CN115078613A - HPLC detection method for folic acid and D-folic acid as impurity thereof - Google Patents
HPLC detection method for folic acid and D-folic acid as impurity thereof Download PDFInfo
- Publication number
- CN115078613A CN115078613A CN202210590278.3A CN202210590278A CN115078613A CN 115078613 A CN115078613 A CN 115078613A CN 202210590278 A CN202210590278 A CN 202210590278A CN 115078613 A CN115078613 A CN 115078613A
- Authority
- CN
- China
- Prior art keywords
- folic acid
- solution
- detected
- sample
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 121
- OVBPIULPVIDEAO-GFCCVEGCSA-N (2r)-2-[[4-[(2-amino-4-oxo-1h-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-GFCCVEGCSA-N 0.000 title claims abstract description 84
- 238000001514 detection method Methods 0.000 title claims abstract description 63
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 229960000304 folic acid Drugs 0.000 title claims abstract description 61
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 61
- 239000011724 folic acid Substances 0.000 title claims abstract description 61
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 38
- 239000012535 impurity Substances 0.000 title claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 82
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 66
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000000523 sample Substances 0.000 claims abstract description 33
- 239000012488 sample solution Substances 0.000 claims abstract description 22
- 239000013558 reference substance Substances 0.000 claims abstract description 11
- 238000005349 anion exchange Methods 0.000 claims abstract description 7
- 239000012088 reference solution Substances 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 24
- 229960000583 acetic acid Drugs 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000005303 weighing Methods 0.000 claims description 16
- 238000007865 diluting Methods 0.000 claims description 14
- 239000003085 diluting agent Substances 0.000 claims description 12
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 238000004364 calculation method Methods 0.000 claims description 4
- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 3
- 238000010268 HPLC based assay Methods 0.000 claims 1
- 238000005220 pharmaceutical analysis Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 238000000926 separation method Methods 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 6
- 239000012085 test solution Substances 0.000 description 5
- 238000011835 investigation Methods 0.000 description 4
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 2
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 2
- 230000007797 corrosion Effects 0.000 description 2
- 238000005260 corrosion Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 229960001404 quinidine Drugs 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- SMZHKGXSEAGRTI-UHFFFAOYSA-N 1,1,1-trichloropropan-2-one Chemical compound CC(=O)C(Cl)(Cl)Cl SMZHKGXSEAGRTI-UHFFFAOYSA-N 0.000 description 1
- 206010002065 Anaemia megaloblastic Diseases 0.000 description 1
- 208000000682 Megaloblastic Anemia Diseases 0.000 description 1
- GADGMZDHLQLZRI-VIFPVBQESA-N N-(4-aminobenzoyl)-L-glutamic acid Chemical compound NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 GADGMZDHLQLZRI-VIFPVBQESA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 201000010829 Spina bifida Diseases 0.000 description 1
- 208000006097 Spinal Dysraphism Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001343 alkyl silanes Chemical group 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 208000003906 hydrocephalus Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 231100001016 megaloblastic anemia Toxicity 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000276 neural tube Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- CPNGPNLZQNNVQM-UHFFFAOYSA-N pteridine Chemical compound N1=CN=CC2=NC=CN=C21 CPNGPNLZQNNVQM-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- RSKNEEODWFLVFF-UHFFFAOYSA-N sulfuric acid;2,5,6-triamino-1h-pyrimidin-4-one Chemical compound OS(O)(=O)=O.NC1=NC(=O)C(N)=C(N)N1 RSKNEEODWFLVFF-UHFFFAOYSA-N 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of pharmaceutical analysis, and particularly provides an HPLC (high performance liquid chromatography) detection method for folic acid and D-folic acid as an impurity thereof, which comprises the following steps: preparing a sample solution to be detected; preparing a reference substance solution; respectively carrying out HPLC detection on the sample solution to be detected and the reference solution; wherein: the HPLC detection conditions are as follows: the chromatographic column adopts a weak anion exchange chiral column; the mobile phase comprises component A methanol and component B acetic acid solution; the volume ratio of the methanol to the acetic acid solution is (88: 12) - (92: 8); calculating the content of the D-folic acid in the sample to be detected: and calculating the content of the D-folic acid in the sample to be detected by using the peak area of the D-folic acid in the sample solution to be detected and the peak area of the D-folic acid in the reference solution by adopting an external standard method. The detection method greatly improves the stability of the folic acid impurity detection method, and can simply, quickly and stably detect folic acid impurities.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, and particularly relates to an HPLC (high performance liquid chromatography) detection method for folic acid and D-folic acid as an impurity thereof.
