CN115902083A - HPLC analysis method suitable for L-carnosine - Google Patents
HPLC analysis method suitable for L-carnosine Download PDFInfo
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- CQOVPNPJLQNMDC-ZETCQYMHSA-N carnosine Chemical compound [NH3+]CCC(=O)N[C@H](C([O-])=O)CC1=CNC=N1 CQOVPNPJLQNMDC-ZETCQYMHSA-N 0.000 title claims abstract description 112
- CQOVPNPJLQNMDC-UHFFFAOYSA-N N-beta-alanyl-L-histidine Natural products NCCC(=O)NC(C(O)=O)CC1=CN=CN1 CQOVPNPJLQNMDC-UHFFFAOYSA-N 0.000 title claims abstract description 111
- 108010087806 Carnosine Proteins 0.000 title claims abstract description 110
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000004458 analytical method Methods 0.000 claims abstract description 26
- 238000004140 cleaning Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000010829 isocratic elution Methods 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 claims description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 239000012071 phase Substances 0.000 abstract description 39
- 230000000694 effects Effects 0.000 abstract description 7
- 239000007791 liquid phase Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 229940044199 carnosine Drugs 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- QRYRORQUOLYVBU-VBKZILBWSA-N Carnosic acid Natural products CC([C@@H]1CC2)(C)CCC[C@]1(C(O)=O)C1=C2C=C(C(C)C)C(O)=C1O QRYRORQUOLYVBU-VBKZILBWSA-N 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical compound CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 238000004801 process automation Methods 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940000635 beta-alanine Drugs 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
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Abstract
The invention relates to an HPLC analysis method suitable for L-carnosine, which is characterized in that: the first step is as follows: preparing a solution of an L-carnosine sample to be detected; the second step is that: setting liquid phase chromatographic conditions according to the prepared L-carnosine sample to be detected; the third step: preparing a mobile phase for analysis; the fourth step: determining the L-carnosine sample to be detected by adopting an analysis program; the fifth step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min; the HPLC analysis method suitable for the L-carnosine is simple to operate, saves a large amount of time cost and economic cost when continuously measuring the L-carnosine, greatly simplifies the complexity of operation, and has good analysis effect.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a High Performance Liquid Chromatography (HPLC) analysis method suitable for L-carnosine.
Background
L-carnosine, known as beta-alanyl-L-histidine, is a dipeptide consisting of two amino acids, beta-alanine and L-histidine. The multi-country research shows that: l-carnosine has strong antioxidant activity and can scavenge Reactive Oxygen Species (ROS). Therefore, the cosmetic has wide application in anti-aging, anti-oxidation and whitening cosmetics.
In the prior art, many reports have been made on an HPLC analysis method of L-carnosine, and the L-carnosine is measured by adopting an amino column by using high performance liquid chromatography (Korea, chengxiaohui, the lattice, the crow shadow, the graduation. China pharmaceutical impurities 2009, 44 (14): 1111-1113.) in the content of L-carnosine bulk drugs and related substances thereof by using the HPLC analysis method, and the linear concentration range of the L-carnosine analyzed by the method is 9.93-99.3 mg/L, and the primary analysis is 10min. It is reported that the theoretical plate number of L-carnosine analyzed by this method is not less than 3000.
L-carnosine is measured by adopting a C18 column in Dailyao and the like (Dailyao, zhuxiana, chenyuyan, zhang Yifei, guojiao, chenmeilan, reversed phase high performance liquid chromatography, ion exchange chromatography for measuring carnosine content in beef, drug analysis impurities 2019, 39 (5): 805-812), acetonitrile-water is used as a mobile phase in the method, and the linear range of the measured carnosine is 0.2-5 mu g/mL.
Suixin (Shenyang pharmaceutical university Master's graduation paper "L-carnosine structure confirmation and in vivo and in vitro pharmacokinetics research" reported the HPLC method for measuring L-carnosine, and this paper adopts NH 2 The column used as an analytical column used was acetonitrile-phosphate buffer (32 mmol. Multidot.L) -1 Potassium dihydrogen phosphate, 8 mmol. Multidot.L -1 Dipotassium hydrogen phosphate) as a mobile phase to the content of L-carnosine in the artificial gastric juice, which can be analyzed by this methodThe linear range of carnosine is 10-120 mug/mL. Meanwhile, the HPLC method for measuring the content of L-carnosine in rat intestinal perfusate is also reported. Also using NH 2 Column using acetonitrile-phosphate buffer (1 mmol. L) -1 Potassium dihydrogen phosphate, 0.2 mmol. Multidot.L -1 Dipotassium phosphate) as a mobile phase, and the linear range of the L-carnosine analyzable by the method is 22.6-158 mu g/mL.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems in the prior art, the invention aims to solve the relevant problem that the conventional mobile phase (methanol-water, acetonitrile-water) cannot reasonably elute the L-carnosine.
