CN117451869A - Method for measuring content of L-carnitine in protein mixed lipid food - Google Patents
Method for measuring content of L-carnitine in protein mixed lipid food Download PDFInfo
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- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 44
- 150000002632 lipids Chemical class 0.000 title claims abstract description 18
- 235000013305 food Nutrition 0.000 title claims abstract description 17
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000003208 petroleum Substances 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 6
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 4
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 4
- 239000010935 stainless steel Substances 0.000 claims abstract description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 29
- 239000008363 phosphate buffer Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
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- 238000001816 cooling Methods 0.000 claims description 3
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- 108010009736 Protein Hydrolysates Proteins 0.000 claims 1
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- 239000012071 phase Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 10
- 238000005903 acid hydrolysis reaction Methods 0.000 abstract description 9
- 238000005119 centrifugation Methods 0.000 abstract description 7
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- 229910019142 PO4 Inorganic materials 0.000 abstract description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 3
- 239000010452 phosphate Substances 0.000 abstract description 3
- 235000012041 food component Nutrition 0.000 abstract description 2
- 239000005417 food ingredient Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 27
- 239000012488 sample solution Substances 0.000 description 18
- 239000012086 standard solution Substances 0.000 description 15
- 230000014759 maintenance of location Effects 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 8
- 101000985493 Mus musculus Hermansky-Pudlak syndrome 3 protein homolog Proteins 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
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- 238000012360 testing method Methods 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229960001518 levocarnitine Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 3
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- 230000003139 buffering effect Effects 0.000 description 3
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- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
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- BQMPVGMHZKZPBH-BYZBDTJCSA-N (3r)-3-hydroxy-4-(trimethylazaniumyl)butanoate Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O.C[N+](C)(C)C[C@H](O)CC([O-])=O BQMPVGMHZKZPBH-BYZBDTJCSA-N 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
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Classifications
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- 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
- G01N30/02—Column chromatography
-
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- 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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of food ingredient detection, and relates to a method for measuring the content of L-carnitine in protein mixed lipid foods. The pretreatment process of the sample comprises the following steps: after acidolysis of the sample, adding petroleum ether, centrifuging at low temperature to remove lipid substances, and then diluting and measuring by using phosphate buffer solution; the liquid chromatography detection conditions are as follows: chromatographic column: 250 mm. Times.4.6mm.times.5 μm stainless steel column, column temperature: 30 ℃, flow rate: 1mL/min, the mobile phase was acetonitrile: phosphate buffer solution volume ratio is 65:35, detection wavelength: 210nm. The method has the advantages that the pretreatment is optimized, the acid hydrolysis and petroleum ether extraction centrifugation are used for separating and extracting the L-carnitine from the complex sample rich in protein fat, the problems of low extraction efficiency, complicated pretreatment process or high consumable materials in other methods are solved, the defect that a single phosphate is used as a mobile phase in the traditional hydrophilic chromatography method for measuring the L-carnitine content is overcome, and the method is suitable for the condition of pretreatment with wider pH range.
Description
Technical Field
The invention belongs to the technical field of food ingredient detection, relates to a method for measuring the content of L-carnitine in protein-mixed lipid food, and particularly relates to a hydrophilic liquid chromatography method for measuring the content of L-carnitine in protein-mixed lipid food.
Background
L-carnitine (L-carnitine) is also called as L-carnitine and vitamin BT, is an amino acid for promoting fat to be converted into energy, has the effects of oxidative decomposition of fat and fatigue resistance, has proved to have good safety, and can be used as a food nutrition enhancer. Therefore, with the research on the chemical components and medical value of the levocarnitine, the levocarnitine is widely applied to foods needing to assist in fat metabolism. However, with the rapid development of food, the food processing forms and processes are continuously updated, and for the novel food protein bars, more products contain the L-carnitine, and in order to increase different tastes, the protein bars are added with lipid substances which form packages on the L-carnitine, so that the detection of the L-carnitine is difficult. Meanwhile, after the treatment by the traditional treatment method, the liquid chromatography detection has a great number of impurity peaks, and the peaks and the peak areas of the L-carnitine are interfered, so that the content of the L-carnitine in the protein mixed lipid sample can not be well detected by the traditional method.
Disclosure of Invention
The invention provides a novel hydrophilic liquid chromatography determination method aiming at the problem of determination of the content of L-carnitine in a traditional protein mixed lipid sample.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
sample treatment process: the protein space structure is destroyed by acid hydrolysis, and the physically-encapsulated L-carnitine is released or the combined L-carnitine is released.
