CN110346471B - High performance liquid chromatography for determining carnosine and anserine contents in fish head - Google Patents
High performance liquid chromatography for determining carnosine and anserine contents in fish head Download PDFInfo
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Abstract
The invention relates to the technical field of detection methods, and discloses a high performance liquid chromatography for determining the content of carnosine and anserine in fish heads, which comprises the following steps: taking soft tissue of the head of the fish, mashing the soft tissue, adding an extracting solution into the crushed soft tissue as a sample, and heating and extracting the sample to obtain a crude extracting solution; cooling the crude extractive solution to room temperature, freezing, centrifuging, and filtering the supernatant with microporous membrane; and measuring the content of carnosine and anserine in the filtrate by using high performance liquid chromatography, wherein mesoporous modified Al-SBA-15 loaded with copper ions is used as a filler in a chromatographic column of the high performance liquid chromatography. The mesoporous modified Al-SBA-15 loaded with copper ions is used as a filler in a chromatographic column during high performance liquid chromatography, so that the adsorption and separation effects on carnosine and anserine are good, the detection accuracy and sensitivity are high, the operation is simple and convenient, a complex pretreatment step is not needed, the peak area and retention time deviation are small when the device is used for detecting on a machine only through simple pretreatment, and the accuracy is high.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to a high performance liquid chromatography for determining the content of carnosine and anserine in fish heads.
Background
Carnosine and anserine are functional dipeptides synthesized from L-histidine and β -alanine, are present in animal skeletal muscle and brain, are usually extracted from poultry skeletal muscle, and have important physiological effects of maintaining body pH stability, resisting oxidation, resisting aging, regulating nerves, and the like. Therefore, it is necessary to establish a rapid and reliable method for detecting carnosine and anserine.
At present, no relevant national standard exists for detecting carnosine and anserine, and the main detection methods comprise high performance liquid chromatography, ion exchange chromatography and capillary electrophoresis. However, the three methods have high requirements on separation and purification of samples, complex pretreatment methods are required, and the steps for determining the content of carnosine and anserine in organisms are complicated and the measurement efficiency is low.
In order to improve the measurement accuracy and simplify the pretreatment method, high performance liquid chromatography and mass spectrometry are generally used to measure carnosine and anserine in vivo, for example, the following documents: UHPLC-ESI-MS/MS method determination animal source food extract in carnosine and anserine content [ J ]. analysis and test reports, 2018. in disclose an ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometry, with molecular ion and fragment ion mass-to-charge ratio qualitative, establish a simple, rapid, can determine carnosine and anserine content method at the same time, in order to meet the analysis requirements of carnosine and anserine content in animal source extracts in the fields such as special medicine food, health food, etc.
However, the detection cost is high by using an expensive and complicated mass spectrum detector, most laboratories do not have detection conditions, and the detection method is not suitable for wide application.
Disclosure of Invention
The invention aims to overcome the defects that when a high performance liquid chromatography and mass spectrometry combined method is used for measuring carnosine and anserine in a living body in the prior art, a mass spectrometry detector has higher detection cost, most laboratories do not have detection conditions, and the detection method is not beneficial to wide application; if only high performance liquid chromatography is adopted for determination, the accuracy is poor, the requirements on separation and purification of samples are high, a complex pretreatment method is needed, the determination steps are complicated, and the measurement efficiency is low.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high performance liquid chromatography for determining carnosine and anserine contents in fish head comprises the following steps:
(1) taking soft tissue of the head of the fish, mashing the soft tissue, adding an extracting solution into the crushed soft tissue as a sample, and heating and extracting the sample to obtain a crude extracting solution;
(2) cooling the crude extractive solution to room temperature, freezing, centrifuging, and filtering the supernatant with microporous membrane;
(3) and measuring the content of carnosine and anserine in the filtrate by using high performance liquid chromatography, wherein mesoporous modified Al-SBA-15 loaded with copper ions is used as a filler in a chromatographic column of the high performance liquid chromatography.