Still another object of the present invention is to solve the technical problem of low concentration range of L-carnosine samples in HPLC analysis in the prior art.
The invention further aims to reasonably use the C18 chromatographic column on the premise of ensuring the stability of the analysis method of the L-carnosine, so that the column efficiency is not influenced by certain damage caused by improper mobile phase.
Means for solving the problems
The present invention relates to:
1. an HPLC analysis method suitable for L-carnosine, which is characterized in that:
the first step is as follows: setting liquid phase chromatographic conditions;
the second step: preparing a mobile phase for analysis, wherein; mobile phase A was 20mM KH 2 PO 4 Aqueous solution (pH = 4.7); the mobile phase B is methanol;
the third step: the analytical procedure was set to isocratic elution, elution concentration: mobile phase A: 85-95%, mobile phase B: 5-15%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more L-carnosine samples to be tested, wherein the L-carnosine samples can be single L-carnosine or a mixture containing the L-carnosine;
the fifth step: determining the solution of one L-carnosine sample to be detected by adopting the analysis program, and recording a chromatogram;
and a sixth step: optionally calculating the content of L-carnosine from a standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
eighth step: and repeating the fifth step and the seventh step, and sequentially determining other undetected L-carnosine samples to be detected.
2. The HPLC analysis method for L-carnosine according to item 1, wherein: the standard curve is established by preparing three or more standard solutions of L-carnosine as required and performing the measurement using the liquid chromatography conditions in the first step.
3. The HPLC analysis method for L-carnosine according to item 1 or 2, wherein: the detection wavelength range is 200-300 nm.
4. The HPLC analysis method for L-carnosine according to item 1 or 2, wherein the column is packed with octadecyl bonding phase and has a size of 5 μm, 250X 4.6mm, and the flow rate is 1mL/min.
5. The HPLC analysis method for L-carnosine according to item 1 or 2, wherein: the temperature of the chromatographic column is room temperature, the sample injection amount is 5-10 mu L, the temperature of the sample injector is the environmental temperature, and the collection time of the sample to be detected is 4-6 min.
6. The HPLC analytical method for L-carnosine according to item 1 or 2, which is characterized by: the concentration of the L-carnosine sample to be detected is 0.4-1.3 g/L.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention discovers a novel HPLC analysis method suitable for L-carnosine, which can realize continuous analysis and determination of the L-carnosine, and the peak is normally distributed, and the theoretical plate number is considerable; secondly, the HPLC analysis method applicable to the L-carnosine can realize the analysis and purity calculation of L-carnosine samples in a higher concentration range. And the method is used for analyzing the L-carnosine with different concentrations, so that good linearity can be obtained.
The HPLC analysis method suitable for the L-carnosine is simple to operate, the primary analysis time is shortened to 5 minutes while the L-carnosine is continuously analyzed and determined, the detectable concentration is greatly improved while the continuous analysis is realized, the linear range is greatly widened, and feasible basis is provided for the detection of the L-carnosine in production. Meanwhile, the chromatographic columns for analyzing the L-carnosine and other peptides are unified, so that the whole-process automation is facilitated, and a large amount of time cost and labor cost can be saved.
Drawings
FIG. 1 is an HPLC chart of L-carnosine in example 1.
FIG. 2 is a linear plot of peak area versus concentration for different concentrations (0.4, 0.5, 0.6, 0.8, 1) of g/L-carnosine samples from example 1.
FIG. 3 is an HPLC chart of L-carnosine in example 2.
FIG. 4 is an HPLC chart of L-carnosine in comparative example 1.
FIG. 5 is an HPLC chart of L-carnosine in comparative example 2.
FIG. 6 is an HPLC chart of L-carnosine in comparative example 3.
