CN114324636A - Method for determining vitamin B6 and related impurities thereof by HPLC method - Google Patents
Method for determining vitamin B6 and related impurities thereof by HPLC method Download PDFInfo
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- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 239000012535 impurity Substances 0.000 title claims abstract description 65
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 239000011726 vitamin B6 Substances 0.000 title claims abstract description 61
- 235000019158 vitamin B6 Nutrition 0.000 title claims abstract description 61
- 229940011671 vitamin b6 Drugs 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 239000000945 filler Substances 0.000 claims abstract description 9
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 9
- HRQDCDQDOPSGBR-UHFFFAOYSA-M sodium;octane-1-sulfonate Chemical compound [Na+].CCCCCCCCS([O-])(=O)=O HRQDCDQDOPSGBR-UHFFFAOYSA-M 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 5
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 20
- 239000012085 test solution Substances 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000010606 normalization Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims 5
- 238000002360 preparation method Methods 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000003908 quality control method Methods 0.000 abstract description 3
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 56
- 239000000047 product Substances 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000012071 phase Substances 0.000 description 10
- 238000006460 hydrolysis reaction Methods 0.000 description 9
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N Butyraldehyde Chemical compound CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 238000010992 reflux Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- HGBOYTHUEUWSSQ-UHFFFAOYSA-N valeric aldehyde Natural products CCCCC=O HGBOYTHUEUWSSQ-UHFFFAOYSA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 238000005698 Diels-Alder reaction Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000005899 aromatization reaction Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013373 food additive Nutrition 0.000 description 2
- 239000002778 food additive Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- ROBLTDOHDSGGDT-UHFFFAOYSA-M sodium;pentane-1-sulfonate Chemical compound [Na+].CCCCCS([O-])(=O)=O ROBLTDOHDSGGDT-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 125000004938 5-pyridyl group Chemical group N1=CC=CC(=C1)* 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- ZCQWOFVYLHDMMC-UHFFFAOYSA-N Oxazole Chemical compound C1=COC=N1 ZCQWOFVYLHDMMC-UHFFFAOYSA-N 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
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Abstract
The invention aims to provide a method for measuring vitamin B6 and related impurities thereof by an HPLC method. The method can simultaneously detect the vitamin B6, the impurity A (number H029-S-I-06) and various related impurities, improve the separation and detection efficiency and ensure the quality control of the vitamin B6 bulk drug and the preparation thereof. The technical scheme of the invention is as follows: the chromatographic column adopted in the method is characterized in that octadecylsilane chemically bonded silica is used as a filler, mobile phase A and mobile phase B are adopted for gradient elution, and the gradient elution enters a detector for detection. Mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20); mobile phase B: and (3) acetonitrile.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining vitamin B6 and related impurities thereof by an HPLC (high performance liquid chromatography) method.
Background
The main commercial form of vitamin B6, pyridoxine hydrochloride, is widely used in the fields of feed additives, food additives, health products, medicines, etc.
Currently, the oxazole method is widely used in industry to synthesize vitamin B6. The method comprises Diels-Alder (Diels-Alder) reaction, aromatization reaction, hydrolysis reaction, and refining to obtain refined vitamin B6.
The presence of impurities can reduce the efficacy and even in some cases produce side effects. The study of new impurities is a dynamically evolving and constantly advancing process.
At present, the content determination methods of vitamin B6, namely the Chinese pharmacopoeia method and the European pharmacopoeia method, cannot well detect newly-found impurities and cannot better control the quality of the product.
Therefore, it is necessary to develop a method for more effectively measuring vitamin B6 and its related impurities and more effectively controlling the quality of vitamin B6 medicine.
Disclosure of Invention
The invention aims to provide a method for measuring vitamin B6 and related impurities thereof by an HPLC method. The method can simultaneously detect the vitamin B6, the impurity H029-S-I-06 and various related impurities, improve the separation and detection efficiency and ensure the quality control of the vitamin B6 bulk drug and the preparation thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for determining vitamin B6 and related impurities thereof by an HPLC method comprises the steps of using octadecylsilane chemically bonded silica as a filler, performing gradient elution by using a mobile phase A and a mobile phase B, and detecting by using a detector; the vitamin B6 and its related impurities are as follows:
the HPLC (high performance liquid chromatography) method comprises the following steps:
solvent: 10% methanol solution
Test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and fix the volume, and diluting to prepare a solution containing about 1mg in each 1 ml.
Chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (preferably Agilent InfinityLab Poroshell120EC-C18, 4.6 x 150mm, 4 μm); mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20); mobile phase B: acetonitrile; the flow rate is 1.0 ml/min; the detection wavelength is 285 nm; the column temperature is 30 ℃; the injection volume was 5. mu.l.