The mesoporous SBA-15 material has an adjustable pore structure, a larger pore diameter and better hydrothermal stability, is easy to functionalize, and forms a hydrophobic or hydrophilic environment, so that the mesoporous SBA-15 material is an ideal raw material for chromatographic column packing. However, the Si-OH bond existing in the SBA-15 hardly can be physically and chemically adsorbed with carnosine and anserine, so that the content of the carnosine and the anserine cannot be well separated and measured by using the SBA-15 as a filler.
The invention modifies SBA-15, adds aluminum element into SBA-15 to form Al-SBA-15, introduces carboxyl and loaded copper ion into silicon oxide framework, the carboxyl can generate nonspecific combination with the amino end of carnosine and anserine to form ionic bond, and then adsorbs carnosine and anserine; the loaded copper ions can be chelated with imidazolyl in carnosine and anserine, so that the adsorption quantity of the carnosine and the anserine in the mesoporous material is further improved; and the aluminum element in the Al-SBA-15 increases the acid sites in the silicon dioxide material, increases the charges on the surface of the material, strengthens the interaction between copper ions and Al-SBA-15 carriers, and ensures that the copper ions are more uniformly dispersed on the surface of the material.
Therefore, when the copper ion-loaded mesoporous modified Al-SBA-15 is used as a filler in a chromatographic column during high performance liquid chromatography analysis, the adsorption and separation effects on carnosine and anserine are good, the detection accuracy and sensitivity are high, the operation is simple and convenient, a complex pretreatment step is not needed, and the detection can be carried out on a machine only by simple pretreatment in the steps (1) and (2).
Preferably, the preparation method of the copper ion loaded mesoporous modified Al-SBA-15 in the step (3) is as follows:
A) dissolving P123 and NaF in 1-2mol/L hydrochloric acid solution, and stirring at 35-45 ℃ until the solutions are completely dissolved;
B) dropwise adding cyanoethyltriethoxysilane into the solution, stirring for 20-30min, dropwise adding ethyl orthosilicate, and stirring at 35-45 deg.C for 6-8 h;
C) adding 1-2mol/L NaAlO into the solution2Hydrochloric acid solution, stirring for 20-30h at 35-45 ℃;
D) adjusting the pH value of the solution to 1.6-1.7 by ammonia water, transferring the solution into a Teflon lining reaction kettle, and activating the reaction kettle in an oven at the temperature of 100-;
E) taking the reaction kettle out of the oven, adjusting the pH value of the solution to 5.5-6.5 by using ammonia water after the temperature is reduced to room temperature, and then placing the solution in an oven at the temperature of 100-;
F) after filtering, washing the product with distilled water and absolute ethyl alcohol, and drying at 60-100 ℃ for 8-12h to obtain cyanated Al-SBA-15;
G) adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 44% -47%, stirring and reacting for 24-36h at 90-100 ℃, filtering, washing, and drying for 8-12h at 70-90 ℃ to obtain the carboxylated Al-SBA-15;
H) dispersing the carboxylated Al-SBA-15 in 0.1-0.2mol/L CuSO4 solution, stirring for reaction for 20-30h, carrying out suction filtration, washing the obtained product, and drying at 50-70 ℃ for 8-10 h;
I) and roasting the obtained product in a muffle furnace at 500-600 ℃ for 4-5h to obtain the copper ion loaded mesoporous modified Al-SBA-15.
In the steps A) to F) of the invention, the polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is taken as a template, NaF is taken as an auxiliary agent, and NaAlO is taken as2Is an aluminum source, and is added with cyanoethyl triethoxyCo-hydrolyzing and polycondensing silane and ethyl orthosilicate, preparing a cyanolated Al-SBA-15 mesoporous material with good stability by a pH adjusting method, and carrying out acidolysis on the cyanolated Al-SBA-15 by sulfuric acid in the step G) to obtain carboxylated Al-SBA-15; in the step H), divalent copper ions are loaded on the surface of the carboxylated Al-SBA-15, and then the template substances are removed through roasting in the step I), so that the copper ion loaded mesoporous modified Al-SBA-15 is obtained.
