CN108459116B - Method for detecting Neu5Gc in fermented red meat product by using fluorescence high performance liquid chromatography - Google Patents
Method for detecting Neu5Gc in fermented red meat product by using fluorescence high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for detecting Neu5Gc in a fermented red meat product by using fluorescence high performance liquid chromatography, which comprises the following steps: preparing a Neu5Gc standard substance solution; drawing a Neu5Gc standard curve; preparing a sample solution of the fermented red meat product; pre-column derivatization; fluorescence high performance liquid chromatography conditions; determination of the content of Neu5Gc in the sample. The invention has accurate detection result, good repeatability, high detection specificity and sensitivity, low cost and low toxicity, and can effectively reduce the detection time and improve the detection efficiency.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for detecting Neu5Gc in a fermented red meat product by using fluorescence high performance liquid chromatography.
Background
Fermented meat products have been long in history, and are popular among people due to their unique flavors, various nutrients produced by microbial fermentation, and convenient storage. In general, fermented meat products are mostly prepared from red meat (beef, mutton, pork, etc.) as a fermentation raw material. Herein, red meat generally refers to animal meat having more red muscle fiber than white muscle fiber, including beef, mutton, pork and processed products thereof (fangdan. red meat, diet Neu5Gc intake and body anti-Neu5Gc antibody relation study [ D ]. university of mansion master academic thesis 2015.). International cancer research institutes of 26 months and 10 months in 2015 listed red meat as a possible carcinogen, which made red meat and its red meat products a hot topic of health problems. Research shows that exogenous N-glycolylneuraminic acid (called Neu5Gc for short) is a specific risk substance for red meat carcinogenicity. Therefore, the detection of the content of Neu5Gc in the fermented red meat product becomes a main index for measuring the safety problem of the fermented red meat product.
Neu5Gc is one of the main structures of sialic acid, and there are many methods for detecting sialic acid at present, and the commonly used methods include a colorimetric method-based resorcinol method, a thiohexaric acid (TBA) method, a high performance liquid chromatography (RP-HPLC), a fluorescent high performance liquid chromatography (HPLC-FLD) and the like. Wherein, the third part (2015 edition) of the Chinese pharmacopoeia includes a resorcinol-hydrochloric acid method (one of spectrophotometry) for measuring the sialic acid content in capsular polysaccharide, and the total sialic acid content can only be detected by the spectrophotometry, but the Neu5Gc content cannot be actually detected. The existing HPLC detection method is mostly used for detecting Neu5Gc in fresh red meat and milk, and no report is found on the determination method of the Neu5Gc content in the fermented red meat product at present. Because the microorganism ferments and decomposes organic matters to generate metabolites, the interference of other impurities in the fermented red meat products is larger than that of fresh red meat and milk, and the detection difficulty is greatly increased. The detection conditions of the conventional fluorescence high performance liquid chromatography are as follows: under the condition that the mobile phase is methanol-acetonitrile-ultrapure water (7: 8: 85, V/V), the peak separation of Neu5Gc such as unfermented red meat, milk and the like is good, but the target peak of the fermented red meat product is superposed with the impurity peak, and the target peak cannot be effectively separated. The detection time is longer under the isocratic elution condition, and the detection efficiency is influenced. Acetonitrile is used as a mobile phase, and the problems of great toxicity, high cost and the like exist.
Disclosure of Invention
The invention aims to overcome the defects and provide the method for detecting Neu5Gc in the fermented red meat product by using the fluorescence high performance liquid chromatography, which has the advantages of accurate detection result, good repeatability, high detection specificity and sensitivity, effective reduction of detection time, improvement of detection efficiency, low cost and low toxicity.