The determination method comprises the following steps: precisely measuring the test solution, injecting into a liquid chromatograph, and recording the chromatogram. Calculated by area normalization.
The method can effectively separate and detect the vitamin B6, the impurity H029-S-I-06 and the main peaks of a plurality of related impurities simultaneously, thereby improving the separation and detection efficiency; the quality of the vitamin B6 bulk drug and the preparation thereof can be more accurately ensured to be controllable, and the safety and the effectiveness of the product can be finally determined.
The Chinese pharmacopoeia 2020(CP2020) and the European pharmacopoeia 10(EP10) cannot simultaneously separate and detect the main peaks of vitamin B6, H029-S-I-06 and a plurality of related impurities. In particular, H029-S-I-06 cannot be effectively separated and detected.
The invention provides a vitamin B6 impurity, named as vitamin B6 impurity A (impurity number: H029-S-I-06), with the following structural formula:
the synthetic route of the impurity A is as follows:
the specific preparation method of the impurity A comprises the following steps:
200ml of toluene, 20.56g of vitamin B6, 38.04g of p-toluenesulfonic acid and 14.40g of butyraldehyde are added into a reaction bottle for reflux and water separation for 3 hours, the temperature is reduced to room temperature, the pH value of a system is adjusted to be neutral by saturated sodium bicarbonate solution, the toluene phase is washed twice by water, the organic phase is evaporated to dryness under reduced pressure to obtain viscous liquid, 100ml of ethyl acetate is added, the mixture is stirred and crystallized, filtered, and washed by ethyl acetate to obtain a white-like solid, namely the impurity A.
The preparation method of the vitamin B6 has the following reaction formula:
wherein R is1Is C1-C4 alkyl, R2Is hydrogen or C1-C4 alkyl;
preferably, the substituent R1Is ethyl, R2Is n-propyl or isopropyl.
The preferred vitamin B6 reaction is as follows:
when vitamin B6 is used as a food additive, a health product, and a pharmaceutical product, it is desirable that the product be high in purity, content, and impurities be low to provide better safety to consumers.
The inventor of the invention researches and discovers that during the hydrolysis reaction, the phenolic hydroxyl and the adjacent alkyl hydroxyl in the structure of the crude vitamin B6 can form a six-membered ring together with butyraldehyde generated by the hydrolysis reaction to generate an impurity A, and the impurity A is difficult to remove in the subsequent reaction.
Impurity A has the structural formula:
the present inventors have studied and found that when a large amount of water is present in the hydrolysis reaction system, the impurity a is easily formed and the amount is relatively large. When the amount of water in the hydrolysis reaction system is relatively small, the impurity a is not easily formed and the amount is relatively small. Therefore, the inventor adjusts the hydrolysis reaction process, thereby reducing the formation of the impurity A and improving the quality of the final product vitamin B6.
The invention provides application of impurity A as a reference substance in detection of vitamin B6 and related substances thereof.
The method for determining vitamin B6 and related impurities by an HPLC method is not provided. The method of the present invention can simultaneously detect vitamin B6 and impurity (8-methyl-2-propyl-4H- [1,3] dioxa [4,5-c ] pyridin-5-yl) methanol (code: H029-S-I-06) and various related impurities
The invention has the beneficial effects that:
the HPLC method provided by the invention can simultaneously detect vitamin B6, impurity H029-S-I-06 and other multiple impurities, and compared with the prior art, the separation detection efficiency is improved; the quality control of vitamin B6 and related substances thereof is significant; the quality of the vitamin B6 bulk drug and the preparation thereof can be more accurately ensured to be controllable, and the safety and the effectiveness of the product can be finally determined.
Drawings
FIG. 1: example 2 detection of crude vitamin B6;
FIG. 2: example 3 vitamin B6 end product testing;
FIG. 3: example 4 detection of crude vitamin B6;
FIG. 4: example 4 vitamin B6 end product testing;
FIG. 5: comparative example 1 an impurity mixture chromatogram under HPLC conditions in chinese pharmacopoeia 2020 edition (CP2020) vitamin B6;
FIG. 6: comparative example 1 according to chromatogram of test solution under HPLC condition of China pharmacopoeia 2020 edition (CP2020) vitamin B6;
FIG. 7: comparative example 2 impurity mixture chromatogram according to HPLC conditions under european pharmacopeia version 10(EP10) vitamin B6;
FIG. 8: comparative example 2 followed the chromatogram of the test solution under HPLC conditions according to European pharmacopoeia 10 edition (EP10) vitamin B6.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Preparation example 1: the preparation method of the impurity A (H029-S-I-06) comprises the following steps:
200ml of toluene, 20.56g of vitamin B6, 38.04g of p-toluenesulfonic acid and 14.40g of butyraldehyde are added into a reaction bottle for reflux and water diversion for 3 hours, the temperature is reduced to room temperature, the pH value of a system is adjusted to be neutral by saturated sodium bicarbonate solution, the toluene phase is washed twice by water, the organic phase is evaporated to dryness under reduced pressure to obtain viscous liquid, 100ml of ethyl acetate is added, stirring and crystallization are carried out, filtering is carried out, and ethyl acetate is washed to obtain white-like solid, namely the impurity A.