The fatty acid is formed by acid hydrolysis of the lipid material, releasing the physically incorporated, adsorbed l-carnitine.
The peptides hydrolyzed from the proteolysis are precipitated by low temperature centrifugation and further lipid material is removed by petroleum ether.
The acid hydrolysate was collected as an extract and diluted with phosphate buffer.
The sample pretreatment steps are as follows:
step S1: thoroughly pulverizing protein-lipid mixture food to obtain large particles with no obvious visual effect, and freeze pulverizing or sea sand dispersing for some products with high fat content or high viscosity.
Step S2: accurately weighing the crushed powder, and adding the powder into the acid hydrolysis liquid.
Step S3: and (3) mixing the sample acid hydrolysis liquid mixture obtained in the step (S2) uniformly by vortex, and heating in a water bath to carry out acidolysis.
Step S4: and (3) cooling the acidolysis solution in the step (S3), adding petroleum ether, rapidly oscillating, and centrifuging.
Step S5: the acidolysis solution in step S4 was diluted with Phosphate Buffer (PB).
Preferably, in the step S2, the hydrolysate is a hydrochloric acid solution (analytically pure) with a sufficient concentration of 0.2 to 1.0mol/L.
Preferably, in the step S3, the vortex speed is 2500r/min, the vortex duration is 5min, the water bath hydrolysis temperature is 70 ℃, and the water bath hydrolysis time is 3h.
Preferably, in the step S4, the petroleum ether is analytically pure, the number of rapid oscillations is 3-5, and the centrifugation conditions are as follows: the temperature was 3℃and the rotational speed was 4000r/min, and the centrifugation time was 10min.
Preferably, in the step S5, the phosphate buffer solution is disodium hydrogen phosphate with the concentration of 0.05 mol/L: sodium dihydrogen phosphate with the concentration of 0.05mol/L is 7.9:92.1, ph=5.8.
The detection conditions were as follows:
instrument: HPLC-UV system.
Chromatographic column: venusil HILIC column, 250mm×4.6mm, 5 μm, stainless steel column.
Column temperature: 30 ℃.
Flow rate: 1mL/min.
Mobile phase: acetonitrile: PB volume ratio = 65:35.
Detection wavelength: 210nm.
The acid hydrolysis technology is based on the principle that acid hydrolysis can destroy macromolecular structure organic compounds, and utilizes the characteristics that the destruction can prevent the target substance from dissociating or chemical bond combination in physical space to prevent the target substance from dissociating, so that the target substance is easy to be extracted completely, and the target substance and lipid substance products are fatty acid substances and are insoluble in water, so that the interference on the target substance can not be increased.
PB is a pH buffer solution, has a certain range of buffering capacity for pH, and because of different acid consumption capacities of acid hydrolyzed samples, a wider pH range appears, firstly, a qualitative retention time can be caused to be different between a sample solution and a standard solution, and secondly, a chromatographic column with a limited pH tolerance range can be damaged, PB is used for buffering to replace adjustment of a pH meter, and manual operation and redundant dilution are reduced.
HILIC (diamido) chromatographic column is a hydrophilic technical chromatographic column, and more acting force is realized by adopting a reverse mode based on normal phase chromatography, so that better separation effect is achieved, and the method has obvious effect on water-soluble and ionic compounds and reduces the harm of mobile phase modification to the chromatographic column.
Compared with the prior art, the invention has the advantages and positive effects that:
the method has the advantages that the pretreatment is optimized, the acid hydrolysis and petroleum ether extraction centrifugation are used for separating and extracting the L-carnitine from the complex sample rich in protein fat, the problems of low extraction efficiency, complicated pretreatment process or high consumable materials in other methods are solved, the defect that a single phosphate is used as a mobile phase in the traditional hydrophilic chromatography method for measuring the L-carnitine content is overcome, and the method is suitable for the condition of pretreatment with wider pH range.
Drawings
Fig. 1 is a residual result graph.
FIG. 2 is a chromatogram of the standard solution in example 2.
FIG. 3 is a chromatogram of the sample solution in example 2.
FIG. 4 is a chromatogram of the standard solution in example 3.
FIG. 5 is a chromatogram of the sample solution in example 3.
