CN117705986A - Detection method and application of total component content of compound tranexamic acid preparation - Google Patents
Detection method and application of total component content of compound tranexamic acid preparation Download PDFInfo
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- CN117705986A CN117705986A CN202311742503.1A CN202311742503A CN117705986A CN 117705986 A CN117705986 A CN 117705986A CN 202311742503 A CN202311742503 A CN 202311742503A CN 117705986 A CN117705986 A CN 117705986A
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- 229960000401 tranexamic acid Drugs 0.000 title claims abstract description 58
- GYDJEQRTZSCIOI-LJGSYFOKSA-N tranexamic acid Chemical compound NC[C@H]1CC[C@H](C(O)=O)CC1 GYDJEQRTZSCIOI-LJGSYFOKSA-N 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 150000001875 compounds Chemical class 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 52
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 43
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims abstract description 28
- 108010024636 Glutathione Proteins 0.000 claims abstract description 26
- 229960003180 glutathione Drugs 0.000 claims abstract description 26
- 235000003969 glutathione Nutrition 0.000 claims abstract description 26
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 25
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims abstract description 25
- 229960002079 calcium pantothenate Drugs 0.000 claims abstract description 25
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 25
- 239000011718 vitamin C Substances 0.000 claims abstract description 25
- 235000013878 L-cysteine Nutrition 0.000 claims abstract description 14
- 239000004201 L-cysteine Substances 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 239000012085 test solution Substances 0.000 claims abstract description 8
- 229960002433 cysteine Drugs 0.000 claims abstract description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 36
- 239000000523 sample Substances 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 22
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 238000002347 injection Methods 0.000 claims description 16
- 239000007924 injection Substances 0.000 claims description 16
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- -1 sodium octane sulfonate ion Chemical class 0.000 claims description 15
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 13
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 12
- 239000011734 sodium Substances 0.000 claims description 12
- 229910052708 sodium Inorganic materials 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 4
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- KWKAKUADMBZCLK-UHFFFAOYSA-N methyl heptene Natural products CCCCCCC=C KWKAKUADMBZCLK-UHFFFAOYSA-N 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 230000005526 G1 to G0 transition Effects 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- LNEJQCNMHWLLNW-UHFFFAOYSA-L potassium sodium dihydrogen phosphate octane-1-sulfonate Chemical compound [Na+].[K+].OP(O)([O-])=O.CCCCCCCCS([O-])(=O)=O LNEJQCNMHWLLNW-UHFFFAOYSA-L 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 23
- 239000003814 drug Substances 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 239000012071 phase Substances 0.000 description 48
- 239000000243 solution Substances 0.000 description 24
- 230000014759 maintenance of location Effects 0.000 description 16
- 239000013558 reference substance Substances 0.000 description 14
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 11
- HRQDCDQDOPSGBR-UHFFFAOYSA-M sodium;octane-1-sulfonate Chemical compound [Na+].CCCCCCCCS([O-])(=O)=O HRQDCDQDOPSGBR-UHFFFAOYSA-M 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000003085 diluting agent Substances 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 238000005070 sampling Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- XFXIJSRNFKHZFW-UHFFFAOYSA-N [Na].CCCCCCCC Chemical compound [Na].CCCCCCCC XFXIJSRNFKHZFW-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- NHOXRBDMOTVJBL-UHFFFAOYSA-M potassium;dihydrogen phosphate;methanol Chemical compound [K+].OC.OP(O)([O-])=O NHOXRBDMOTVJBL-UHFFFAOYSA-M 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical compound CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- FKSLYSSVKFYJKE-UHFFFAOYSA-N n,n-diethylethanamine;methanol Chemical compound OC.CCN(CC)CC FKSLYSSVKFYJKE-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 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
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a detection method and application of total component content of a compound tranexamic acid preparation, and belongs to the technical field of medicine analysis. The method adopts high performance liquid chromatography for detection, and comprises the following steps: and (3) feeding the compound tranexamic acid test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the contents of tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate by an external standard method. The detection method can detect 5 components in the compound tranexamic acid tablet at the same time. And the main peak symmetry, the separation degree, the stability and the content accuracy of each component obtained by the detection method are all good.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for detecting the total component content of a compound tranexamic acid preparation and application thereof.