The invention is realized by the following technical scheme:
the invention discloses a method for detecting Neu5Gc in a fermented red meat product by using fluorescence high performance liquid chromatography, which comprises the following steps:
(1) neu5Gc Standard solution preparation
Preparing a 1mmol/L Neu5Gc standard solution;
(2) drawing a Neu5Gc standard curve
Sucking 1mmol/LNeu5Gc standard solution to prepare Neu5Gc standard solution of 10, 25, 50, 100, 200, 300, 400 mu mol/L, taking 900 mu L of Neu5Gc standard solution with different concentrations, respectively adding 100 mu L of 4,5-methylenedioxy-1, 2-phthalic diamine salt (4,5-methylenedioxy-1,2-phenylenediamine dihydrate, DMB) derivative reagent, deriving in a water bath kettle at 50 ℃ in a dark place for 2h, putting into ice water mixed solution, rapidly cooling through 0.22 membrane, and then carrying out sample detection, wherein the concentration of the standard solution is taken as a horizontal axis, and the chromatographic peak area is taken as a vertical axis to prepare a linear standard curve;
(3) preparation of sample solution of fermented red meat product
Taking fermented red meat products (the traditional fermented salted meat in China, fermented sausage, ham and the like), and dividing the fermented red meat products into massive or minced meat-shaped fermented red meat products according to different shapes of raw materials of the fermented red meat products;
a. meat paste-like fermented red meat product sample solution
Accurately weighing 1g of minced meat, homogenizing the minced meat by using 10ml of 30% saturated ammonium sulfate solution to precipitate protein, freeze-drying to remove ammonium sulfate, adding 10ml of 2mol/L glacial acetic acid into a freeze-dried sample to hydrolyze for 3h in a water bath kettle at 80 ℃, centrifuging at the rotating speed of 12000r/min for 10min to take supernatant, filtering by using a 0.45 mu m membrane, carrying out secondary freeze-drying to remove the glacial acetic acid, dissolving the freeze-dried powder in 1ml of 0.1mol/LNaOH, carrying out water bath at 37 ℃ for 30min, and filtering by using a 0.45 mu m membrane to obtain a sample solution in order to dissociate Neu5 Gc;
b. sample solution of block-shaped fermented red meat product
Selecting a lean meat part of a blocky fermented red meat product, adding 1g of an intermediate meat sample below 1cm into 10ml of 30% saturated ammonium sulfate solution for homogenizing to precipitate protein, freeze-drying to remove ammonium sulfate, adding a freeze-dried sample into 10ml of 2mol/L glacial acetic acid, hydrolyzing in a water bath kettle at 80 ℃ for 3h, centrifuging at a rotating speed of 12000r/min for 10min to obtain a supernatant, filtering by using a 0.45 mu m membrane, performing secondary freeze-drying to remove the glacial acetic acid, dissolving the freeze-dried powder in 1ml of 0.1mol/LNaOH, performing water bath at 37 ℃ for 30min, and filtering by using a 0.45 mu m membrane to obtain a sample solution;
(4) precolumn derivatization
Accurately absorbing 900 mu L of sample solution by fluorescence high performance liquid chromatography, adding 100 mu L of LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, then putting the mixture into ice water mixed solution, and rapidly cooling the mixture through a 0.22 film to be detected;
(5) fluorescence high performance liquid chromatography conditions
C18 reverse chromatographic column with octadecylsilane chemically bonded filler is adopted, the excitation wavelength of a fluorescence detector is 373nm, the emission wavelength is 448nm, the column temperature is 30 ℃, the flow rate is 0.9m L/min, and the sample injection volume is 10 mu L. Wherein the mobile phase A is ultrapure water, the mobile phase B is methanol, and the gradient elution procedure is as follows: 80% of A and 20% of B at 0-18 min; 100% B for 18.1-21 min; 80% of A and 20% of B at 21.1-28 min;
(6) determination of the content of Neu5Gc in the sample
Detecting the sample solution obtained in the step (4) according to the condition of the step (5), measuring the peak area of Neu5Gc, and calculating the content of Neu5Gc in the sample according to the following formula corresponding to the Neu5Gc standard curve drawn in the step (2):
in the formula: a is the content of Neu5Gc in the sample/(mu g/g or mu g/mL); m is1The mass/microgram of Neu5Gc corresponding to the peak area ratio of the Neu5Gc chromatographic peak and the external standard chromatographic peak in the sample; r is sample dilution multiple; m is the sample volume/(g or mL).
Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can show that: the pretreatment method of different meat samples is carried out according to the characteristics of the fermented red meat products, and Neu5Gc is effectively extracted. Through gradient elution, the detection time of the sample is reduced, and the detection efficiency is improved. The mobile phase adopts ultrapure water and methanol, and has low cost and low toxicity. The method can effectively separate impurity peaks and main component peaks in the fermented red meat product, and the separation degree R is more than 1.5. The detection result is accurate, the repeatability is good, the detection specificity and the sensitivity are high, the Relative Standard Deviation (RSD) is below 1.6 percent, the detection Limit (LOD) is 0.001 mu mol/L, and the quantification Limit (LOQ) is 0.003 mu mol/L.
The advantageous effects of the present invention will be further described below by way of specific embodiments.
Drawings
FIG. 1 is a Neu5Gc standard chromatogram;
FIG. 2 is a Neu5Gc standard curve;
FIG. 3 is a sample map.
