CN117269408A - Method for simultaneously detecting contents of four organic acids in food additive and application thereof - Google Patents
Method for simultaneously detecting contents of four organic acids in food additive and application thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 76
- 235000013373 food additive Nutrition 0.000 title claims abstract description 17
- 239000002778 food additive Substances 0.000 title claims abstract description 17
- 150000007524 organic acids Chemical class 0.000 title claims abstract description 14
- 235000005985 organic acids Nutrition 0.000 title claims abstract description 9
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims abstract description 72
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims abstract description 71
- 235000011090 malic acid Nutrition 0.000 claims abstract description 66
- 239000001630 malic acid Substances 0.000 claims abstract description 61
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 claims abstract description 57
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims abstract description 54
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 50
- 239000002253 acid Substances 0.000 claims abstract description 49
- 239000000243 solution Substances 0.000 claims abstract description 38
- 239000001530 fumaric acid Substances 0.000 claims abstract description 28
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 27
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims abstract description 27
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000011976 maleic acid Substances 0.000 claims abstract description 26
- 239000012488 sample solution Substances 0.000 claims abstract description 26
- 239000013582 standard series solution Substances 0.000 claims abstract description 22
- 239000001384 succinic acid Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000005259 measurement Methods 0.000 claims abstract description 11
- 239000012496 blank sample Substances 0.000 claims abstract description 7
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims abstract description 7
- 229940099690 malic acid Drugs 0.000 claims description 55
- 239000000523 sample Substances 0.000 claims description 51
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 46
- 239000000126 substance Substances 0.000 claims description 30
- 239000011550 stock solution Substances 0.000 claims description 25
- 238000004458 analytical method Methods 0.000 claims description 22
- 238000012360 testing method Methods 0.000 claims description 15
- 238000005303 weighing Methods 0.000 claims description 13
- 239000001394 sodium malate Substances 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 12
- 238000004090 dissolution Methods 0.000 claims description 8
- 239000000654 additive Substances 0.000 claims description 6
- 230000000996 additive effect Effects 0.000 claims description 6
- 238000005341 cation exchange Methods 0.000 claims description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 229940116298 l- malic acid Drugs 0.000 claims description 5
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 claims description 3
- 239000012490 blank solution Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 3
- 239000007788 liquid Substances 0.000 description 20
- 150000007513 acids Chemical class 0.000 description 11
- 125000005842 heteroatom Chemical group 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 5
- 238000004448 titration Methods 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000004454 trace mineral analysis Methods 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 238000002479 acid--base titration Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- WPUMTJGUQUYPIV-JIZZDEOASA-L disodium (S)-malate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](O)CC([O-])=O WPUMTJGUQUYPIV-JIZZDEOASA-L 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000019265 sodium DL-malate Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical group [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000003918 potentiometric titration Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/96—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 using ion-exchange
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention relates to the technical field of organic acid detection, in particular to a method for simultaneously detecting the contents of four organic acids in a food additive and application thereof. The method comprises the following steps: preparing a malic acid standard series solution and a mixed acid standard series solution; preparing a sample solution to be tested; respectively carrying out liquid chromatography measurement on malic acid standard series solutions with different concentrations and mixed acid standard series solutions with different concentrations, measuring corresponding peak areas, and respectively drawing standard curves of malic acid, succinic acid, fumaric acid and maleic acid; respectively carrying out liquid chromatography measurement on the sample solution and the blank sample solution to obtain peak areas, and obtaining mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be measured according to the standard curve; and calculating the content of the target object in the sample solution to be detected according to the mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be detected. The invention realizes the accurate detection of the malic acid and the mixed acid of the food additive.