FIG. 6 is a chromatogram of the sample solution in comparative example 1.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be provided with reference to specific examples. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as described herein, and therefore the present invention is not limited to the specific embodiments of the disclosure that follow.
Example 1
The test object of this example is a commercially available cocoa protein stick.
1. Instruments and reagents.
High performance liquid chromatograph (e 2695 type, UV/Vis detector, watthour limited); centrifuges (LX-155T 500R type, qingdao sea biomedical science, inc.); laboratory pH meter (model FE20, meltrel-tolidor instruments limited); multitube vortex mixer (model BC-1000, shenzhen comma biotechnology Co., ltd.); digital display thermostatic water bath (HH-4 type, gold south Instrument manufacturing Co., ltd.); pipette gun (Transferpette S variabel-200. Mu.l, BRAND; transferpette Svariabel-100. Mu.l, BRAND).
Acetonitrile (chromatographic purity), sodium dihydrogen phosphate (analytical purity), disodium hydrogen phosphate (analytical purity), petroleum ether (analytical purity, boiling range 30-60 ℃); l-carnitine standard (CAS: 541-15-1); PB buffer: 0.05mol/L disodium hydrogen phosphate: volume ratio of 0.05mol/L sodium dihydrogen phosphate=7.9: 92.1, ph=5.8; extracting solution: 0.5mol/L hydrochloric acid solution; the water used in the experiment is primary water.
2. Detection method
2.1 Preparation of standard stock solution of L-carnitine
Accurately weighing 19.0mg of L-carnitine standard substance, dissolving in 10mL PB buffer solution, and preparing into standard stock with a concentration of 1900 μg/mL for later use.
2.2 Preparation of sample solutions
A protein stick (cocoa) sample was crushed and thoroughly mixed, 1.00g of the sample was precisely weighed into a 50ml centrifuge tube, and 25.0ml of 0.5mol/L HCl aqueous solution was added for dissolution. After dissolution, vortex for 5min at 2500r/min followed by a thermostatic water bath at 70 ℃ for 3h. After cooling to room temperature, 20.0 ml petroleum ether is added, the mixture is rapidly vibrated for 5 times, and centrifuged at 4000r/min at 3 ℃ for 10min, and after centrifugation, the supernatant liquid is sucked into the container to be 1ml (the total volume of the supernatant liquid is the volume of hydrochloric acid solution, in this example, 25.0 ml) and then the supernatant liquid is filtered through a 0.45 mu m polyethersulfone microporous filter membrane. 500.0 μl of the solution after film coating is accurately sucked by a pipette, and the volume is fixed to a scale by PB solution in a 10.00 mL volumetric flask for standby.
2.3 Chromatographic conditions
Chromatographic column: venusil HILIC column (250 mm. Times.4.6 mm,5 μm, stainless steel column); detection wavelength: UV210nm; column temperature: 30 ℃; flow rate: 1 ml/min; sample injection amount: 10 μl; the mobile phase is acetonitrile to (0.05 mol/L disodium hydrogen phosphate to 0.05mol/L sodium dihydrogen phosphate volume ratio=7.9:92.1) volume ratio=65:35. Qualitative, external standard peak area method was used to quantify the retention time.
2.4 Standard Curve
Precisely sucking the stock solution of the standard substance, diluting with PB solution, performing linear regression analysis with concentration as abscissa and peak area as ordinate, measuring concentration and peak area to obtain good linear relationship, randomly distributing residual errors, and obtaining linear equation of y= 341.19x-154.04 according to linear regression result, and determining coefficient r, wherein the intercept is smaller than 2% of 100% response value of marked concentration 2 0.99921, correlation r= 0.99961, formulation procedure and test results are shown in table 1, residual is shown in fig. 1, and fig. 1 illustrates that standard curve fitting is reasonably effective.
TABLE 1 Standard solution preparation Process and test results
2.5 calculation method
And calculating the peak area of the sample according to a standard curve fitting formula to obtain the sample concentration.
The formula is:wherein x is the concentration of the sample solution in μg.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the y is the peak area of the sample solution; alpha is the intercept of the standard curve and beta is the slope of the standard curve.
And obtaining the mass fraction of the L-carnitine in the sample through the conversion of the concentration of the sample solution obtained by the calculation of the formula by the fixed volume, the dilution multiple and the sampling amount. In this example, the dilution ratio and the sampling amount were converted to 25× (10×0.2) =500. The calculation formula is as follows:
，
wherein ω represents the mass fraction of L-carnitine in the sample in units of μ g.g -1 ；
X is the concentration of the sample solution in μg.mL -1 ；
V is the volume of constant volume, mL;
f is dilution multiple;
m is the sampling amount, g.