Background
Folic acid consists of pteridine, p-aminobenzoic acid and L-glutamic acid, also called pteroylglutamic acid, which is one of the B vitamins. Folic acid is a water-soluble vitamin, and is one of essential nutrients for human body. The main function of folic acid is to enhance the immunity of the organism and participate in hematopoiesis. If the human body is lack of folic acid, megaloblastic anemia and leukopenia can be caused, in addition, folic acid can be taken in pregnancy to prevent the fetus from generating neural tube malformation, such as hydrocephalus and spina bifida, and folic acid taken by the old can be helpful for improving memory and mental symptoms. In addition, folic acid also has the function of anti-tumor, can cause cancer cell apoptosis and has certain influence on the gene expression of cancer cells.
Folic acid has high popularization rate in daily application due to important clinical effects, and particularly has the key quality when being taken as a medicine for pregnant women.
The traditional folic acid production method comprises the steps of preparing p-aminobenzoyl glutamic acid, trichloroacetone and 2,4, 5-triamino-6-hydroxypyrimidine sulfate, and performing cyclization reaction for 6 hours at the pH value of 3.4-3.6 and the temperature of 36-40 ℃ in the presence of sodium acetate and sodium metabisulfite to obtain an orange folic acid crude product. And refining the folic acid crude product with acid and alkali to obtain the folic acid finished product.
Folic acid and D-folic acid are chiral isomers, have similar polarities, and cannot be effectively detected in actual work, so that a chromatographic detection method capable of effectively controlling the impurities of the D-folic acid is urgently needed to be developed, so that an important basis is provided for obtaining qualified folic acid raw material medicines, and evaluation and verification of folic acid quality are facilitated.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the HPLC detection method for folic acid and D-folic acid as the impurity thereof, which can effectively separate folic acid and D-folic acid, and greatly improves the stability, accuracy and sensitivity of the impurity detection method for folic acid, so that folic acid impurities can be simply, quickly, sensitively, accurately and stably detected, and the quality stability of folic acid is effectively ensured.
In order to realize the purpose, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for HPLC detection of folic acid and D-folic acid as an impurity thereof, comprising the following steps:
preparing a sample solution to be detected;
preparing a reference substance solution by using a D-folic acid standard substance;
respectively carrying out HPLC detection on the prepared sample solution to be detected and the prepared reference substance solution; wherein: the HPLC detection conditions were as follows: the chromatographic column adopts a weak anion exchange chiral column; the mobile phase comprises a component A and a component B; the component A is methanol, and the component B is acetic acid solution; the volume ratio of the methanol to the acetic acid solution is (88: 12) - (92: 8) (for example, 8:1, 9:1, 10:1, 11: 1);
calculating the content of the D-folic acid in the sample to be detected: and calculating the content of the D-folic acid in the sample to be detected by using the peak area of the D-folic acid in the sample solution to be detected and the peak area of the D-folic acid in the reference solution by adopting an external standard method.
In the method for detecting folic acid and D-folic acid as an impurity thereof by HPLC, as a preferred embodiment, the volume ratio of the methanol to the acetic acid solution is 90: 10.
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, the weak anion exchange chiral column is CHIRLPAK QD-AX,4.6mm x150mm and 5 μm as a preferred embodiment.
O-9- (tert-butyl ester carbamoyl) quinidine is covalently bonded on the silica gel surface of a chromatographic column CHIRLPAK QD-AX,4.6mmX150mm and 5 mu m.