Description
Technical Field
The invention relates to the technical field of organic acid detection, in particular to a method for simultaneously detecting the contents of four organic acids in a food additive and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Malic acid is an important food additive and has the function of acidity regulator. At present, the national standard of a plurality of food additives such as L-malic acid, DL-malic acid, L-sodium malate, DL-sodium malate and the like is approved in China, and domestic manufacturers of related products have more than ten, so that the food additive has a certain market scale. The production of such food additives is often carried out by fermentation, and therefore, some by-products of hetero acids, including succinic acid, fumaric acid and maleic acid, which have similar structures are inevitably produced during the production process. In the current standard method and other detection methods, the following method is often adopted for detecting the main component of malic acid (sodium): one is to directly measure the total acid in a sample by an acid-base titration method or a perchloric acid potentiometric titration method to be used as the content of malic acid, but the method has poor accuracy when the content of the mixed acid is higher, and can not be analyzed by the same method as the content of the mixed acid; the other is to detect the content of malic acid and other mixed acids simultaneously by adopting a liquid chromatography, but the ultraviolet detector has the defect of 'measuring constant substances by adopting a trace analysis method' due to higher sensitivity, and the detection result of the main component of the sample is seriously unreal after the error in the detection process is amplified, so that the simultaneous measurement of the malic acid and the mixed acids is difficult to realize.
For analysis of mixed acid such as maleic acid, fumaric acid and the like, the existing detection method does not comprise determination of succinic acid, and the actual sample analysis of the product in recent years finds that succinic acid is the mixed acid with the highest content in the fermentation production process; secondly, the liquid chromatographic separation system of the phosphate buffer solution and the C18 chromatographic column which are commonly adopted at present cannot effectively separate the mixed acid and the interfering substances, so that the measurement results of the mixed acid such as fumaric acid, succinic acid and the like are inaccurate; in addition, the sensitivity of the ultraviolet detector to several organic acids is greatly different, so that different concentrations of each material standard series are required to be prepared, and the requirements on pretreatment operation are high.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention discloses a method for simultaneously detecting the contents of four organic acids in a food additive and application thereof, wherein the four organic acids comprise: after a sample to be tested is treated, each component in the sample solution is separated through a hydrogen type cation exchange chromatographic column, and is detected by a differential refraction detector according to the density difference of a separating substance and a mobile phase, and the external standard method is used for quantification, so that the accurate detection of malic acid and hetero acid in a food additive is realized.
Specifically, the technical scheme of the invention is as follows:
a method for simultaneously detecting the contents of four organic acids in a food additive comprises the following steps:
(1) Preparing a malic acid standard stock solution, and mixing a fumaric acid, maleic acid and succinic acid mixed standard stock solution;
(2) Preparing a malic acid standard series solution and a mixed acid standard series solution;
(3) Preparing a sample solution to be tested: weighing a sample to be measured, drying and cooling to room temperature; accurately weighing the dried test sample, dissolving with sulfuric acid solution, fixing the volume, shaking uniformly, filtering, and analyzing and measuring by an instrument; preparing a blank sample solution according to the same processing steps as the sample to be tested;
(4) Respectively carrying out liquid chromatography measurement on malic acid standard series solutions with different concentrations and mixed acid standard series solutions with different concentrations, measuring corresponding peak areas, and respectively drawing standard curves of malic acid, succinic acid, fumaric acid and maleic acid by taking the mass concentration of the mixed standard solutions as an abscissa and the peak areas as an ordinate;
respectively carrying out liquid chromatography measurement on the sample solution and the blank sample solution to obtain peak areas, and obtaining mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be measured according to the standard curve;
(5) And calculating the content of the target object in the sample solution to be detected according to the mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be detected.
In one or more embodiments of the present invention, the step of preparing the malic acid standard stock solution in step (1) comprises: accurately weighing a malic acid standard substance, adding sulfuric acid solution for dissolution and fixing the volume, and preparing the malic acid standard stock solution with the mass fraction of 1%.
In one or more embodiments of the present invention, the step of preparing the mixed standard stock solution of fumaric acid, maleic acid and succinic acid in step (1) includes: and respectively and accurately weighing a fumaric acid standard substance, a maleic acid standard substance and a succinic acid standard substance, adding sulfuric acid solution for dissolution and volume fixing, and preparing into mixed standard stock solution of mixed acid with mass fraction of 0.1%.