Preferably, in steps A) to C), NaF, P123, cyanoethyltriethoxysilane, tetraethoxysilane, NaAlO are added to the solution2The mass ratio of HCl is 1 (170-) (180): (40-45): (500-) (550): (20-50): 2600-) (2800). The copper ion loaded mesoporous modified Al-SBA-15 prepared by adopting the proportion has larger pores, moderate silicon-aluminum ratio, easy loading of divalent copper ions and strong adsorbability on carnosine and anserine.
Preferably, the extract in step (1) is one of distilled water, 0.1mol/L NaOH solution and 0.6mol/L hydrochloric acid.
Preferably, the mass of the sample added to the extract liquid in the step (1) is (0.8-1.2g)/10 mL.
Preferably, the heating extraction temperature in the step (1) is 75-85 ℃, and the extraction time is 10-20 min.
The extraction rate of carnosine and anserine is high under appropriate reaction conditions by adopting the extracting solution and the using amount of the extracting solution, and the subsequent analysis and determination are facilitated
Preferably, the temperature of the freezing and centrifuging in the step (2) is 0-4 ℃, the time is 10-20min, and the aperture of the microporous filter membrane is 0.45 μm. The method can effectively retain the carnosine and the anserine in the sample, remove other impurities and facilitate the subsequent sample injection analysis of high performance liquid chromatography.
Preferably, in the step (3), 3.8 to 4g of the mesoporous modified Al-SBA-15 filler supporting copper ions is loaded into a stainless steel column tube of 250mm × 4.6mm by using a homogenization method to obtain the chromatographic column, wherein a solution in the homogenization method is n-hexane-isopropanol (V: V ═ 90:10), and a loading pressure is 40 to 50 MPa. The chromatographic column stationary phase filled by the method has moderate dosage and good adsorption performance on carnosine and anserine.
Preferably, the high performance liquid chromatography in the step (3) is carried out by taking 20mmol/L phosphate buffer solution with the pH of 8-9 and methanol as mobile phases and carrying out elution with equal gradient, wherein the mass fraction of the methanol is 1-5%. The eluent can effectively elute the carnosine and the anserine, and is beneficial to the determination of the carnosine and the anserine.
Preferably, in the step (3), the measurement is carried out by using an ultraviolet detector at a wavelength of 230nm, a flow rate of 1.0mL/min and a column temperature of 35 ℃. When the parameters are adopted to measure the carnosine and the anserine, the deviation of peak area and retention time is small, and the method has high accuracy.
Therefore, the invention has the following beneficial effects: the mesoporous modified Al-SBA-15 loaded with copper ions is used as a filler in a chromatographic column during high performance liquid chromatography, so that the adsorption and separation effects on carnosine and anserine are good, the detection accuracy and sensitivity are high, the operation is simple and convenient, a complex pretreatment step is not needed, the peak area and retention time deviation are small when the device is used for detecting on a machine only through simple pretreatment, and the accuracy is high.
Drawings
FIG. 1 is a chromatogram of a standard sample (250 mg/L);
FIG. 2 is a chromatogram of a sample of the fish head of example 1.
Detailed Description
The invention is further described with reference to the following detailed description and accompanying drawings.
Example 1:
preparation of the filler: dissolving P123 and NaF in 1.5mol/L hydrochloric acid solution, and stirring at 40 ℃ until the solutions are completely dissolved; dropwise adding cyanoethyltriethoxysilane into the solution, stirring for 25min, dropwise adding tetraethoxysilane, and stirring at 40 deg.C for 7 hr; then adding 1.5mol/L NaAlO2Hydrochloric acid solution, stirring at 40 deg.C for 24h, in which NaF, P123, cyanoethyltriethoxysilane, tetraethoxysilane, NaAlO2The mass ratio of HCl is 1:175:42:520:30: 2700; adjusting the pH value of the solution to 1.65 by using ammonia water, transferring the solution into a Teflon lining reaction kettle, and putting the reaction kettle in a drying oven at 150 ℃ for activation for 40 hours; drying the reaction kettleTaking out the solution from the box, adjusting the pH of the solution to 6 by using ammonia water after the solution is cooled to room temperature, and then putting the solution in an oven at 150 ℃ for activation for 40 hours; after filtration, washing the product with distilled water and absolute ethyl alcohol, and drying at 80 ℃ for 10h to obtain cyanated Al-SBA-15; adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 45%, stirring and reacting for 30 hours at 95 ℃, filtering, washing, and drying for 10 hours at 80 ℃ to obtain the carboxylated Al-SBA-15; dispersing the carboxylated Al-SBA-15 in 0.15mol/L CuSO4And stirring the solution for reaction for 24 hours, carrying out suction filtration, washing the obtained product, drying the product at 60 ℃ for 9 hours, and roasting the product in a muffle furnace at 550 ℃ for 4.5 hours to obtain the copper ion loaded mesoporous modified Al-SBA-15 filler.