The test peak area of the sample of this example was 15426.
Fitting function is y= 341.19x-154.04;
the carry-over calculation is as follows:
(15426+154.04)÷341.19×500÷1.00÷10 6 ×100=2.28g·100g -1 。
2.6 accuracy verification
The correctness was verified using background-free samples and reagent labeling, 1.00g of samples, 3 portions were placed in 50mL centrifuge tubes. Precisely measuring 1mL, 2mL and 4mL of standard solution in a 25mL volumetric flask, using 0.5mol/L hydrochloric acid to fix the volume to scale, respectively sucking 5.0mL into three blank centrifuge tubes as a sample three-level standard adding, respectively sucking 5.0mL into three blank centrifuge tubes as a reagent three-level standard adding, and processing according to the method of step 2.2. The measurement results show that the sample labeling recovery rate of different types and different levels is 98.2% -101.6%, and the reagent labeling recovery rate is 99.6% -100.2%, which indicates that the accuracy of the method is higher.
2.7 method precision verification
6 parts of cocoa protein bars of the embodiment are accurately weighed, 1.0g of each part is placed into a 50mL centrifuge tube, 1.0mL of the cocoa protein bars are taken to be constant to 5.0mL of the cocoa protein bars after the cocoa protein bars are subjected to film coating, the rest sample solutions are prepared according to the method in the step 2.2, and the cocoa protein bars are respectively put on a machine at different time periods, so that the RSD of the result is less than 2%. The reproducibility of the method is shown to be better, and the results are shown in Table 3.
TABLE 3 repeatability test results
Example 2
In this example, acidolysis was carried out using a 0.2mol/L hydrochloric acid solution, and the rest of the procedure was identical to that in example 1. The chromatogram of the standard solution is shown in fig. 2, and the chromatogram of the sample solution is shown in fig. 3. The peak pattern of the target is preferable as can be seen from fig. 2 and 3. Therefore, the 0.2mol/L hydrochloric acid solution also has better acidolysis capacity, and can meet the detection requirement. In addition, it should be noted that, because the chromatographic column is sensitive to the pH, in order to ensure the accuracy of detection, the pH of the solution to be detected should preferably not differ too much from the pH of the solution in the detection of the standard curve, but because the PB solution has a certain buffering capacity, the standard curve is applicable in the range of 0.2-0.5mol/L of the hydrochloric acid solution, and no operation of adjusting the pH is required, so that the acidolysis solution in the range, the pH value in the detection of the machine presents almost consistent qualitative capacity on the chromatographic column, which also verifies the precision of the method of the present invention from the side.
Example 3
In the embodiment, 1.0mol/L hydrochloric acid solution is adopted for acidolysis, and because the concentration of the hydrochloric acid solution is high, in order to ensure that the pH values of the standard solution and the sample solution are in a relatively consistent state, the sample and the standard solution are subjected to the same pretreatment. The specific operation is as follows: 1.0g of the sample was weighed, 25mL of 1.0mol/L hydrochloric acid solution was added, 1.0mL of the standard solution was aspirated, 24mL of 1.0mol/L hydrochloric acid solution was added, and the rest of the steps for preparing the standard solution and the sample solution were the same as those in step 2.2 of example 1. Because the HILIC chromatographic column is sensitive to pH, when the pH value of acidolysis solution slightly exceeds the buffer range of PB solution, the retention time can be obviously advanced, if the standard solution is not synchronously detected, the approximation of the pH value is difficult to ensure without increasing the step of regulating the pH value, the retention time of the standard solution is greatly different from that of the sample solution and cannot be determined, but if the standard solution and the sample solution are synchronously treated, the defect can be overcome, and the standard solution is accurately determined, and the method is shown in Table 4, figure 4 and figure 5.
TABLE 4 high concentration acidolysis retention time
Comparative example 1
This comparative example omits the hydrochloric acid solution acidolysis step, and the remaining steps are identical to those of example 1. As shown in fig. 6, the sample detection results are shown in fig. 6, and it can be seen that the sample impurities are complex, the target peak separation effect is poor, the extraction efficiency is low, and the detection cannot be performed.