In one or more embodiments of the present invention, the step of preparing the malic acid standard series solution in step (2) includes: accurately absorbing the malic acid standard stock solution, adding sulfuric acid solution for dissolution and fixing the volume to prepare standard series solutions with the mass fractions of 0.1%, 0.2% and 0.5% of malic acid respectively; and taking sulfuric acid solution and standard stock solution as standard solutions with the mass fractions of 0% and 1% of malic acid respectively.
In one or more embodiments of the present invention, the step of preparing the mixed standard series solution of the hetero-acid in the step (2) includes: accurately sucking mixed standard stock solution of the mixed acid, and fixing the volume by using sulfuric acid solution to prepare three mixed standard solutions with mass fractions of 0.0005%, 0.002%, 0.005%, 0.01% and 0.02%.
In one or more embodiments of the present invention, the conditions of the liquid chromatography determination in step (4) are: the chromatographic column adopts a hydrogen type cation exchange chromatographic column Aminex HPX-87H,300 mm multiplied by 7.8mm with polystyrene-divinylbenzene resin as filler.
In one or more embodiments of the present invention, the conditions of the liquid chromatography determination in step (4) are: column temperature: 60. the temperature is lower than the temperature; mobile phase: a sulfuric acid solution; flow rate: 0.7 mL/min.
In one or more embodiments of the present invention, the conditions of the liquid chromatography determination in step (4) are: sample injection amount: 20. mu L; detecting the temperature of the cell: 40. the temperature is lower than the temperature; analysis time: 20 minutes.
In one or more embodiments of the present invention, the method for calculating the content of the target object in the solution to be tested according to the mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the solution to be tested in the step (5) includes:
the content of each target in the sample to be measured is calculated according to the mass fraction by adopting the following formula:
wherein:
W-the mass fraction of the substance to be measured in the sample to be measured, in percent (%);
c-the mass fraction of the substance to be measured in the solution of the sample to be measured, obtained from the standard curve, in percentage (%);
c 0 -the mass fraction of the substance to be measured in the sample blank solution obtained from the standard curve, in percent (%);
V-the volumetric volume of the solution to be tested in milliliters (mL);
m-the mass of the test sample in grams (g);
f-conversion coefficient, wherein the L-sodium malate and the DL-sodium malate are 1.66, and the L-malic acid and the DL-malic acid are 1.
In a second aspect of the invention, there is provided the use of the method of the first aspect for simultaneous detection of four organic acid levels in a food additive in additive detection.
The invention has the following beneficial effects:
the method can synchronously analyze the main components and several kinds of hetero acids in the food additives L-malic acid, DL-malic acid, L-sodium malate and DL-sodium malate products, omits the step of titrating and analyzing the content of malic acid (sodium) by the existing method, greatly improves the detection efficiency and reduces the requirements on instrument consumables and operation capacity;
the invention uses liquid chromatography to analyze, which solves the problems that the existing method uses total acid or total acid radical content as main component content directly when adopting titration method to analyze, and the content of hetero acid is not deducted or considered, thus leading to higher detection result with different degrees;
the invention adopts a chromatographic separation system which is more suitable for organic acid separation, comprises a hydrogen type cation exchange chromatographic column and sulfuric acid solution, obviously improves the separation effect of a target object peak and a impurity peak, and improves the detection accuracy;
the invention adopts the differential refraction detector to exert the detection advantage of the differential refraction detector on constant substances, greatly reduces the influence of different and oversensitive responses of various substances caused by the analysis of the ultraviolet detector, and remarkably improves the analysis precision and accuracy.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a liquid chromatogram of a mixed standard sample of mixed acids analyzed by the liquid chromatography method (C18 column, ultraviolet detector) disclosed in the prior art in the embodiment of the invention;
FIG. 2 is a liquid chromatogram of a mixed sample of mixed acids analyzed by the liquid chromatography method (C18 column, UV detector) disclosed in the prior art;
FIG. 3 is a second liquid chromatogram of a mixed sample of mixed acids analyzed by the presently disclosed liquid chromatography method (C18 column, UV detector) in an embodiment of the invention;
FIG. 4 is a liquid chromatogram third of an embodiment of the present invention using the presently disclosed liquid chromatography method (C18 column, UV detector) for analyzing a mixed sample of hybrid acids;
FIG. 5 is a liquid chromatogram of a malic acid standard sample detected by the method of the invention in an embodiment of the invention;
FIG. 6 is a liquid chromatogram of a mixed standard sample of hybrid acids detected by the method of the present invention in an embodiment of the present invention;
FIG. 7 is a liquid chromatogram of a practical test sample 1 of a malic acid additive tested by the method of the invention in an embodiment of the invention;
FIG. 8 is a liquid chromatogram of practical test sample 2 of the malic acid additive tested by the method according to the invention in the example of the invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In one embodiment of the invention, there is provided the use of the recombinant human 3D skin model described above in skin-related basic research.