Filling a chromatographic column: 3.9g of the prepared mesoporous modified Al-SBA-15 filler loading copper ions is filled into a stainless steel column tube with the thickness of 250mm multiplied by 4.6mm by adopting a homogenization method to obtain a chromatographic column, the solution in the homogenization method is n-hexane-isopropanol (V: V ═ 90:10), and the loading pressure is 45 MPa.
Sample pretreatment: taking soft tissue of fish head, mashing with a high-speed tissue mashing machine, and storing in a refrigerator at-20 deg.C; weighing 1.0G of the above sample in a 50mL centrifuge tube, adding 10mL of distilled water extract, shaking for 1min, extracting in 80 deg.C water bath for 15min, cooling to room temperature, shaking for 1min, freezing and centrifuging at 4 deg.C and 10000G for 15min, and filtering the supernatant with 0.45 water phase microporous membrane.
Determination of carnosine and anserine: a sample size of 20. mu.L, at 20mmol/L phosphate buffer salt (potassium dihydrogen phosphate and disodium hydrogen phosphate configuration) at pH 9: methanol 99:1 as mobile phase, and gradient elution; detecting with ultraviolet detector at wavelength of 230 nm; the flow rate is 1.0 mL/min; the column temperature was 35 ℃. Carnosine and anserine can be separated from other components to achieve baseline separation.
Example 2:
preparation of the filler: dissolving P123 and NaF in 2mol/L hydrochloric acid solution, and stirring at 45 ℃ until the solutions are completely dissolved; dropwise adding cyanoethyltriethoxysilane into the solution, stirring for 30min, dropwise adding tetraethoxysilane, and stirring at 45 deg.C for 6 hr; then adding 2mol/L NaAlO2Hydrochloric acid solution, stirring for 20h at 45 ℃, and adding NaF, P123 and cyanoethyl triethoxy in the solutionSilane, tetraethoxysilane, NaAlO2The mass ratio of HCl is 1:180:45:550:50: 2800; adjusting the pH value of the solution to 1.7 by using ammonia water, transferring the solution into a Teflon lining reaction kettle, and putting the reaction kettle in a 200 ℃ oven to activate for 36 hours; taking the reaction kettle out of the oven, adjusting the pH of the solution to 6.5 by using ammonia water after the temperature of the reaction kettle is reduced to room temperature, and then putting the reaction kettle in the oven at 200 ℃ for activation for 36 hours; after filtering, washing the product with distilled water and absolute ethyl alcohol, and drying at 100 ℃ for 8h to obtain cyanated Al-SBA-15; adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 47%, stirring and reacting for 24 hours at 100 ℃, filtering, washing, and drying for 8 hours at 90 ℃ to obtain the carboxylated Al-SBA-15; dispersing carboxylated Al-SBA-15 in 0.2mol/L CuSO4And stirring the solution for reaction for 20 hours, carrying out suction filtration, washing the obtained product, drying the product at 70 ℃ for 8 hours, and roasting the product in a muffle furnace at 600 ℃ for 4 hours to obtain the copper ion loaded mesoporous modified Al-SBA-15 filler.