Background
The compound tranexamic acid tablet is a compound preparation product consisting of tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate, and has the effects of whitening and fading spots. As a compound preparation, the components have certain compatibility and proportioning relationship, so that the activity of the medicine is effectively improved, and the convenience of administration is improved.
Currently, the method for testing the contents of tranexamic acid, vitamin C, glutathione and L-cysteine is mainly titration in pharmacopoeias of various countries. For a compound preparation, the method for testing the content of each component by adopting a titration method has no specificity, and the content of each component cannot be accurately determined. It is particularly important to find a convenient, rapid, accurate and efficient analysis method for testing the content of each component in the compound preparation.
However, the structures of the components in the tranexamic acid compound tablet are as follows:
it can be seen that the 5 main components structurally contain less conjugated double bonds, most of the 5 main components contain amino, carboxyl and hydroxyl groups, the molecular polarity is large, the retention is weak, the retention and separation difficulty of 5 components by using the reversed-phase high-efficiency chromatography is large, and the method is a technical problem to be solved by the content test of the compound tranexamic acid tablet at present.
Disclosure of Invention
Aiming at the problem that the conventional technology does not utilize a reversed-phase high-efficiency chromatography to detect 5 components of the compound tranexamic acid tablet simultaneously, the invention provides a method for detecting the total component content of the compound tranexamic acid preparation, and the conventional high-efficiency liquid chromatograph and a reverse-phase chromatographic column can be utilized to detect 5 components in the compound tranexamic acid tablet. And the main peak symmetry, the separation degree, the stability and the content accuracy of each component obtained by the detection method are all good.
In one aspect, the invention provides a method for detecting the total component content of a compound tranexamic acid preparation, which adopts a high performance liquid chromatography for detection and comprises the following steps: feeding a compound tranexamic acid sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of each component, wherein the total components comprise tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate, and the detection conditions of the high performance liquid chromatograph are as follows:
stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: the mobile phase A is buffer solution containing sodium octane sulfonate ion pair reagent and phosphate, the mobile phase B is methanol or acetonitrile, and the flow rate is 0.5-1mL/min;
detection wavelength: 200-230 nm.
In the research and development process, in order to improve the retention of each component, through a plurality of growths and attempts, the inventor adopts a buffer solution containing phosphate and octane sodium sulfonate ion pair reagent as a mobile phase A, and is matched with an organic phase of methanol or acetonitrile, so that the retention of each component can be improved.
It will be appreciated that the above reagents, such as methanol or acetonitrile, are generally understood in the art to be analytically pure reagents having a purity of 99.9% or more. For the buffer solution of the mobile phase, the solvent is usually water, and the water used is usually purified water such as deionized water.
In some embodiments, the chromatographic column is Agilent ZORBAX SB-C18,3.0mm by 150mm,3.5 μm in size. The chromatographic column is matched with the mobile phase system of the invention, so that better detection performance can be realized.
In some embodiments, mobile phase a is a sodium 1-octanesulfonate-potassium dihydrogen phosphate buffer solution having a pH of 2.4-2.9 and mobile phase B is methanol.
In order to realize better separation degree of the components, the invention also screens the influence of the flowing relative separation systems with different pH values, and the result shows that the components have stronger dependence on the pH value, and the pH value of the flowing phase is more ideal between 2.4 and 2.9. Meanwhile, the inventor also finds that 5 main components are more stable in aqueous solution than 0.1% phosphoric acid aqueous solution in the research and development process, so that 1-octane sodium sulfonate-potassium dihydrogen phosphate buffer solution with pH of 2.4-2.9 is selected as mobile phase A.
It will be appreciated that the above pH will generally have an error of + -0.05 during actual operation, and that the pH described in the present invention is the pH measured at the time of formulation using a pH meter, and the temperature measured at the pH is 25 ℃.
In some embodiments, the mobile phase A has a concentration of 1.+ -. 0.1g/L sodium 1-octanesulfonate and a concentration of 50.+ -. 5mM potassium dihydrogen phosphate.
In some embodiments, the mobile phase a is pH adjusted to 2.5 with phosphoric acid.