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified.
Example 1
1. The method is as follows: after the sample is treated, each component in the sample solution is separated through a hydrogen cation exchange chromatographic column, and the sample solution is detected by a differential refraction detector according to the density difference between the separated substance and the mobile phase, and is quantified by an external standard method.
2. Reagents and materials: primary water, sulfuric acid solution c (clay H 2 SO 4 ) =0.01 mol/L, malic acid standard (C 4 H 6 O 4 CAS number: 97-67-6, purity not less than 99.0%), fumaric acid standard (C) 4 H 4 O 4 CAS number: 110-17-8, purity not less than 99.0%), maleic acid standard (C) 4 H 4 O 4 CAS number: 110-16-7, purity not less than 99.0%), succinic acid standard (C) 4 H 6 O 4 CAS number: 110-15-6, the purity is more than or equal to 99.0 percent), and a microporous filter membrane.
3. Instrument and apparatus: high performance liquid chromatograph (equipped with differential refraction detector), electronic analytical balance (precision 0.01 mg), electrothermal constant temperature drying oven (temperature precision 1 deg.C).
4. Instrument analysis reference conditions:
4.1 chromatography column: a hydrogen type cation exchange chromatographic column Aminex HPX-87H,300 mm×7.8mm or other chromatographic columns with equivalent analysis effect is filled with polystyrene-divinylbenzene resin.
4.2 column temperature: 60. DEG C.
4.3 mobile phase: sulfuric acid solution.
4.4 flow rate: 0.7 mL/min.
4.5 sample injection amount: 20. mu L.
4.6 detecting cell temperature: 40. DEG C.
4.7 analysis time: 20 minutes.
5. Preparing a standard solution:
5.1 preparation of standard stock solution: accurately weighing a malic acid standard product 1 g to 1 mg, adding sulfuric acid solution for dissolution and fixing the volume to 100 mL, and preparing a malic acid standard stock solution with the mass fraction of 1% (the mass concentration is 10 mg/mL); respectively accurately weighing 100 mg of fumaric acid standard substance, 100 mg of maleic acid standard substance and 100 mg of succinic acid standard substance, accurately obtaining 0.1 mg, dissolving with sulfuric acid solution, and fixing volume to 100 mL to prepare mixed standard stock solution of mixed acid with mass fraction of 0.1% (mass concentration of 1 mg/mL). The stock solution is stored in a refrigerator at the temperature of 4 ℃ and has the effective period of 6 months.
5.2 preparation of malic acid Standard series solution: taking 3 10 mL volumetric flasks, respectively and accurately sucking 1.00 mL, 2.00 mL and 5.00 mL of malic acid standard stock solution, adding sulfuric acid solution for dissolution, and fixing the volume to scale to prepare standard series solutions with the mass fractions of 0.1%, 0.2% and 0.5% of malic acid respectively. And taking sulfuric acid solution and standard stock solution as standard solutions with the mass fractions of 0% and 1% of malic acid respectively. Storing in refrigerator at 4deg.C for 1 month.