Filling a chromatographic column: 4g of the prepared mesoporous modified Al-SBA-15 filler loading copper ions is filled into a stainless steel column tube with the thickness of 250mm multiplied by 4.6mm by adopting a homogenization method to obtain a chromatographic column, the solution in the homogenization method is n-hexane-isopropanol (V: V is 90:10), and the loading pressure is 50 MPa.
Sample pretreatment: taking soft tissue of fish head, mashing with a high-speed tissue mashing machine, and storing in a refrigerator at-20 deg.C; weighing 1.2G of the above sample in a 50mL centrifuge tube, adding 10mL of distilled water extract, shaking for 1min, extracting in 85 deg.C water bath for 20min, cooling to room temperature, shaking for 1min, freezing and centrifuging at 2 deg.C and 10000G for 20min, and filtering the supernatant with 0.45 water phase microporous membrane.
Determination of carnosine and anserine: a sample size of 20. mu.L, at 20mmol/L phosphate buffer salt (potassium dihydrogen phosphate and disodium hydrogen phosphate configuration) at pH 9: methanol is 95:5 as a mobile phase, and the gradient elution is carried out; detecting with ultraviolet detector at wavelength of 230 nm; the flow rate is 1.0 mL/min; the column temperature was 35 ℃. Carnosine and anserine can be separated from other components to achieve the purpose of baseline separation.
Example 3:
preparation of the filler: dissolving P123 and NaF in 1mol/L hydrochloric acid solution, and stirring at 35 ℃ until the solutions are completely dissolved; into the above solutionDropwise adding cyanoethyltriethoxysilane, stirring for 20min, dropwise adding ethyl orthosilicate, and stirring at 35 deg.C for 8 hr; then adding 1mol/L NaAlO2Hydrochloric acid solution, stirring for 30h at 35 ℃, and adding NaF, P123, cyanoethyl triethoxysilane, tetraethoxysilane and NaAlO in the solution2The mass ratio of HCl is 1:170:40:500:20: 2600; adjusting the pH value of the solution to 1.6 by using ammonia water, transferring the solution into a Teflon lining reaction kettle, and putting the reaction kettle in a drying oven at 100 ℃ for activation for 48 hours; taking the reaction kettle out of the oven, adjusting the pH of the solution to 5.5 by using ammonia water after the temperature of the reaction kettle is reduced to room temperature, and then putting the solution in the oven with the temperature of 100 ℃ for activation for 48 hours; after filtration, washing the product with distilled water and absolute ethyl alcohol, and drying at 60 ℃ for 12h to obtain cyanated Al-SBA-15; adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 44%, stirring and reacting for 36 hours at 90 ℃, filtering, washing, and drying for 12 hours at 70 ℃ to obtain the carboxylated Al-SBA-15; dispersing carboxylated Al-SBA-15 in 0.1mol/L CuSO4And stirring the solution for reaction for 30h, then carrying out suction filtration, washing the obtained product, drying the washed product at 50 ℃ for 10h, and roasting the dried product in a muffle furnace at 500 ℃ for 5h to obtain the copper ion loaded mesoporous modified Al-SBA-15 filler.
Filling a chromatographic column: 3.8g of the prepared mesoporous modified Al-SBA-15 filler loading copper ions is filled into a stainless steel column tube with the thickness of 250mm multiplied by 4.6mm by adopting a homogenization method to obtain a chromatographic column, the solution in the homogenization method is n-hexane-isopropanol (V: V ═ 90:10), and the loading pressure is 40 MPa.
Sample pretreatment: taking soft tissue of fish head, mashing with a high-speed tissue mashing machine, and storing in a refrigerator at-20 deg.C; weighing 0.8G of the sample, putting the sample into a 50mL centrifuge tube, adding 10mL of distilled water extract, shaking the rotary nest for 1min, extracting in 75 ℃ water bath for 10min, cooling to room temperature, shaking the rotary nest for 1min, freezing and centrifuging at 0 ℃ and 10000G for 10min, and taking supernatant and filtering with a 0.45 aqueous phase microporous membrane.