In some embodiments, the mobile phase a is formulated as follows: weighing 6.8g of monopotassium phosphate and 1.0g of 1-octane sodium sulfonate, putting into 1000mL of water, dissolving and shaking uniformly, adjusting the pH to 2.5 by using phosphoric acid, and carrying out suction filtration to obtain the product.
In some embodiments, the elution is performed according to the following gradient elution procedure:
the time of gradient elution is changed from 3min to 15min, and the flowing phase changes into uniform speed.
The above-mentioned proportion of mobile phases refers to the volume of each mobile phase as a percentage of the total volume of the eluent. It will be appreciated that the gradient elution procedure described above refers to an initial eluent of 95% by volume of mobile phase a and 5% by volume of mobile phase B at 0 min; gradient elution is carried out within 0-3 min according to the volume ratio of the mobile phase B of 5%; the volume ratio of the mobile phase A in the eluent is reduced to 70% in 15min, and the volume ratio of the mobile phase B is increased to 30%; and (3) isocratic elution is carried out within 15-18 min according to the volume ratio of the mobile phase B of 30%.
Wherein, the conversion rate of the mobile phase A from 95% to 70% is a uniform change process within 3-15 min. Correspondingly, the conversion rate of mobile phase B from 5% to 30% is also a constant rate change process.
In some embodiments, the component content is calculated by an external standard method.
In some embodiments, the flow rate is 0.5-0.9mL/min, preferably 0.7mL/min.
In some embodiments, the detection wavelength is 205 to 220nm, preferably 205 to 215nm, more preferably 210nm.
In consideration of the spectral characteristics of tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate, the detection wavelength is end absorption, so in the high performance liquid chromatography, the detection wavelength can be selected to be 200-230 nm, and factors such as the signal intensity of a target object and the fluctuation stability of a detection baseline are comprehensively considered, and the detection wavelength can be selected to be 205-220nm, preferably 205-215 nm, and more preferably 210nm.
In some embodiments, the external standard method is performed according to a conventional procedure in the art, and standard substances and the like are used to prepare reagents with different concentration gradients, so as to establish a standard curve for quantitative detection.
In some embodiments, the detection conditions further comprise a column temperature of 35 to 45 ℃, such as 40 ℃.
In some schemes, the compound tranexamic acid test solution is aqueous solution, and the sample injection amount is 5-10 mu L, for example 10 mu L; wherein, in the compound tranexamic acid test solution, the concentration of tranexamic acid is 1.0+/-0.2 mg/mL, the concentration of vitamin C is 0.54+/-0.108 mg/mL, the concentration of glutathione is 0.068+/-0.0136 mg/mL, the concentration of L-cysteine is 0.1+/-0.02 mg/mL, and the concentration of calcium pantothenate is 0.04+/-0.008 mg/mL.
On the other hand, the invention also provides application of the detection method in quality control of the compound tranexamic acid preparation.
In some embodiments, the compound tranexamic acid formulation is a tablet.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
the method for detecting the total component content of the compound tranexamic acid preparation solves the problems that the specificity of a titration method is poor and the content of each component of the tranexamic acid compound preparation cannot be accurately measured, can detect the total component content in the compound tranexamic acid tablet by using a reverse high performance liquid chromatography, solves the problem that a small molecular compound with large polarity remains weak in the reverse chromatography, has low requirements on equipment, and can finish detection by using a common liquid chromatograph and matching with a chromatographic column with high cost performance.
Meanwhile, the main peak symmetry, the separation degree, the stability and the content accuracy of each component obtained by the detection method are all good, and the content of 5 components can be synchronously measured.
Drawings
FIG. 1 is a chart showing the retention and separation of components using an aqueous phosphoric acid-acetonitrile system.
FIG. 2 is a chart showing the retention and separation of components of an acetonitrile system using 1g/L of ion pair reagent (sodium 1-octanesulfonate) -0.1% phosphoric acid in water.
FIG. 3 is a graph showing the retention and separation of components of a methanol system using 1g/L of ion pair reagent (sodium 1-octanesulfonate) -20mM potassium dihydrogen phosphate solution.
FIG. 4 is a graph showing the retention and separation of components of a methanol system using 1g/L of ion pair reagent (sodium 1-octanesulfonate) -20mM potassium dihydrogen phosphate solution-0.1% triethylamine (pH 2.5 adjusted by phosphoric acid).
