample (fig. 13), 11 organic acid standard solutions of 3 different concentrations (specific concentrations are shown in table 2) were added, and each addition level was measured repeatedly 6 times to calculate the average normalized recovery (R) and precision (RSD).
2 results of the test
The results are shown in Table 2.
TABLE 2 recovery rate and precision test results of the simultaneous determination method for the content of organic acids for feeding
As can be seen from Table 2, the recovery rate of each organic acid component is between 92.9% and 112.2%, and the relative standard deviation is 0.2% to 2.4%, which indicates that the detection method established by the invention has good repeatability and high accuracy.
As can be seen from figure 13, the content of fumaric acid, formic acid, propionic acid and sorbic acid in the samples of organic acidulants was successfully determined using the method of the present invention.
Claims (10)
1. A method for simultaneously measuring the content of 11 feeding organic acids in organic acidifiers is characterized by comprising the following steps: (1) Preparing standard working solutions of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid; (2) Performing chromatographic analysis on the standard working solution by adopting an ion chromatograph to obtain an ion chromatogram of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid; (3) Respectively drawing standard curves of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid according to the area of the peak of the response chromatogram of each organic acid in the ion chromatogram to the concentration of each organic acid in the standard working solution; (4) Pretreating a non-coated or coated organic acidifying agent sample to be measured, and then carrying out chromatographic analysis by using an ion chromatograph to obtain a sample solution ion chromatogram; (5) And calculating the content of each organic acid by a standard curve according to the chromatographic peak area corresponding to each organic acid in the ion chromatogram of the sample solution.
2. The simultaneous assay method according to claim 1, wherein the standard working solutions of formic acid, acetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid and malic acid in step (1) are prepared by a method comprising: weighing each organic acid standard substance in a volumetric flask, adding water or ethanol to a constant volume to a scale, and preparing an organic acid standard solution mother solution; accurately transferring the organic acid standard solution mother liquor with the corresponding volume into the same volumetric flask, and diluting the volume with the mobile phase to prepare a mixed standard solution.
3. The simultaneous assay method according to claim 1, wherein the chromatographic conditions for the chromatographic analysis by ion chromatography in step (2) or step (4) comprise a mobile phase and an ion chromatographic column, wherein the mobile phase consists of an aqueous solution of sulfuric acid, acetone and acetonitrile; the ion chromatographic column is an organic acid column; wherein the mobile phase of the sulfuric acid aqueous solution consists of 0.5 mmol/L sulfuric acid aqueous solution, acetone and acetonitrile.
4. The simultaneous measurement method according to claim 3, characterized in that, in terms of volume ratio, 0.5 mmol/L aqueous sulfuric acid solution: acetone: acetonitrile = 84:6:10.
5. the simultaneous assay method according to claim 2, wherein the Organic acid column is Metrosep Organic Acids 250/7.8.
6. The simultaneous determination method according to claim 1, wherein the chromatographic conditions for the chromatographic analysis by the ion chromatograph in the step (2) or the step (4) further comprise:
flow rate: 0.5 mL/min;
sample introduction volume: 20. mu.L;
column temperature: 30 o C；
A detector: suppressor-conductivity detector.
7. The simultaneous assay method according to claim 1, wherein the step (4) of pretreating the non-coated organic acidifier sample to be assayed comprises: adding a mobile phase into a non-coated organic acidifier sample, performing ultrasonic extraction, centrifuging, and filtering the supernatant with a microporous filter membrane.
8. The simultaneous assay method according to claim 1, wherein the step (4) of pretreating the coated organic-like acidifying agent sample to be assayed comprises: adding petroleum ether into a coated organic acidifier sample, performing ultrasonic treatment, adding ultrapure water until the sample is completely dissolved, separating liquid in a separation funnel, collecting the lower layer solution, centrifuging, and filtering the supernatant with a microporous filter membrane to obtain the organic acidifier.
9. The simultaneous determination method according to claim 1, wherein the method of calculating the content of each organic acid by plotting a standard curve on the chromatographic peak area corresponding to each organic acid in the ion chromatogram of the sample solution in step (5) is a method of quantifying by using an external standard method of a standard curve.
10. The simultaneous determination method according to claim 9, characterized in that the calculation formula for quantification by the standard curve external standard method is as follows:
in the formula:
Xcontent of organic acids in the sample, unit: mg/g;
c- concentration of organic acid determined in the sample solution, unit: mg/L;
V-extract volume, unit: mL;
m- sample weighing mass, unit: g;
k-dilution factor.
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| CN101650347A (en) * | 2009-09-15 | 2010-02-17 | 安徽泰格生物技术股份有限公司 | Method for simultaneously measuring contents of multiple organic acids in feeder acidulant |
| US20130095571A1 (en) * | 2010-12-08 | 2013-04-18 | China Tobacco Guangdong Industrial Co., Ltd. | The method for quick and simultaneous determination of 16 inorganic anions and organic acids in tobacco |
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