5.3 preparation of mixed standard series solutions of the mixed acid: taking 5 volumetric flasks of 100 mL, respectively and accurately sucking the mixed standard stock solutions of the mixed acid of 0.50 mL, 2.00 mL, 5.00 mL, 10.0mL and 20.0 mL, and fixing the volumes to scales by using sulfuric acid solution. The mixed standard solution with the mass fractions of the three hetero acids of 0.0005%, 0.002%, 0.005%, 0.01% and 0.02% is prepared. Storing in refrigerator at 4deg.C for 1 month.
6. Preparation of sample solution: 1) Weighing about 2g of a test sample in a glass dish, placing the test sample in an electrothermal constant-temperature drying oven, drying for 3 hours at 105 ℃, taking out the test sample, and placing the test sample in a dryer for cooling to room temperature; 2) Weighing about 0.5g (accurate to 0.01 mg) of the dried test sample, dissolving with sulfuric acid solution, fixing the volume to 100 mL, shaking uniformly, filtering with a 0.22 μm filter membrane, and waiting for analysis and measurement by an instrument; 3) The preparation of the blank sample solution was carried out according to the same procedure as for the sample in 2).
7. The instrument analysis step:
7.1 Under the analysis condition of the instrument, respectively injecting 5 malic acid standard series solutions with different concentrations and 5 mixed acid standard series solutions with different concentrations into a liquid chromatograph, measuring the corresponding peak areas, and respectively drawing standard curves of malic acid, succinic acid, fumaric acid and maleic acid by taking the mass concentration of the mixed standard solutions as an abscissa and the peak areas as an ordinate.
7.2 And respectively injecting the sample solution and the blank sample solution into a liquid chromatograph to obtain peak areas, and obtaining mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be detected according to a standard curve.
8. Calculation of target content in sample solution
The content of each target in the sample is calculated according to the mass fraction by adopting the following formula:
wherein:
W-the mass fraction of the substance to be measured in the sample, in percent (%);
c-the mass fraction of the substance to be measured in the sample solution, in percent (%), derived from the standard curve;
c 0 -the mass fraction of the substance to be measured in the sample blank solution obtained from the standard curve, in percent (%);
V-the volumetric volume of the sample solution in milliliters (mL);
m-mass of the sample in grams (g);
f-conversion coefficient, wherein the L-sodium malate and the DL-sodium malate are 1.66, and the L-malic acid and the DL-malic acid are 1.
Example 2
In the embodiment of the invention, the analysis methods of malic acid and sodium malate in the food additive product in the prior art are compared with the method in the invention, and the results are shown in table 1:
TABLE 1 characterization of the inventive method versus the prior art method
The method of the invention has higher analysis accuracy and precision to the main component of malic acid than the existing method. In the prior art, a sodium hydroxide acid-base titration method is mostly adopted for the main component analysis of the malic acid product, a perchloric acid titration method and a potentiometer are adopted for the detection of sodium malate in GB 30508-2015, and the titration results are adopted for directly calculating the content of the main component during calculation, so that serious misalignment of the measurement result can be caused when the content of the hetero acid is higher; in the publication of Wei Jian Commission 2018, a liquid chromatography ultraviolet detector is adopted, and superposition calculation of the content of the hetero acid can be avoided, but the adoption of a trace analysis means such as liquid chromatography to analyze the constant substances can lead to the amplification of errors in the weighing and diluting process, so that the precision and the accuracy of the detection result are poor. The method of the invention optimizes and verifies on the basis of the liquid chromatography, adjusts chromatographic column, mobile phase, detector and instrument parameters, solves the problem that the titration method can not deduct the content of the mixed acid, and improves the stability and precision of the analysis of the main component of the malic acid by adopting the liquid chromatography, which is obviously superior to the prior method.
Table 2 comparison of laboratory analysis data for the method of the invention with the prior art representative method
Note that: the method 1 is GB 1886.40-2015, the method 2 is the bulletin method of 2018 of Ministry of health, and the method is adopted for detecting the hetero acid.