FIG. 5 is a graph showing the retention and separation of components of a methanol system using 1g/L of an ion-pairing reagent (sodium 1-octanesulfonate) -50mM potassium dihydrogen phosphate solution (pH 2.5 adjusted by phosphoric acid).
FIG. 6 is a graph showing the retention and separation of components of a methanol system using 1g/L of an ion-pairing reagent (sodium 1-octanesulfonate) -50mM potassium dihydrogen phosphate solution (pH 2.3 adjusted by phosphoric acid).
FIG. 7 is a chart showing the retention and separation of the components of a methanol system using 1g/L of ion pair reagent (sodium 1-octanesulfonate) -50mM potassium dihydrogen phosphate solution (pH 2.4-2.9 adjusted by phosphoric acid).
FIG. 8 is a chart showing the retention and separation of components using a 1g/L ion pair reagent (sodium 1-octanesulfonate) -50mM potassium dihydrogen phosphate solution (pH 2.5 adjusted with phosphoric acid), methanol system, and flow rate of 0.5-0.9 ml/min.
FIG. 9 is a chart showing the retention and separation of the components using a 1g/L ion pair reagent (sodium 1-octanesulfonate) -50mM potassium dihydrogen phosphate solution (pH 2.5 adjusted with phosphoric acid) in a methanol system at a column temperature of 35℃and 40℃and 45 ℃.
Fig. 10 is a chart showing the retention and separation of components in a proprietary assay and mixed standard solution.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the examples below, were selected according to conventional methods and conditions, or according to the commercial specifications
Example 1
The method for measuring the contents of five components including tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate in the compound tranexamic acid preparation is explored and optimized.
1. Reagents and apparatus
1. Reference substance
1.1 reference sources
The double content standard assignment is carried out on each raw material by using a reference substance (table 1) of the Chinese food and drug verification institute to be used as a working reference substance (used as a secondary reference substance).
Table 1.5 comparative examples of national food and drug institute for testing
1.2 preparation of control solution
1.2.1 Single reference substance configuration
About 50mg of L-cysteine, about 50mg of glutathione, about 50mg of calcium pantothenate, about 50mg of vitamin C and about 50mg of tranexamic acid are respectively taken, precisely weighed, respectively placed in 20ml measuring bottles, diluted by a diluent, fixed in volume to scale, and uniformly shaken to obtain each single product reference solution.
1.2.2 Mixed control configuration
About 108mg of L-cysteine, about 68mg of glutathione and 40mg of calcium pantothenate are precisely weighed, placed in the same 100ml measuring flask, diluted by a diluent, fixed to a scale, shaken uniformly to prepare two parts of mixed Stock solution in parallel, and marked as Stock-1 and Stock-2 respectively to prepare the mixed Stock solution.
Taking about 50mg of tranexamic acid and about 27mg of vitamin C, precisely weighing, placing into a same 50ml measuring flask, diluting and dissolving by using a diluent, precisely transferring 5ml of the mixed stock solution into the 50ml measuring flask, fixing the volume to a scale by using the diluent, shaking uniformly, and preparing two parts of the mixed stock solution serving as a reference substance solution in parallel, wherein the two parts of mixed stock solution are respectively marked as STD-1 and STD-2.
2. Test article
2.1 sample Source
The test sample is tranexamic acid compound tablet which is developed and produced by Shanghai pharmaceutical research and development Co.
2.2 preparation of sample solution
Taking 10 tablets of tranexamic acid compound tablets, and grinding the tablets into fine powder. Weighing 1150mg of ground fine powder, precisely weighing, adding a proper amount of diluent into a 100ml measuring flask, performing ultrasonic treatment for 2min, taking out, cooling, diluting with the diluent, fixing the volume to a scale, shaking uniformly, standing, centrifuging at 5000rpm for 5min, taking supernatant, filtering (filter membrane: aqueous phase needle filter, 0.45 μm), discarding 2ml of primary filtrate, taking filtrate, performing sample injection analysis, preparing two parts in parallel, respectively marked as SPL1-1 and SPL1-2, and using the two parts as a sample solution 1 for measuring the calcium pantothenate content.