Detailed Description
Example 1
A method for detecting Neu5Gc in a fermented red meat product by using fluorescence high performance liquid chromatography comprises the following steps:
(1) neu5Gc Standard solution preparation
Accurately weighing 0.0825g of Neu5Gc standard substance, dissolving in 250ml of volumetric flask with ultrapure water, and fixing the volume to the scale, namely 1mmol/L of Neu5Gc standard substance solution.
(2) Drawing a Neu5Gc standard curve
Sucking 1mmol/L Neu5Gc standard solution to prepare 10, 25, 50, 100, 200, 300 and 400 mu mol/L Neu5Gc standard solution, taking 900 mu L Neu5Gc standard solution with different concentrations, respectively adding 100 mu LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2 hours, putting the obtained product into ice water mixed solution, rapidly cooling the obtained product to pass through a 0.22 membrane, and performing sample detection, wherein a linear standard curve is prepared by taking the concentration of the standard solution as the horizontal axis and the chromatographic peak area as the vertical axis;
as shown by the Neu5Gc standard chromatogram (FIG. 1), the peak appearance time of Neu5Gc was 14.484min, and the peak shape was good. Linear calibration curves were prepared with standard concentration as axis and corresponding chromatographic peak area as axis, as shown in (fig. 2), linear calibration curve equation for Neu5 Gc: 3.83964x-16.33805, R2＝0.99941。
(3) Sample solution preparation
Selecting lean meat part of fermented ham, collecting 1g of intermediate meat sample below 1cm, adding 10ml of 30% saturated ammonium sulfate solution, homogenizing to precipitate protein, and freeze drying to remove ammonium sulfate. To free Neu5Gc, the lyophilized sample was hydrolyzed in a water bath at 80 ℃ for 3 hours by adding 10ml of 2mol/L glacial acetic acid, the supernatant was centrifuged at 12000r/min for 10min, and the filtrate was filtered through a 0.45 μm membrane and lyophilized again to remove the glacial acetic acid. Dissolving the lyophilized powder in 1ml of 0.1mol/LNaOH, performing water bath at 37 ℃ for 30min, and filtering with a 0.45 mu m membrane to obtain a sample solution.
(4) Precolumn derivatization
Accurately sucking 900 mu L of sample solution, adding 100 mu L of LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, then putting the mixture into ice-water mixed liquid, and rapidly cooling the mixture through a 0.22 film to be detected.
(5) Fluorescence high performance liquid chromatography conditions
An Agilent 1260 liquid chromatograph is adopted to be combined with a fluorescence detector, an Agilent ZOTBAX Eclipse XDB-C18 reverse chromatographic column (250mm multiplied by 4.6mm,5 mu m) is selected, the excitation wavelength of the fluorescence detector is 373nm, the emission wavelength is 448nm, the column temperature is 30 ℃, the flow rate is 0.9m L/min, and the sample injection volume is 10 mu. Wherein the mobile phase A is ultrapure water, the mobile phase B is methanol, and the gradient elution procedure is as follows: 80% of A and 20% of B for 0-18 min; 100% B for 18.1-21 min; 80% of A and 20% of B for 21.1-28 min;
(6) determination of Neu5Gc content in fermented ham
Actually, the average value of the peak area of Neu5Gc in the traditional fermented ham is 286.7, and the Neu5Gc standard curve is drawn according to the step (2) and the following formula is shown:
in the formula: a is the content of Neu5Gc in the sample/(mu g/g or mu g/mL); m is1The mass/microgram of Neu5Gc corresponding to the peak area ratio of the Neu5Gc chromatographic peak and the external standard chromatographic peak in the sample; r is sample dilution multiple; m is the sample volume/(g or mL).
The content of Neu5Gc in the traditional fermented ham is calculated as follows: 25.67. mu.g/g.
Example 2
Fluorescence high performance liquid detection of Neu5G content in traditional fermented salted meat with acid for 30 days:
(1) sample solution preparation
Selecting lean meat part of acid preserved meat fermented for 30 days, collecting 1g of intermediate meat sample below 1cm, adding 10ml of 30% saturated ammonium sulfate solution, homogenizing to precipitate protein, and freeze drying to remove ammonium sulfate. To free Neu5Gc, the lyophilized sample was hydrolyzed in a water bath at 80 ℃ for 3 hours by adding 10ml of 2mol/L glacial acetic acid, the supernatant was centrifuged at 12000r/min for 10min, and the filtrate was filtered through a 0.45 μm membrane and lyophilized again to remove the glacial acetic acid. Dissolving the lyophilized powder in 1ml of 0.1mol/LNaOH, performing water bath at 37 ℃ for 30min, and filtering with a 0.45 mu m membrane to obtain a sample solution.