Determination of carnosine and anserine: a sample size of 20. mu.L, at 20mmol/L phosphate buffer salt (potassium dihydrogen phosphate and disodium hydrogen phosphate configuration) at pH 8: methanol is 95:5 as a mobile phase, and the gradient elution is carried out; detecting with ultraviolet detector at wavelength of 230 nm; the flow rate is 1.0 mL/min; the column temperature was 35 ℃. Carnosine and anserine can be separated from other components to achieve the purpose of baseline separation.
Comparative example 1:
comparative example 1 differs from example 1 in that the packing for the column in comparative example 1 uses unmodified SBA-15 and is prepared by: dissolving P123 and NaF in 1.5mol/L hydrochloric acid solution, and stirring at 40 ℃ until the solutions are completely dissolved; dropwise adding tetraethoxysilane into the solution, and stirring for 7 hours at the temperature of 40 ℃, wherein the mass ratio of NaF, P123, tetraethoxysilane and HCl in the solution is 1:175:520: 2700; transferring into a Teflon lining reaction kettle, and placing the reaction kettle in a drying oven at 150 ℃ for activation for 40 h; and after filtering, washing the product with distilled water and absolute ethyl alcohol, drying at 80 ℃ for 10h, and roasting at 550 ℃ in a muffle furnace for 4.5h to obtain the SBA-15 filler. The rest is the same as in example 1.
During determination, carnosine and anserine cannot be separated from other components to achieve the aim of baseline separation.
Comparative example 2:
comparative example 2 differs from example 1 in that the packing for the column in comparative example 2 uses carboxylated Al-SBA-15 not loaded with copper ions, and is prepared by: dissolving P123 and NaF in 1.5mol/L hydrochloric acid solution, and stirring at 40 ℃ until the solutions are completely dissolved; dropwise adding cyanoethyltriethoxysilane into the solution, stirring for 25min, dropwise adding tetraethoxysilane, and stirring at 40 deg.C for 7 hr; then adding 1.5mol/L NaAlO2Hydrochloric acid solution, stirring at 40 deg.C for 24h, in which NaF, P123, cyanoethyltriethoxysilane, tetraethoxysilane, NaAlO2The mass ratio of HCl is 1:175:42:520:30: 2700; adjusting the pH value of the solution to 1.65 by using ammonia water, transferring the solution into a Teflon lining reaction kettle, and putting the reaction kettle in a drying oven at 150 ℃ for activation for 40 hours; taking the reaction kettle out of the oven, adjusting the pH of the solution to 6 by using ammonia water after the temperature of the reaction kettle is reduced to room temperature, and then putting the reaction kettle in the oven at 150 ℃ for activation for 40 hours; after filtration, washing the product with distilled water and absolute ethyl alcohol, and drying at 80 ℃ for 10h to obtain cyanated Al-SBA-15; adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 45%, stirring and reacting for 30 hours at 95 ℃, filtering, washing, and drying for 10 hours at 80 ℃ to obtain the carboxylated Al-SBA-15; washing the carboxylated Al-SBA-15, drying at 60 ℃ for 9h, and then putting the dried product in a muffle furnace at 550 DEG CThen roasting for 4.5h to obtain the carboxylated Al-SBA-15 filler. The rest is the same as in example 1.
During determination, carnosine and anserine cannot be completely separated from other components to achieve the aim of baseline separation.
Linear range measurement of the method:
respectively and accurately weighing 50mg of carnosine and anserine standard products, dissolving with ultrapure water, and diluting to a constant volume of 50mL volumetric flask to prepare 1000mg/L of carnosine and anserine standard stock solutions. The standard stock solution was diluted to a mixed standard solution of carnosine and anserine at a mass concentration of 1.0, 5.0, 10.0, 20.0, 100.0, 200.0, 500.0mg/L, and the mixture was subjected to measurement under the chromatographic conditions described in example 1, and a linear regression was performed using the standard solution concentration as abscissa and the peak area as ordinate, and the linear regression equation, correlation coefficient, and linear range thereof are shown in table 1. The measurement result shows that the mass concentration of carnosine and anserine has a good linear relation with the peak area under the chromatographic condition.