Taking 5ml of the sample solution 1 to 25ml of a measuring flask, diluting to a constant volume to a scale, shaking uniformly, preparing two parts in parallel, and respectively marking the two parts as SPL2-1 and SPL2-2 as the sample solution 2 for measuring the content of the rest components.
3. Instrument for measuring and controlling the intensity of light
The high performance liquid chromatograph used in the method is the Siemens FeiteMate 3000.
2. Chromatographic conditions
Sample injection is carried out under different chromatographic conditions, and chromatograms of the compound tranexamic acid preparation (tablet) are inspected so as to fumbly and optimize detection conditions.
1. Phosphoric acid aqueous solution-acetonitrile system
Chromatographic column: agilent ZORBAX Eclipse Plus C18,4.6 mm. Times.250 mm,5 μm.
Mobile phase a:0.1% phosphoric acid in water.
Mobile phase B: acetonitrile.
Flow rate: 1.0mL/min.
Column temperature: 40 ℃.
Sample injection amount: 5. Mu.L.
Detection wavelength: 210nm.
The gradient elution procedure was as follows:
TABLE 2 gradient elution procedure
| Time (min) | Mobile phase A (% v/v) | Mobile phase B (% v/v) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 15 | 5 | 95 |
| 15.1 | 95 | 5 |
| 20 | 95 | 5 |
Taking the single-product reference substance solutions, respectively sampling, and obtaining a chromatogram shown in figure 1, wherein L-cysteine, vitamin C, tranexamic acid and glutathione are relatively weak in retention under the system.
2. Sodium octanesulphonate-phosphoric acid-acetonitrile system
Chromatographic column: waters Symmetry C18,4.6 mm. Times.250 mm,5 μm.
Mobile phase a:1g/L sodium 1-octanesulfonate-0.1% phosphoric acid aqueous solution.
Mobile phase B: acetonitrile.
Flow rate: 1.0mL/min.
Column temperature: 40 ℃.
Sample injection amount: 5. Mu.L.
Detection wavelength: 210nm of
The gradient elution procedure was as follows:
TABLE 3 gradient elution procedure
| Time (min) | Mobile phase A (% v/v) | Mobile phase B (% v/v) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 15 | 70 | 30 |
| 15.1 | 95 | 5 |
| 20 | 95 | 5 |
The single-product reference substance solutions are taken and respectively injected, the obtained chromatograms are shown in figure 2, and the figure shows that the components in the system are retained and enhanced and can be separated from each other, but the peak-to-peak type is poor, and the introduced ions cause fluctuation to the system.
3. Sodium octanesulfonate-20 mM potassium dihydrogen phosphate-methanol system
Chromatographic column: waters Symmetry C18,4.6 mm. Times.250 mm,5 μm.
Mobile phase a:1g/L sodium 1-octanesulfonate-20 mM potassium dihydrogen phosphate aqueous solution.
Mobile phase B: methanol.
Flow rate: 1.0mL/min.
Column temperature: 40 ℃.
Sample injection amount: 5. Mu.L.
Detection wavelength: 210nm.
The gradient elution procedure was as follows:
TABLE 4 gradient elution procedure
Taking the mixed reference substance solution, and carrying out sample injection detection, wherein the obtained chromatogram is shown in figure 3, and the figure shows that under the system, vitamin C and glutathione cannot be separated, and the separation degree and the peak type of each peak do not meet the requirements.
4. Sodium octane sulfonate-20 mM potassium dihydrogen phosphate-0.1% triethylamine-methanol system (pH 2.5)
Chromatographic column: agilent ZORBAX SB-C18,3.0 mm. Times.150 mm,3.5 μm.
Mobile phase a:1g/L sodium 1-octanesulfonate-20 mM potassium dihydrogen phosphate aqueous solution-0.1% triethylamine (pH 2.5 adjusted by phosphoric acid).
Mobile phase B: methanol.
Flow rate: 1.0mL/min.
Column temperature: 40 ℃.
Sample injection amount: 5. Mu.L.
Detection wavelength: 210nm.