(2) Precolumn derivatization
Accurately sucking 900 mu L of sample solution, adding 100 mu L of LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, then putting the mixture into ice-water mixed liquid, and rapidly cooling the mixture through a 0.22 film to be detected.
(3) Fluorescence high performance liquid chromatography conditions
An Agilent 1260 liquid chromatograph is adopted to be combined with a fluorescence detector, an Agilent ZOTBAX Eclipse SB-C18 reverse chromatographic column (250mm multiplied by 4.6mm,5 mu m) is selected, the excitation wavelength of the fluorescence detector is 373nm, the emission wavelength is 448nm, the column temperature is 30 ℃, the flow rate is 0.9m L/min, and the sample injection volume is 10 mu. Wherein the mobile phase A is ultrapure water, the mobile phase B is methanol, and the gradient elution procedure is as follows: 80% of A and 20% of B for 0-18 min; 100% B for 18.1-21 min; 80% of A and 20% of B for 21.1-28 min;
(7) determination of Neu5Gc content in acid preserved meat
The chromatogram of the acid preserved meat detected by fluorescence high performance liquid chromatography (FIG. 3) shows that the obtained peak has good shape and high separation degree (R >1.5) with impurities.
The mean value of the peak area of Neu5Gc in acid preserved meat is 303.095, according to Neu5Gc standard curve in example 1, and the following formula:
in the formula: a is the content of Neu5Gc in the sample/(mu g/g or mu g/mL); m is1The mass/microgram of Neu5Gc corresponding to the peak area ratio of the Neu5Gc chromatographic peak and the external standard chromatographic peak in the sample; r is sample dilution multiple; m is the sample volume/(g or mL).
Calculating to obtain Neu5Gc content in the acid preserved meat fermented for 30 days in the traditional way: 27.06. mu.g/g.
Example 3
Detecting the content of Neu5G in the salami by using a fluorescence high performance liquid chromatography:
(1) sample solution preparation
The casings were peeled off, 1g of the meat emulsion was accurately weighed, homogenized with 10ml of 30% saturated ammonium sulfate solution to precipitate the protein, and freeze-dried to remove ammonium sulfate. To free Neu5Gc, the lyophilized sample was hydrolyzed in a water bath at 80 ℃ for 3 hours by adding 10ml of 2mol/L glacial acetic acid, the supernatant was centrifuged at 12000r/min for 10min, and the filtrate was filtered through a 0.45 μm membrane and lyophilized again to remove the glacial acetic acid. Dissolving the lyophilized powder in 1ml of 0.1mol/LNaOH, performing water bath at 37 ℃ for 30min, and filtering with a 0.45 mu m membrane to obtain a sample solution.
(2) Precolumn derivatization
Accurately sucking 900 mu L of sample solution, adding 100 mu L of LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, then putting the mixture into ice-water mixed liquid, and rapidly cooling the mixture through a 0.22 film to be detected.
(3) Fluorescence high performance liquid chromatography conditions
An Agilent 1260 liquid chromatograph is adopted to be combined with a fluorescence detector, an Agilent ZOTBAX Eclipse XDB-C18 reverse chromatographic column (250mm multiplied by 4.6mm,5 mu m) is selected, the excitation wavelength of the fluorescence detector is 373nm, the emission wavelength is 448nm, the column temperature is 30 ℃, the flow rate is 0.9m L/min, and the sample injection volume is 10 mu. Wherein the mobile phase A is ultrapure water, the mobile phase B is methanol, and the gradient elution procedure is as follows: 80% of A and 20% of B for 0-18 min; 100% B for 18.1-21 min; 80% of A and 20% of B for 21.1-28 min;
(8) determination of Neu5Gc content in salami
The mean peak area of the salami Neu5G was found to be 172.6, based on the Neu5Gc standard curve in example 1 and the following formula:
in the formula: a is the content of Neu5Gc in the sample/(mu g/g or mu g/mL); m is1The mass/microgram of Neu5Gc corresponding to the peak area ratio of the Neu5Gc chromatographic peak and the external standard chromatographic peak in the sample; r is sample dilutionMultiple; m is the sample volume/(g or mL).
The content of Neu5Gc in the salami is calculated as follows: 16.01 μ g/g.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.