As can be seen from the comparative analysis data of Table 2, when the main component analysis is carried out by adopting the titration method (method 1), the total acid content sum is within the range of 100.5% -102.14%, which is obviously higher than the ranges of 98.00% -99.99% and 99.87% -100.03% of the method 2 and the method of the invention, and is also obviously higher than 100%, which shows poor accuracy and unreasonable test calculation logic of the method; from the aspect of precision, the RSD of the method is in the range of 0.02% -0.09%, and is remarkably superior to the range of 0.09% -0.35% and 0.40% -1.60% of the method 1. This shows that the method of the invention significantly improves precision and accuracy compared with the existing analytical methods.
The method has higher analysis precision on the mixed acid such as maleic acid, fumaric acid and the like than the prior method. In the prior art, phosphate buffer solution is mostly adopted as a mobile phase, a C18 chromatographic column is used for separation, and an ultraviolet detector is used for detection, although the method can better perform quantitative analysis on the mixed acid, the succinic acid and the fumaric acid of a plurality of products cannot be effectively separated from interference peaks under the given instrument condition when the comparison test is carried out in the method, the quantitative accuracy is obviously interfered, and the requirement of the substance separation degree of the liquid chromatography detection is difficult to be met, as shown in fig. 1-4, wherein fig. 1 is a liquid chromatogram of a mixed acid mixed standard sample analyzed by adopting the liquid chromatography method (C18 column and the ultraviolet detector) disclosed in the prior art in the embodiment of the invention; FIG. 2 is a liquid chromatogram of a mixed sample of mixed acids analyzed by the liquid chromatography method (C18 column, UV detector) disclosed in the prior art; FIG. 3 is a second liquid chromatogram of a mixed sample of mixed acids analyzed by the presently disclosed liquid chromatography method (C18 column, UV detector) in an embodiment of the invention; FIG. 4 is a liquid chromatogram of a mixed sample of hybrid acids analyzed by the presently disclosed liquid chromatography method (C18 column, UV detector) in accordance with an embodiment of the present invention. In addition, as the response of fumaric acid and maleic acid in the ultraviolet detector is more than hundred times higher than that of succinic acid, the three kinds of mixed acid are not suitable for adopting mixed standard series with the same concentration, the preparation difficulty of standard solution is improved, and the error risk is increased.
The method adopts the differential refraction detector to analyze, utilizes the characteristics of taking density and refraction as electric signal analysis, reduces the sensitivity of instrument signals on the basis of solving the problem of the separation degree of the mixed acid, shields the chromatographic peaks of trace interfering substances, can provide higher analysis accuracy and precision, and effectively overcomes the defect of 'constant substance trace analysis'; meanwhile, the defect that signals of maleic acid and fumaric acid are obviously higher than those of succinic acid is overcome, standard series solution preparation with the same concentration can be realized, and the pretreatment operation is greatly simplified. The chromatograms of the standard solution and the actual measurement sample are shown in fig. 5-8, wherein fig. 5 is a liquid chromatogram of a malic acid standard sample detected by the method according to the embodiment of the invention; FIG. 6 is a liquid chromatogram of a mixed standard sample of hybrid acids detected by the method of the present invention in an embodiment of the present invention; FIG. 7 is a liquid chromatogram of a practical test sample 1 of a malic acid additive tested by the method of the invention in an embodiment of the invention; FIG. 8 is a liquid chromatogram of practical test sample 2 of the malic acid additive tested by the method according to the invention in the example of the invention.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for simultaneously detecting the contents of four organic acids in a food additive, which is characterized by comprising the following steps:
(1) Preparing a malic acid standard stock solution, and mixing a fumaric acid, maleic acid and succinic acid mixed standard stock solution;
(2) Preparing a malic acid standard series solution and a mixed acid standard series solution;
(3) Preparing a sample solution to be tested: weighing a sample to be measured, drying and cooling to room temperature; accurately weighing the dried test sample, dissolving with sulfuric acid solution, fixing the volume, shaking uniformly, filtering, and analyzing and measuring by an instrument; preparing a blank sample solution according to the same processing steps as the sample to be tested;
(4) Respectively carrying out liquid chromatography measurement on malic acid standard series solutions with different concentrations and mixed acid standard series solutions with different concentrations, measuring corresponding peak areas, and respectively drawing standard curves of malic acid, succinic acid, fumaric acid and maleic acid by taking the mass concentration of the mixed standard solutions as an abscissa and the peak areas as an ordinate;
respectively carrying out liquid chromatography measurement on the sample solution and the blank sample solution to obtain peak areas, and obtaining mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be measured according to the standard curve;
(5) And calculating the content of the target object in the sample solution to be detected according to the mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be detected.