The gradient elution procedure was as follows:
TABLE 5 gradient elution procedure
| Time (min) | Mobile phase A (% v/v) | Mobile phase B (% v/v) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 15 | 70 | 30 |
| 15.1 | 95 | 5 |
| 20 | 95 | 5 |
The conditions are adopted for two sample injection, the first needle is used for injecting the mixed reference substance of the 5 components, but the tranexamic acid in the chromatogram can not be detected, and the second needle is used for independently injecting the tranexamic acid standard substance solution with high concentration (2.5 mg/ml). The chromatogram is shown in fig. 4 (the first needle is arranged below the base line, and the second needle is arranged above the base line), and it can be seen from the figure that in the system, after the mixed reference substance is injected, the vitamin C and the L-cysteine are not separated, and the tranexamic acid response is weaker.
5. Sodium octanesulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5)
Chromatographic column: agilent ZORBAX SB-C18,3.0 mm. Times.150 mm,3.5 μm.
Mobile phase a:1g/L sodium 1-octanesulfonate-50 mM potassium dihydrogen phosphate aqueous solution (pH 2.5 adjusted by phosphoric acid).
Mobile phase B: methanol.
Flow rate: 0.7mL/min.
Column temperature: 40 ℃.
Sample injection amount: 10 mu L.
Detection wavelength: 210nm.
The gradient elution procedure was as follows:
TABLE 6 gradient elution procedure
| Time (min) | Mobile phase A (% v/v) | Mobile phase B (% v/v) |
| 0 | 95 | 5 |
| 3 | 95 | 5 |
| 15 | 70 | 30 |
| 18 | 70 | 30 |
| 18.1 | 95 | 5 |
| 25 | 95 | 5 |
Taking the mixed reference substance solution, sampling and detecting, and obtaining a chromatogram shown in figure 5, wherein the chromatogram is shown in the figure, and the system has better base line, better retention of each component and good separation degree and peak type among peaks.
6. Sodium octanesulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.3)
Mobile phase a:1g/L sodium 1-octanesulfonate-50 mM potassium dihydrogen phosphate aqueous solution (pH 2.3 adjusted by phosphoric acid).
Mobile phase B: methanol.
The other chromatographic conditions were the same as those of the above-mentioned 5 th octanesulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5).
Taking the mixed reference substance solution, and carrying out sample injection detection, wherein the obtained chromatogram is shown in figure 6, and the graph shows that glutathione and calcium pantothenate cannot be separated well under the condition of pH 2.3.
7. System pH adjustment
Referring to the conditions of the above-mentioned sodium octane sulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5), the pH of mobile phase A was adjusted to 2.4, 2.5, 2.7 and 2.9 with phosphoric acid alone, and chromatograms were recorded after respective sample injections.
As shown in FIG. 7, the chromatogram shows that the components can be well reserved under the condition of pH of 2.4-2.9, and the separation degree and the peak type between the peaks are good.
8. System flow rate adjustment
With reference to the conditions of the above 5 th octanesulfonate-50 mM potassium dihydrogen phosphate-methanol system (pH 2.5), the flow rates were adjusted to 0.5mL/min, 0.7mL/min, 0.8mL/min and 0.9mL/min, and the chromatograms were recorded after sample injection, respectively.
As shown in the graph in FIG. 8, the components can be well reserved under the condition of 0.5-0.9mL/min, and the separation degree and the peak type among the peaks are good.
9. Column temperature adjustment
Referring to the conditions of the above 5 th octanesulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5), the column temperatures were adjusted to 35℃only, 40℃only and 45℃only, and the chromatograms were recorded after respective sample injections.
The chromatogram is shown in FIG. 9, and the chromatogram shows that the components can be well reserved at the column temperature of 35-45 ℃, and the separation degree and the peak type between the peaks are good.
Example 2
This example demonstrates methodology for the detection method screened in example 1.
The detection conditions used were carried out in accordance with the "sodium octane sulfonate-50 mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5)" under item 5 in example 1.
1. Specificity and System applicability
1.1 blank solvent and blank adjuvant solution
Taking diluent purified water as a blank solution, weighing blank auxiliary materials except active ingredients of the compound tranexamic acid tablet, placing the blank auxiliary materials into a volumetric flask, dissolving, diluting, fixing the volume, shaking uniformly, and filtering to obtain a blank auxiliary material solution; the blank solution and the blank auxiliary material solution are respectively injected into a high performance liquid chromatograph for detection, and the result is shown in fig. 10, and the blank solution and the blank auxiliary material solution have no interference at the positions of 5 component peak positions from the graph.