Table 1: linear range of the method.
| Composition (I) | Linear equation of equations | Coefficient of correlation (R)2) | Linear Range (mg/L) |
| Carnosine | y＝1303.9x+1838.2 | 0.9999 | 5.0-500.0 |
| Anserine | y＝3002.9x-1190.7 | 1 | 1.0-500.0 |
Selection of high performance liquid chromatography mobile phase:
the sample was injected at 250mg/L, the pH of the mobile phase and the ratio of methanol in the mobile phase were varied, and the degree of separation of the sample was measured under the same chromatographic conditions as in example 1, and the results are shown in Table 2. The results show that the separation effect gradually becomes better with the increase of the pH and the decrease of the methanol ratio, and the sample separation effect is the best when pure water phase is used. But in order to reduce the loss of the chromatographic column and enhance the service life of the chromatographic column, the invention finally selects 20mmol/L phosphate buffer solution with the pH of 8-9 and methanol as mobile phases, wherein the mass fraction of the methanol is 1-5%.
Table 2: degree of separation of standards under different mobile phase conditions.
| Mobile phase | pH 75% methanol | pH 85% methanol | pH 95% methanol | pH 9 pure water phase | pH 91% methanol |
| Degree of separation (R) | 1.88 | 3.01 | 3.33 | 5.57 | 4.67 |
Precision measurement of the method:
and continuously injecting sample 5 times within the same day by using the same concentration of carnosine and anserine standard solution to determine the precision. The retention time and relative standard deviation of the peak area were calculated. Standard deviations of retention time from peak area for carnosine standards were 0.114% and 0.618%; the standard deviation of retention time and peak area of anserine standard was 0.0426% and 0.514%. The retention time of carnosine and anserine and the RSD of peak area are both less than 1%, which shows that the method has good precision.
Calculation of detection limit and quantification limit:
according TO the detection limit and quantification limit calculation formula given in the integral TO modified LIQUID requirement book:
detection limit:
and (4) quantitative limit:
where σ represents the standard error of the standard curve after linear regression and S' represents the slope of the standard curve.
Calculating the detection limit of carnosine of the invention to be 3.12mg/L and the quantification limit to be 9.46mg/L according to a formula; the detection limit of anserine is 1.07mg/L, and the quantification limit is 3.24 mg/L.
Determination of recovery:
the results of measuring the recovery rates of carnosine and anserine using the chromatographic conditions in example 1 are shown in table 3, which indicates that the recovery rates of carnosine and anserine in the present invention are high.
Table 3: and (5) determining the recovery rate.
| Adding quantity (mg) | Carnosine recovery (%) | Recovery rate of anserine (%) |
| 0 | / | / |
| 1.0 | 101 | 101 |
| 1.5 | 102 | 103 |
| 2.0 | 114 | 115 |
Claims (10)
1. A high performance liquid chromatography for measuring the content of carnosine and anserine in fish heads is characterized by comprising the following steps:
(1) taking soft tissue of the head of the fish, mashing the soft tissue, adding an extracting solution into the crushed soft tissue as a sample, and heating and extracting the sample to obtain a crude extracting solution;
(2) cooling the crude extractive solution to room temperature, freezing, centrifuging, and filtering the supernatant with microporous membrane;
(3) measuring the content of carnosine and anserine in the filtrate by using high performance liquid chromatography, wherein mesoporous modified Al-SBA-15 loaded with copper ions is used as a filler in a chromatographic column of the high performance liquid chromatography; and during the high performance liquid chromatography detection, an ultraviolet detector is used for detection.