Detailed Description
The structure of the L-carnosine is special, and in order to obtain a standard chromatographic peak shape and obtain an accurate peak area when the purity of the L-carnosine is analyzed, an amino column is mostly adopted as a chromatographic column in the prior art, however, the adoption of the amino column not only increases the cost of the column per se in actual production, but also needs to replace the chromatographic column when other products are continuously produced, so that the labor cost and the time cost are increased. Through intensive research, the invention discovers an HPLC analysis method suitable for L-carnosine, which can obtain an excellent chromatographic peak shape, completely separate a target peak from other peaks, has narrow half-peak width, sharp peak shape, stable base line and higher theoretical plate number and is particularly suitable for the L-carnosine as follows:
the HPLC analysis method suitable for the L-carnosine comprises the following steps:
the first step is as follows: setting liquid chromatography conditions;
the second step is that: preparing a mobile phase for analysis, wherein; the mobile phase A is 20mM KH 2 PO 4 Aqueous solution (pH = 4.7); the mobile phase B is methanol;
the third step: the analytical procedure was set to isocratic elution, elution concentration: mobile phase A: 85-95%, mobile phase B: 5-15%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more L-carnosine samples to be tested, wherein the L-carnosine samples can be single L-carnosine or a mixture containing the L-carnosine;
the fifth step: determining the solution of one L-carnosine sample to be detected by adopting the analysis program, and recording a chromatogram;
and a sixth step: optionally calculating the content of L-carnosine from a standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
eighth step: and repeating the fifth step and the seventh step, and sequentially determining other undetected L-carnosine samples to be detected.
The above standard curve is established by preparing three or more, preferably five or more, standard solutions of L-carnosine at different concentrations as necessary and performing the measurement using the liquid chromatography conditions in the above first step.
The mobile phase A is 20mM KH 2 PO 4 An aqueous solution (pH 4.7); the mobile phase B is methanol. The analytical procedure was isocratic elution, elution concentration: mobile phase A: 85-95%, mobile phase B: 5-15%, selecting mobile phase A:90%, mobile phase B:10 percent. The range is selected to obtain the best analysis results. The flow rate of the mobile phase is 0.8 to 1.2mL/min, preferably 1mL/min. The flow rate selection is very critical, the analysis period can be shortened by increasing the flow rate, the time is saved, however, if the flow rate is not properly selected, the flow rate is too high, two peaks are possibly separated very closely or even overlapped, integration cannot be carried out, the flow rate is too low, retention loss of a small part of components is possibly caused, and the analysis precision is influenced. In order to enable the peak of the target peptide to be completely displayed, the concentration of the peptide is not high enough, otherwise, the target peak is capped, and therefore, even if a mixed peak appears at the peak top, the judgment cannot be carried out; but the concentration cannot be too low, otherwise even if there is a stray peak, the peak height is too low and the baseline is misjudged to be uneven. The concentrations of the L-carnosine samples to be tested were therefore: 0.4 to 1.3g/L, preferably 0.4 to 1.0g/L.
The detection wavelength is determined by the nature of the peptide to be detected and is not influenced by the analysis conditions. However, the wavelength range is preferably 200 to 300nm, more preferably 210 to 230nm, still more preferably 260 to 280nm, and particularly preferably 210nm. The reason is that: the L-carnosine solution was scanned at full wavelength and showed that L-carnosine had the strongest light absorption at 210nm, so 210nm was most preferred as the detection wavelength for HPLC analysis.
The column is a conventionally used column, preferably one packed with an octadecyl bonded phase, such as, for example, an Ulipret Sinochrom ODS-AP
The column temperature of the chromatographic column is 25 ℃, and the sample injection amount is 5-10 mu L, preferably 5 mu L.
The temperature of the sample injector is the ambient temperature, and the collection time of the sample to be detected is 4-6 min, preferably 6min.
The technical solution of the present invention is further described below by means of specific examples.
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. It should be understood, however, that these examples are illustrative only and are not intended to limit the present invention. Unless otherwise specified, the raw materials used in the examples of the present invention are all those commonly used in the art, and the methods used in the examples are all those conventional in the art.
Examples
Example 1.
Instruments and conditions:
an Agilent1260InfinityII LC high performance liquid chromatograph and an OpenLabCDS2 software system are adopted; in an elett Sinochrom ODS-AP
The column (250X 4.6 mm) is an analytical column, and the column temperature is 25 ℃; the ultraviolet detection wavelength is 210nm.