2. The method of claim 1, wherein the step of formulating the standard stock solution of malic acid in step (1) comprises: accurately weighing a malic acid standard substance, adding sulfuric acid solution for dissolution and fixing the volume, and preparing the malic acid standard stock solution with the mass fraction of 1%.
3. The method of claim 1, wherein the step of preparing a mixed standard stock solution of fumaric acid, maleic acid and succinic acid in step (1) comprises: and respectively and accurately weighing a fumaric acid standard substance, a maleic acid standard substance and a succinic acid standard substance, adding sulfuric acid solution for dissolution and volume fixing, and preparing into mixed standard stock solution of mixed acid with mass fraction of 0.1%.
4. The method of claim 1, wherein the step of preparing a standard series of solutions of malic acid in step (2) comprises: accurately absorbing the malic acid standard stock solution, adding sulfuric acid solution for dissolution and fixing the volume to prepare standard series solutions with the mass fractions of 0.1%, 0.2% and 0.5% of malic acid respectively; and taking sulfuric acid solution and standard stock solution as standard solutions with the mass fractions of 0% and 1% of malic acid respectively.
5. The method of claim 1, wherein the step of preparing the mixed standard series of mixed acid solutions in step (2) comprises: accurately sucking mixed standard stock solution of the mixed acid, and fixing the volume by using sulfuric acid solution to prepare three mixed standard solutions with mass fractions of 0.0005%, 0.002%, 0.005%, 0.01% and 0.02%.
6. The method of claim 1, wherein the conditions of the liquid chromatography assay in step (4) are: the chromatographic column adopts a hydrogen type cation exchange chromatographic column Aminex HPX-87H,300 mm multiplied by 7.8mm with polystyrene-divinylbenzene resin as filler.
7. The method of claim 1, wherein the conditions of the liquid chromatography assay in step (4) are: column temperature: 60. the temperature is lower than the temperature; mobile phase: a sulfuric acid solution; flow rate: 0.7 mL/min.
8. The method of claim 1, wherein the conditions of the liquid chromatography assay in step (4) are: sample injection amount: 20. mu L; detecting the temperature of the cell: 40. the temperature is lower than the temperature; analysis time: 20 minutes.
9. The method of claim 1, wherein the method for calculating the content of the target in the sample solution to be measured according to the mass fractions of malic acid, fumaric acid, maleic acid and succinic acid in the sample solution to be measured in the step (5) comprises the following steps:
the content of each target in the sample to be measured is calculated according to the mass fraction by adopting the following formula:
wherein:
W-the mass fraction of the substance to be measured in the sample to be measured, in percent (%);
c-the mass fraction of the substance to be measured in the solution of the sample to be measured, obtained from the standard curve, in percentage (%);
c 0 -the mass fraction of the substance to be measured in the sample blank solution obtained from the standard curve, in percent (%);
V -the volumetric volume of the solution to be tested in milliliters (mL);
m -the mass of the test sample in grams (g);
f-conversion coefficient, wherein the L-sodium malate and the DL-sodium malate are 1.66, and the L-malic acid and the DL-malic acid are 1.
10. Use of the method according to any one of claims 1 to 9 for simultaneous detection of four organic acid contents in a food additive in additive detection.
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