2. The high performance liquid chromatography for measuring the carnosine and anserine contents in fish heads according to claim 1, wherein the preparation method of the copper ion-loaded mesoporous modified Al-SBA-15 in the step (3) is as follows:
A) dissolving P123 and NaF in 1-2mol/L hydrochloric acid solution, and stirring at 35-45 ℃ until the solutions are completely dissolved;
B) dropwise adding cyanoethyltriethoxysilane into the solution, stirring for 20-30min, dropwise adding ethyl orthosilicate, and stirring at 35-45 deg.C for 6-8 h;
C) adding 1-2mol/L NaAlO into the solution2Hydrochloric acid solution, stirring for 20-30h at 35-45 ℃;
D) adjusting the pH value of the solution to 1.6-1.7 by ammonia water, transferring the solution into a Teflon lining reaction kettle, and activating the reaction kettle in an oven at the temperature of 100-;
E) taking the reaction kettle out of the oven, cooling to room temperature, adjusting the pH value of the solution to 5.5-6.5 with ammonia water, and then placing the solution in an oven at the temperature of 100-;
F) after filtering, washing the product with distilled water and absolute ethyl alcohol, and drying at 60-100 ℃ for 8-12h to obtain cyanated Al-SBA-15;
G) adding the cyanized Al-SBA-15 into a sulfuric acid solution with the mass fraction of 44% -47%, stirring and reacting for 24-36h at 90-100 ℃, filtering, washing, and drying for 8-12h at 70-90 ℃ to obtain the carboxylated Al-SBA-15;
H) dispersing the carboxylated Al-SBA-15 in 0.1-0.2mol/L CuSO4Stirring the solution for reaction for 20-30h, then carrying out suction filtration, washing the obtained product, and drying the product at 50-70 ℃ for 8-10 h;
I) and roasting the obtained product in a muffle furnace at 500-600 ℃ for 4-5h to obtain the copper ion loaded mesoporous modified Al-SBA-15.
3. The method of claim 2The high performance liquid chromatography for determining the content of carnosine and anserine in the fish head is characterized in that in the steps A) to C), NaF, P123, cyanoethyltriethoxysilane, tetraethoxysilane and NaAlO are contained in the solution2The mass ratio of HCl is 1 (170-) (180): (40-45): (500-) (550): (20-50): 2600-) (2800).
4. The high performance liquid chromatography for determining the content of carnosine and anserine in fish heads according to claim 1, wherein the extract in step (1) is one of distilled water, 0.1mol/L NaOH solution and 0.6mol/L hydrochloric acid.
5. The high performance liquid chromatography for measuring the carnosine and anserine contents in fish heads according to claim 1 or 4, wherein the mass of the sample added to the extract in the step (1) is (0.8 g-1.2 g)/10 mL.
6. The high performance liquid chromatography for determining the carnosine and anserine contents in fish heads according to claim 1, wherein the heating extraction temperature in the step (1) is 75-85 ℃, and the extraction time is 10-20 min.
7. The high performance liquid chromatography for determining the carnosine and anserine contents in fish heads as claimed in claim 1, wherein the temperature in the step (2) of refrigerated centrifugation is 0-4 ℃, the time is 10-20min, and the pore diameter of the microporous filter membrane is 0.45 μm.
8. The high performance liquid chromatography for measuring the carnosine and anserine contents in the fish heads as claimed in claim 1 or 2, wherein 3.8-4g of mesoporous modified Al-SBA-15 filler loaded with copper ions is loaded into a stainless steel column tube of 250mm x 4.6mm by using a homogenization method in step (3) to obtain the chromatographic column, the solution in the homogenization method is n-hexane-isopropanol with a volume ratio of 90:10, and the loading pressure is 40-50 MPa.
9. The HPLC method according to claim 1 or 2, wherein the HPLC method in step (3) comprises eluting with a constant gradient of 20mmol/L phosphate buffer solution (pH 8-9) and methanol (mass fraction of methanol is 1-5%), as mobile phase.
10. The HPLC method according to claim 1 or 2, wherein the wavelength of the UV detector used in step (3) is 230nm, the flow rate is 1.0mL/min, and the column temperature is 35 ℃.
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| CN111220733A (en) * | 2020-02-24 | 2020-06-02 | 江苏大学 | Method for determining L-carnosine by efficient capillary electrophoresis and application of method to quality evaluation of polaprezinc |
| CN112592315B (en) * | 2020-12-11 | 2021-11-23 | 山东瑞芝生物科技股份有限公司 | Method for preparing high-content tuna anserine by membrane separation and application thereof |
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