The experimental steps are as follows:
five groups of samples were weighed out in amounts of 0.004g, 0.005g, 0.006g, 0.008g, 0.01g of L-carnosine. Will be weighedThe L-carnosine samples are respectively dissolved in ultrapure water to 10mL, and L-carnosine solutions with the concentrations of 0.4g/L, 0.5g/L, 0.6g/L, 0.8g/L and 1.0g/L are obtained. Mobile phase: a:20mM KH 2 PO 4 An aqueous solution (pH 4.7); b: methanol.
Flow rate: 1.0mL/min.
The temperature is 25 ℃.
Sample introduction amount: 5 μ L.
And (3) analysis program: isocratic elution; elution concentration: a mobile phase A:90%, mobile phase B:10 percent.
And continuously carrying out high performance liquid chromatography analysis on the L-carnosine solution with each concentration according to the chromatographic conditions, recording a chromatogram, wherein the flow matching ratio can almost completely elute the L-carnosine without residues in the sample measurement process, and if slightly soluble or insoluble impurities cannot elute in the detection process, the method can be used for cleaning a chromatographic column by using high-proportion methanol.
The HPLC analysis results of the L-carnosine solutions with concentrations of 0.4g/L, 0.5g/L, 0.6g/L, 0.8g/L and 1.0g/L are shown in FIG. 1, and the linear standard curve is shown in FIG. 2. The retention time and the number of theoretical plates for each concentration of L-carnosine are shown in the following table.
| L-carnosine concentration (g/L) | Retention time(s) | Number of theoretical plate |
| 0.4 | 3.130 | 9705.10104 |
| 0.5 | 3.131 | 9625.38600 |
| 0.6 | 3.131 | 9464.66561 |
| 0.8 | 3.132 | 9207.31718 |
| 1 | 3.132 | 8789.40496 |
The concentration-peak area linear regression equation for L-carnosine is shown below:
y＝ax+b
wherein: y: peak area
Concentration of L-carnosine
a:10250.02617
b:89.37449
Example 2.
HPLC analysis of the L-carnosine sample solution was carried out in the same manner as in example 1 except that the concentration of the L-carnosine solution was changed to 0.7g/L, and as a result, as shown in FIG. 3, the retention time was 3.122min, the tailing factor was 1.15176, and the theoretical plate number was 9109.58263. And (3) verifying the linear relation between the peak area and the concentration of the sample, integrating the chromatographic peak of the L-carnosine, substituting the obtained peak area into a standard curve shown in figure 2, and calculating to obtain a concentration of 0.673g/L with an error of less than 5%.
Comparative example 1.
An L-carnosine sample was analyzed in the same manner as in example 1, except that methanol in the mobile phase was changed to acetonitrile, the buffered salt solution was changed to water, and the column was changed to an amino column, and as a result, as shown in FIG. 4, the retention time was 1.803min, the tail factor was 5.79302, and the theoretical plate number was 769.42173. It can be seen that the tailing of the chromatographic peak is serious, the half-peak width is large, and the chromatographic peak is connected with the solvent peak, presumably due to improper selection of analysis conditions, thereby reducing the analysis effect and affecting the accuracy of purity calculation.
Comparative example 2.
The HPLC profile of the L-carnosine sample was analyzed and determined in the same manner as in example 1, except that the buffer salt solution in the mobile phase was changed to water, and as a result, as shown in fig. 5, it can be seen that the chromatographic peak was changed to a double peak and the elution was not smooth enough, presumably due to improper selection of the mobile phase, thereby reducing the analysis effect.
Comparative example 3.
Except that the ratio of mobile phase was adjusted to mobile phase a:80%, mobile phase B:20% and the concentration of the L-carnosine solution was changed to 0.7g/L, and HPLC analysis was performed on a 0.7g/L L-carnosine sample in the same manner as in example 1, and the HPLC chart is shown in FIG. 6, in which the retention time was 3.086min, the tail factor was 1.20551, and the theoretical plate number was 8401.16. From the results it can be seen that: comparative example 3 also gave a good separation effect, but the theoretical plate number was lowered from the recommended mobile phase ratio, and therefore the mobile phase ratio recommended in the present invention was considered to be the optimum ratio.