1.2 degree of separation
Taking the STD-1, injecting into a high performance liquid chromatograph for detection, wherein the separation degree between L-cysteine and vitamin C, between glutathione and L-cysteine, between calcium pantothenate and glutathione, and between tranexamic acid and calcium pantothenate is respectively 8.8, 11.5, 6.7 and 34.8.
The result shows that the method has good specificity.
1.3 System applicability
Firstly, taking STD-1, continuously and repeatedly sampling 5 needles, continuously sampling 2 needles of STD-2, starting to detect samples, every sampling 12 needles of samples, inserting the sample into the sample, and finally ending with the sample into the sample STD-1 at the end of the experiment.
The relative standard deviation of the peak areas of the continuous 5-needle STD-1 is respectively 0.2 percent of vitamin C, 0.2 percent of L-cysteine, 0.1 percent of glutathione, 0.5 percent of calcium pantothenate and 0.3 percent of tranexamic acid; the recovery rate of STD-2 relative to STD-1 response is 99.3% of vitamin C, 99.2% of L-cysteine, 99.3% of glutathione, 99.4% of calcium pantothenate and 99.4% of tranexamic acid respectively; the recovery rate of the interlude sample injection STD-1 relative to the average response of the continuous 5-needle STD-1 is 100.7% -101.6% of vitamin C, 100.1% -100.9% of L-cysteine, 100.6% -101.5% of glutathione, 99.9% -101.4% of calcium pantothenate and 100.3% -101.2% of tranexamic acid respectively.
The result shows that the method has good system applicability.
2. Linearity and range
According to the range of 50% -150% of marked content (specifically selecting five concentration points of 50%, 80%,100%, 120% and 150%), respectively preparing a series of standard linear solutions with the concentration of vitamin C of 0.27-0.81 mg/ml, the concentration of L-cysteine of 0.054-0.162 mg/ml, the concentration of glutathione of 0.034-0.102 mg/ml, the concentration of calcium pantothenate of 0.02-0.06 mg/ml and the concentration of tranexamic acid of 0.5-1.5 mg/ml, and injecting the solutions into a high performance liquid chromatograph for detection.
Drawing each component standard curve according to the obtained chromatogram, wherein each component linear correlation coefficient is respectively as follows: 0.9996 vitamin C, 0.9999L-cysteine, 1.0000 glutathione, 1.0000 calcium pantothenate and 1.0000 tranexamic acid.
The result shows that the detection method has high correlation coefficient of the standard curve and good linear result.
3. Accuracy of
A series of accuracy solutions were prepared at 80%,100% and 120% of the indicated concentration levels, respectively, with 3 portions prepared in parallel for each concentration level. The corresponding blank auxiliary materials and mixed reference substance solutions are respectively added into the solutions with the accuracy, the corresponding concentrations at the concentration levels are 0.432mg/ml, 0.540mg/ml, 0.648mg/ml, 0.08mg/ml, 0.10mg/ml, 0.12mg/ml of L-cysteine, 0.0544mg/ml, 0.068mg/ml, 0.0816mg/ml, 0.032mg/ml, 0.04mg/ml, 0.048mg/ml of calcium pantothenate, 0.8mg/ml, 1.0mg/ml and 1.2mg/ml of tranexamic acid are injected into a high performance liquid chromatograph for detection, and the recovery rate is calculated according to the obtained chromatograms.
The average recovery of vitamin C in 9 parts of the accurate solution was 100.5%, RSD was 0.9%, L-cysteine was 99.7%, RSD was 0.3%, glutathione was 100.9%, RSD was 0.4%, calcium pantothenate was 100.9%, RSD was 0.2%, tranexamic acid was 99.7%, and RSD was 0.6%.
The result shows that the method has good accuracy, and the linear range of 0.432-0.648 mg/ml of vitamin C, 0.08-0.12 mg/ml of L-cysteine, 0.0544-0.0816 mg/ml of glutathione, 0.032-0.048 mg/ml of calcium pantothenate and 0.8-1.2 mg/ml of tranexamic acid can be accurately detected.