Example 1: the HPLC (high performance liquid chromatography) method of the invention
Solvent: 10% methanol solution
Test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and fix the volume, and diluting to prepare a solution containing about 1mg in each 1 ml.
Chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (preferably Agilent InfinityLab Poroshell120EC-C18, 4.6 x 150mm, 4 μm); mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20); mobile phase B: acetonitrile; the flow rate is 1.0 ml/min; the detection wavelength is 285 nm; the column temperature is 30 ℃; the injection volume was 5. mu.l.
The determination method comprises the following steps: precisely measuring the test solution, injecting into a liquid chromatograph, and recording the chromatogram. Calculated by area normalization.
Example 2: preparation of crude vitamin B6 (hydrolysis reaction)
Adding 800ml of dichloromethane and 229g of compound 4 into a reaction bottle, adding 130ml of 32 wt% concentrated hydrochloric acid, refluxing for 3 hours at about 40 ℃ under normal pressure under the condition of stirring, adding a water separator into a reflux device, draining water out of the reaction system, returning an organic phase into the reaction bottle, and refluxing and water separating until the reaction is finished; cooling to 5 ℃, growing the crystal for 1h, filtering, washing a filter cake by 95 wt% of ethanol water solution, and drying in vacuum at 40-70 ℃ to obtain 6g of crude vitamin B, 207 g. The hydrolysis reaction molar yield was 98%.
FIG. 1 shows the purity, content, impurity A content and HPLC chart of crude vitamin B6 obtained in this example; the purity of the vitamin B6 crude product (H029-S) is 99.88 percent, and the content of the impurity A (H029-S-I-06) is 0.06 percent.
The HPLC (high performance liquid chromatography) method is shown in example 1.
Example 3: preparation of final product vitamin B6 (refined)
Adding 300g of purified water and 150g of crude product B6 into a reaction bottle, heating to dissolve, adding 7.5g of activated carbon, stirring and decoloring for 60min at 70 ℃, filtering while hot, washing an activated carbon filter cake with 75g of hot water at 80 ℃, distilling the filtrate under reduced pressure until the residual weight of the system is 285-300 g, slowly cooling to room temperature, cooling to 5 ℃, growing crystals for 1h, performing suction filtration, washing the filter cake with 150ml of 95 wt% ethanol aqueous solution, and performing vacuum drying at 40-70 ℃ to obtain refined products of vitamin B6 and 135 g. The purification step gave a molar yield of 90%.
FIG. 2 is the purity, content, impurity A content and HPLC chart of the final vitamin B6 product obtained in this example; the purity of the vitamin B6(H029-S) is 99.99 percent, and the content of the impurity A (H029-S-I-06) is 0.01 percent.
The HPLC (high performance liquid chromatography) method is shown in example 1.
Example 4: according to the improvement of the synthesis process of vitamin B6 in journal literature, Chentianhao, China journal of medical industry, 2004' preparation of crude vitamin B6 and final product
Experiments were conducted according to the improvement of the synthesis process of vitamin B6 in journal literature, the aromatization reaction, hydrolysis reaction and refining method in chentianhao, journal of chinese medical industry, 2004.
FIG. 3 is the purity, content, impurity A content and HPLC chart of the crude vitamin B6 obtained in this example; the purity of the crude vitamin B6(H029-S) is 98.88 percent, and the content of the impurity A (H029-S-I-06) is 0.94 percent.
The HPLC (high performance liquid chromatography) method is shown in example 1.
FIG. 4 is the purity, content, impurity A content, HPLC chart of the final vitamin B6 product obtained in this example; the purity of the final product of vitamin B6(H029-S) is 99.87%, and the content of impurity A (H029-S-I-06) is 0.08%.
The HPLC (high performance liquid chromatography) method is shown in example 1.
Comparative example 1: detecting according to HPLC condition of China pharmacopoeia 2020 edition (CP2020) vitamin B6
Solvent: 0.04% sodium pentanesulfonate solution (pH3.0) -methanol (85-15).
Test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and fix the volume, and diluting to prepare a solution containing about 2mg in each 1 ml.