The comparison results of the examples and the comparative examples show that the HPLC analysis method suitable for the L-carnosine can continuously analyze and determine the L-carnosine with different concentrations, and the chromatographic peak shape of the L-carnosine is clear. The technology can fully separate the L-carnosine from impurities with good effect, thereby being more beneficial to the realization of whole-process automation by utilizing the method.
In conclusion, the HPLC analysis method suitable for the L-carnosine is simple to operate, saves a large amount of time cost and economic cost when continuously analyzing and determining the L-carnosine, greatly simplifies the complexity of operation and has good analysis effect.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. An HPLC analysis method suitable for L-carnosine, which is characterized in that:
the first step is as follows: setting liquid chromatography conditions;
the second step: preparing a mobile phase for analysis, wherein; mobile phase A was 20mM KH 2 PO 4 Aqueous solution (pH = 4.7); the mobile phase B is methanol;
the third step: the analysis program was set to isocratic elution, elution concentration: mobile phase A: 85-95%, mobile phase B: 5-15%, flow rate: 1mL/min;
the fourth step: preparing a solution of one or more L-carnosine samples to be tested, wherein the L-carnosine samples can be single L-carnosine or a mixture containing the L-carnosine;
the fifth step: determining the solution of one L-carnosine sample to be detected by adopting the analysis program, and recording a chromatogram;
and a sixth step: optionally calculating the content of L-carnosine from a standard curve;
the seventh step: cleaning the chromatographic column by adopting a mobile phase B for 1-5 min;
eighth step: and repeating the fifth step and the seventh step, and sequentially determining other undetected L-carnosine samples to be detected.
2. An HPLC analysis method suitable for L-carnosine according to claim 1, characterized in that: the standard curve is established by preparing three or more standard solutions of L-carnosine as required and performing the measurement using the liquid chromatography conditions in the first step.
3. An HPLC analysis method suitable for L-carnosine according to claim 1, characterized in that: the wavelength range is 200-300 nm.
4. An HPLC analysis method suitable for L-carnosine according to claim 1 or 2, characterized in that: the column was packed with octadecyl bonded phase and was 5 μm,250 × 4.6mm, and the flow rate was 1mL/min.
5. An HPLC analysis method suitable for L-carnosine according to claim 1 or 2, characterized in that: the temperature of the chromatographic column is room temperature, the sample injection amount is 5-10 mu L, the temperature of the sample injector is the environmental temperature, and the collection time of the sample to be detected is 4-6 min.
6. An HPLC analysis method suitable for L-carnosine according to claim 1 or 2, characterized in that: the concentration of the L-carnosine sample is 0.4-1.3 g/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018121583A (en) * | 2017-02-01 | 2018-08-09 | 三谷産業株式会社 | Method for producing carnosine and novel microorganism |
| 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 |
| CN111606856A (en) * | 2020-06-08 | 2020-09-01 | 浙江大学衢州研究院 | Method for separating carnosine and histidine |
| CN111679028A (en) * | 2020-07-24 | 2020-09-18 | 上海市食品药品检验所 | High performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics |
-
2022
- 2022-12-26 CN CN202211682423.7A patent/CN115902083A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018121583A (en) * | 2017-02-01 | 2018-08-09 | 三谷産業株式会社 | Method for producing carnosine and novel microorganism |
| 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 |
| CN111606856A (en) * | 2020-06-08 | 2020-09-01 | 浙江大学衢州研究院 | Method for separating carnosine and histidine |
| CN111679028A (en) * | 2020-07-24 | 2020-09-18 | 上海市食品药品检验所 | High performance liquid chromatography tandem mass spectrometry detection method for four peptides in cosmetics |
Non-Patent Citations (3)
| Title |
|---|
| SHENGYING ZHOU 等: "Identification of Hydrazine in Commercial Preparations of Carnosine and Its Influence on Carnosine’s Antioxidative Properties", ANALYTICAL BIOCHEMISTRY, vol. 261, pages 79 - 86 * |
| 张梅秀 等: "薄层色谱和高效液相色谱联用测定暗纹东方肌肉中肌肽和谷胱甘肽的含量", 天然产物研究与开发, vol. 24, pages 928 - 932 * |
| 王雅丽 等: "HPLC法测定聚普瑞锌颗粒中L-肌肽的含量", 中国药房, vol. 21, no. 01, pages 71 - 72 * |
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