4. Repeatability of
According to the preparation method of the test sample in example 1, 6 parts of the sample are prepared in parallel, and the sample is injected into a high performance liquid chromatograph to be detected, and the content and the relative standard deviation of the content of each component are calculated according to the obtained chromatogram.
The results showed that the average of the labeled amounts of vitamin C was 98.9%, RSD was 0.4%, L-cysteine was 95.9%, RSD was 0.3%, glutathione was 91.3%, RSD was 0.4%, calcium pantothenate was 95.2%, RSD was 0.4%, tranexamic acid was 96.8%, and RSD was 0.3%.
The results show that the method of the invention has good repeatability.
5. Stability.
STD-1 and test solutions (test preparation method in example 1) were taken and placed at 4deg.C, and injected into high performance liquid chromatograph at intervals of 4, 8, 14, 24 and 28 hours, respectively, for detection. The relative standard deviation of the peak areas of STD-1 and the test solution at each time point was calculated from the obtained chromatograms.
The results showed that the relative standard deviation of peak areas at each time point of STD-1 was 0.6% for vitamin C, 0.3% for L-cysteine, 0.6% for glutathione, 0.6% for calcium pantothenate, and 0.6% for tranexamic acid, respectively; the relative standard deviation of peak area of the test solution at each time point is 0.8% of vitamin C, 0.7% of L-cysteine, 0.8% of glutathione, 0.8% of calcium pantothenate and 0.6% of tranexamic acid
The results show that the control and test solutions are stable for at least 28 hours at 4 ℃.
The verification result of the embodiment shows that the method has good system applicability, specificity, linearity and range, accuracy, repeatability and stability.
Claims (10)
1. The method for detecting the total component content of the compound tranexamic acid preparation is characterized by adopting a high performance liquid chromatography method for detection, and comprises the following steps: feeding a compound tranexamic acid sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the content of each component, wherein the total components comprise tranexamic acid, vitamin C, glutathione, L-cysteine and calcium pantothenate, and the detection conditions of the high performance liquid chromatograph are as follows:
stationary phase: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: the mobile phase A is buffer solution containing sodium octane sulfonate ion pair reagent and phosphate, the mobile phase B is methanol or acetonitrile, and the flow rate is 0.5-1mL/min;
detection wavelength: 200-230 nm.
2. The method according to claim 1, wherein the chromatographic column is Agilent ZORBAX SB-C18,3.0mm by 150mm,3.5 μm.
3. The method according to claim 2, wherein the mobile phase A is a sodium 1-octanesulfonate-potassium dihydrogen phosphate buffer solution having a pH of 2.4 to 2.9, and the mobile phase B is methanol.
4. The method according to claim 3, wherein the concentration of 1-octane sodium sulfonate in the mobile phase A is 1.+ -. 0.1g/L and the concentration of potassium dihydrogen phosphate is 50.+ -. 5mM.
5. The method according to claim 4, wherein the mobile phase A is adjusted to pH2.5 with phosphoric acid.
6. The method of claim 1, wherein the elution is performed according to the following gradient elution procedure:
the time of gradient elution is changed from 3min to 15min, and the flowing phase changes into uniform speed.
7. The method according to claim 1, wherein the detection condition meets at least one of the following conditions:
1) Calculating the content of each component by an external standard method;
2) The flow rate is 0.5-0.9mL/min, preferably 0.7mL/min;
3) The detection wavelength is 205 to 220nm, preferably 205 to 215nm, more preferably 210nm.
8. The method of claim 1, wherein the detection conditions further comprise a column temperature of 35-45 ℃, such as 40 ℃.
9. The detection method according to claim 1, wherein the compound tranexamic acid sample solution is an aqueous solution, and the sample injection amount is 5-10 μl, for example 10 μl; wherein, in the compound tranexamic acid test solution, the concentration of tranexamic acid is 1.0+/-0.2 mg/mL, the concentration of vitamin C is 0.54+/-0.108 mg/mL, the concentration of glutathione is 0.068+/-0.0136 mg/mL, the concentration of L-cysteine is 0.1+/-0.02 mg/mL, and the concentration of calcium pantothenate is 0.04+/-0.008 mg/mL.
10. The use of the detection method according to any one of claims 1-9 in quality control of a compound tranexamic acid preparation.
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