The method for measuring the content of the L-carnitine in the high-protein fat sample by the hydrophilic effect chromatography is characterized in that the L-carnitine is separated and extracted from the complex sample rich in protein fat by acidolysis and petroleum ether extraction centrifugation through optimizing pretreatment, so that the blank of the hydrophilic chromatography for measuring the content of the L-carnitine in the protein fat sample is filled, and the defect that a single phosphate is used as a mobile phase in the conventional hydrophilic chromatography for measuring the content of the L-carnitine is overcome, and the method is suitable for the condition of more pretreatment.
In the direct extraction method of pure water solution which is commonly used, because of the complex nature of the sample, part of the sample is blocked by the space structure of protein, part of the sample is blocked by the high viscosity of lipid, the L-carnitine cannot be completely released, the extraction efficiency is low, the space structure of protein is destroyed by acid hydrolysis to release the L-carnitine, lipid substances generate water-insoluble higher fatty acid, so that the influence of the high viscosity of the fat on the extraction is avoided, and nonpolar impurities are further removed by petroleum ether, so that the reduction of impurities in the extract and the increase of the extraction efficiency of a target object can be obviously seen.
The problem of inconsistent retention time caused by pH difference can be solved to a certain extent by using PB buffer solution for sample dilution before detection and application in a mobile phase, and pH adjustment treatment is not needed for the sample solution, but when the increase of hydrochloric acid acidity exceeds the PB buffer capability, the retention time can be advanced, and the consistency of the retention time of the L-carnitine can be ensured by approaching the pH value of the standard solution and the sample solution.
When the pH value is controlled between 4.5 and 6.0, the L-carnitine is not easy to decompose, the pH value is suitable for the acid-base tolerance range of most chromatographic columns, and the relative extremely poor retention time floating can be controlled within 0.2 percent. The PB solution and the HILIC chromatographic column are matched to achieve good separation degree of impurities, and the separation degree in the sample solution can reach a range of 3-7%.
The method has the characteristics of high separation, convenience, short time consumption and accurate result, and can be used as a method for controlling the quality of the L-carnitine in the protein fat sample. In addition, for the appearance and development of more and more L-carnitine health-care food products in the future, the method can also provide a reference method for controlling the content of L-carnitine in other L-carnitine health-care foods.
The present invention is not limited to the above-mentioned embodiments, and any equivalent embodiments which can be changed or modified by the technical content disclosed above can be applied to other fields, but any simple modification, equivalent changes and modification made to the above-mentioned embodiments according to the technical substance of the present invention without departing from the technical content of the present invention still belong to the protection scope of the technical solution of the present invention.
Claims (4)
1. A method for measuring the content of L-carnitine in protein-lipid mixture food is characterized by that the liquid-phase chromatography is used to detect the L-carnitine after the pretreatment of sample,
the pretreatment process of the sample comprises the following steps:
after acidolysis of the sample, adding petroleum ether, centrifuging at low temperature to remove lipid substances, and then diluting and measuring by using phosphate buffer solution;
the liquid chromatography detection conditions are as follows:
chromatographic column: 250 mm. Times.4.6mm.times.5 μm stainless steel column, column temperature: 30 ℃, flow rate: 1mL/min, the mobile phase was acetonitrile: phosphate buffer solution volume ratio is 65:35, detection wavelength: 210nm.
2. The method for measuring the content of L-carnitine in protein-containing mixed lipid foods according to claim 1, wherein the pretreatment process of the sample is that the protein-containing mixed lipid foods are thoroughly crushed until large particles which are not obvious to the naked eye are obtained, the crushed powder is accurately weighed to a certain mass, added into acid hydrolysate, and uniformly mixed by vortex and heated in water bath for acidolysis; cooling the acidolysis solution, adding petroleum ether, rapidly oscillating, centrifuging, and diluting the supernatant with phosphate buffer solution.
3. The method for measuring the content of L-carnitine in a protein-containing food according to claim 2, wherein the acidolysis solution used for acidolysis in the pretreatment of the sample is a hydrochloric acid solution having a concentration of 0.2 to 1.0mol/L.
4. The method for determining the content of L-carnitine in a protein-containing mixed lipid food according to claim 1, wherein the phosphate buffer used in the pretreatment of the sample and the detection of the sample is 0.05mol/L disodium hydrogen phosphate aqueous solution and 0.05mol/L sodium dihydrogen phosphate aqueous solution, and the volume ratio is 7.9:92.1.
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