Impurity mixed solution: taking a proper amount of each impurity reference substance, adding a solvent to dissolve and fix the volume, and diluting to prepare a mixed solution containing about 5 mu g of each impurity in each 1 ml.
Chromatographic conditions are as follows: bonding silica gel with octadecylsilane as filler (Agilent ZORBAX SB-C184.6 x 250mm, 5 μm); mobile phase: 0.04% sodium pentane sulfonate solution (pH adjusted to 3.0 with glacial acetic acid) -methanol (85-15); the flow rate is 1.0 ml/min; the detection wavelength is 291 nm; the column temperature was 25 ℃; the injection volume was 10. mu.l.
The determination method comprises the following steps: precisely measuring the test solution, injecting into a liquid chromatograph, and recording the chromatogram.
FIG. 5: impurity mixed chromatogram
The impurity mixed solution has 8 impurities in total, namely H029-S-I-01, H029-S-I-02, H029-S-I-04, H029-S-I-06, H029-S-I-09, H029-S-I-13, H029-S-I-15 and H029-02, wherein the impurities H029-02, H029-S-I-13 and H029-S-I-06 do not show peaks in the method.
FIG. 6: chromatogram of test solution
H029-S-I-06 does not show a peak.
Comparative example 2: detecting according to HPLC condition of European pharmacopoeia 10 edition (EP10) vitamin B6
Solvent: ultrapure water.
Test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve, fixing the volume, and diluting to prepare a solution containing 2.5mg in each 1 ml.
Test-impurity mixed solution: taking a proper amount of the test sample and each impurity reference substance, adding a solvent to dissolve and fix the volume, and diluting to prepare a mixed solution containing about 2.5mg of the test sample and 2.5 mu g of each impurity in every 1 ml.
Chromatographic conditions are as follows: using octadecylsilane bonded silica gel as filler (Thermo Hypersil BDS C184.6 x 250mm, 5 μm); mobile phase: 0.02mol/l KH2PO4(adjusting the pH to 3.0 with phosphoric acid); the flow rate is 1.0 ml/min; the detection wavelength is 210 nm; the column temperature was 25 ℃; the injection volume was 5. mu.l.
The determination method comprises the following steps: precisely measuring the test solution, injecting into a liquid chromatograph, and recording the chromatogram.
FIG. 7: impurity mixed chromatogram
The impurity mixed solution contains 8 impurities in total, namely H029-S-I-01, H029-S-I-02, H029-S-I-04, H029-S-I-06, H029-S-I-09, H029-S-I-13, H029-S-I-15 and H029-02, wherein the impurities H029-S-I-09, H029-S-I-13, H029-S-I-06 and H029-02 do not produce peaks.
FIG. 8: chromatogram of test solution
H029-S-I-06 does not show a peak.
Claims (10)
1. A method for measuring vitamin B6 and related impurities thereof by an HPLC method is characterized by comprising the following steps:
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for a chromatographic column; mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20); mobile phase B: acetonitrile; gradient elution.
2. The assay of claim 1, wherein the vitamin B6 and impurities are as follows:
3. the assay method according to claim 1, wherein the gradient elution is set as follows:
4. the assay according to claim 1, wherein the chromatographic column is preferably an Agilent InfinityLab Poroshell120 EC-C18; the specification is preferably 4.6 x 150mm, 4 μm.
5. The method of measuring according to claim 1, wherein the detection wavelength is 285 nm.
6. The method according to claim 1, wherein the column temperature is 30 ℃.
7. The assay according to claim 1, wherein the mobile phase flow rate is 1.0 ml/min.
8. The method of claim 1, wherein the sample is a 10% methanol solution.
9. The assay method according to claim 1, wherein the detection step is as follows:
test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and fix the volume, and diluting to prepare a solution containing about 1mg in each 1 ml;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for a chromatographic column; mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20); mobile phase B: acetonitrile; the flow rate is 1.0 ml/min;
the determination method comprises the following steps: precisely measuring a test solution, injecting the test solution into a liquid chromatograph, and recording a chromatogram; calculated by area normalization.
10. The method of measuring according to claim 1, wherein the method is as follows:
solvent: 10% methanol solution;
test solution: taking a proper amount of the product, precisely weighing, adding a solvent to dissolve and fix the volume, and diluting to prepare a solution containing about 1mg in each 1 ml;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: 0.01mol/l sodium octane sulfonate solution (pH2.5) -acetonitrile (80-20) mobile phase B: acetonitrile; the flow rate is 1.0 ml/min; the detection wavelength is 285 nm; the column temperature is 30 ℃; the sample injection volume is 5 mu l;
the determination method comprises the following steps: precisely measuring a test solution, injecting the test solution into a liquid chromatograph, and recording a chromatogram; calculated by area normalization.
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