Claims (1)
1. A method for detecting Neu5Gc in a fermented red meat product by using fluorescence high performance liquid chromatography comprises the following steps:
(1) neu5Gc Standard solution preparation
Preparing a 1mmol/L Neu5Gc standard solution;
(2) drawing a Neu5Gc standard curve
Sucking 1mmol/LNeu5Gc standard solution to prepare Neu5Gc standard solution of 10, 25, 50, 100, 200, 300 and 400 mu mol/L, taking 900 mu L of Neu5Gc standard solution with different concentrations, respectively adding 100 mu L of derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, putting the obtained product into ice water mixed solution, rapidly cooling the obtained product, passing the obtained product through a 0.22 membrane, and performing sample detection, wherein a linear standard curve is prepared by taking the standard solution concentration as a horizontal axis and the chromatographic peak area as a vertical axis;
(3) preparation of sample solution of fermented red meat product
Taking the fermented red meat product, and dividing the fermented red meat product into blocky or meat paste-shaped fermented red meat products according to different shapes of raw materials of the fermented red meat product;
a. meat paste-like fermented red meat product sample solution
Accurately weighing 1g of minced meat, homogenizing the minced meat by using 10ml of 30% saturated ammonium sulfate solution to precipitate protein, freeze-drying to remove ammonium sulfate, adding 10ml of 2mol/L glacial acetic acid into a freeze-dried sample to hydrolyze for 3h in a water bath kettle at 80 ℃, centrifuging at the rotating speed of 12000r/min for 10min to take supernatant, filtering by using a 0.45 mu m membrane, carrying out secondary freeze-drying to remove the glacial acetic acid, dissolving the freeze-dried powder in 1ml of 0.1mol/LNaOH, carrying out water bath at 37 ℃ for 30min, and filtering by using a 0.45 mu m membrane to obtain a sample solution in order to dissociate Neu5 Gc;
b. sample solution of block-shaped fermented red meat product
Selecting a lean meat part of a blocky fermented red meat product, adding 1g of an intermediate meat sample below 1cm into 10ml of 30% saturated ammonium sulfate solution for homogenizing to precipitate protein, freeze-drying to remove ammonium sulfate, adding a freeze-dried sample into 10ml of 2mol/L glacial acetic acid, hydrolyzing in a water bath kettle at 80 ℃ for 3h, centrifuging at a rotating speed of 12000r/min for 10min to obtain a supernatant, filtering by using a 0.45 mu m membrane, performing secondary freeze-drying to remove the glacial acetic acid, dissolving the freeze-dried powder in 1ml of 0.1mol/LNaOH, performing water bath at 37 ℃ for 30min, and filtering by using a 0.45 mu m membrane to obtain a sample solution;
(4) precolumn derivatization
Accurately absorbing 900 mu L of sample solution by fluorescence high performance liquid chromatography, adding 100 mu L of LDMB derivative reagent, performing light-shielding derivation at 50 ℃ in a water bath kettle for 2.5h, then putting the mixture into ice water mixed solution, and rapidly cooling the mixture through a 0.22 film to be detected;
(5) fluorescence high performance liquid chromatography conditions
C18 reverse chromatographic column with octadecylsilane chemically bonded filler is adopted, the excitation wavelength of a fluorescence detector is 373nm, the emission wavelength is 448nm, the column temperature is 30 ℃, the flow rate is 0.9mL/min, and the sample injection volume is 10 muL; wherein the mobile phase A is ultrapure water, the mobile phase B is methanol, and the gradient elution procedure is as follows: 80% of A and 20% of B at 0-18 min; 100% B for 18.1-21 min; 80% of A and 20% of B at 21.1-28 min;
(6) determination of the content of Neu5Gc in the sample
Detecting the sample solution obtained in the step (4) according to the condition of the step (5), measuring the peak area of Neu5Gc, and calculating the content of Neu5Gc in the sample according to the following formula corresponding to the Neu5Gc standard curve drawn in the step (2):
in the formula: alpha is the content of Neu5Gc in the sample/. mu.g/g or/. mu.g/mL; m is1The mass/microgram of Neu5Gc corresponding to the peak area ratio of the Neu5Gc chromatographic peak and the external standard chromatographic peak in the sample; r is sample dilution multiple; m is the sampling amount/g or mL;
wherein: the fermented red meat product in step (3) is sour preserved meat, fermented sausage or ham.
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| CN103149312A (en) * | 2013-02-05 | 2013-06-12 | 东北农业大学 | Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry |
| CN107045019A (en) * | 2016-09-30 | 2017-08-15 | 中国医学科学院输血研究所 | The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG |
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| CN103149312A (en) * | 2013-02-05 | 2013-06-12 | 东北农业大学 | Analyzing method of sialic acid in infant milk powders with ultra-high performance liquid chromatography tandem quadrupole mass spectrometry |
| CN107045019A (en) * | 2016-09-30 | 2017-08-15 | 中国医学科学院输血研究所 | The assay method of IgG Fab fragments and Fc fragment sialic acid contents in a kind of IVIG |
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