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, the concentration of the acetic acid solution is 0.1mol/L as a preferred embodiment; preferably, the acetic acid solution is prepared by diluting glacial acetic acid to 0.1mol/L with water.
The glacial acetic acid is too high in concentration, and pipelines of the detection instrument are not tolerant, so that the service life of the detection instrument can be greatly shortened, and the glacial acetic acid is diluted to 0.1mol/L for use, so that the error is reduced, the detection result is more stable and accurate, and the corrosion to the pipelines of the detection instrument is reduced.
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the pH of the acetic acid solution is 4.0; further preferably, the pH of the acetic acid solution is adjusted to 4.0 using ammonia water.
The component B cannot effectively elute the D-folic acid when the pH value is too large, so that the retention time is prolonged; the pH value is too small, and the pipeline of the detection instrument is not tolerant and is easy to corrode. Other alkaline substances such as diethylamine or triethylamine are used for adjusting the pH value, solvent residue is easily generated in the chromatographic column, and the chromatographic column is easily deteriorated and polluted after long-term standing, so that the separation effect is influenced. In the invention, the volume ratio of acetic acid solution: ammonia water (5-7): 1, the pH value of the acetic acid solution is adjusted to be 4.0 as the component B, so that the D-folic acid can be effectively eluted, and the corrosion to an instrument or the residue in a chromatographic column can be avoided.
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, a preferable embodiment is one in which the HPLC detection conditions are such that the column temperature is 25 to 35 ℃ (e.g., 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃).
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the flow rate of the mobile phase under the HPLC detection conditions is 1.0-1.2mL/min (e.g., 1.05mL/min, 1.1mL/min, 1.15 mL/min).
In the method for HPLC detection of folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the detection wavelength under the HPLC detection conditions is 254nm to 280nm (e.g., 260nm, 265nm, 270nm, 275 nm).
In the method for detecting folic acid and D-folic acid as an impurity thereof by HPLC, the detector in the HPLC detection condition is a PDA detector as a preferred embodiment.
In the method for detecting folic acid and D-folic acid as an impurity thereof by HPLC, as a preferred embodiment, the sample volume of the sample solution to be detected and the control solution in the HPLC detection condition is 20 μ L.
In the HPLC detection method of folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the preparation step of the sample solution to be detected includes: weighing a sample to be detected with the weight m, dissolving the sample to be detected by using a solvent, and diluting the sample to be detected to a constant volume V by using a diluent; preferably, the solvent is a 28.6g/L sodium carbonate solution; preferably, the diluent is water.
The folic acid is dissolved in a sodium carbonate solution and then diluted by water, the whole system is an alkali liquor water system, the folic acid can be completely dissolved in the alkali solution, if other organic solvents such as methanol are used as a diluent, the folic acid cannot be completely dissolved, the accuracy of the solution is easily affected, and the folic acid can be improved in solubility and has the advantages of cost reduction, safety, no pollution and the like by using water as the diluent.
In the method for HPLC detection of folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the step of preparing the control solution comprises: precisely weighing a proper amount of D-folic acid standard substance, firstly dissolving the D-folic acid standard substance by using a solvent used in the preparation of a sample solution to be detected, and then diluting by using a diluent used in the preparation of the sample solution to be detected to obtain a solution with the concentration of C 0 The control solution of (4); preferably, the concentration of D-folic acid in the control solution is 1 mug/mL.
In the HPLC method for detecting folic acid and D-folic acid as an impurity thereof, as a preferred embodiment, the calculation formula for calculating the content of D-folic acid in the sample to be detected is:
in the formula, the first step is that,
x: the mass content of the D-folic acid in the sample to be detected is as follows: percent;
a: peak area of D-folic acid in the sample solution to be detected;
A 0 : peak area of D-folic acid in the control solution;
C 0 : control solution concentration, unit: mu g/mL;
v: the final volume of constant volume after the sample to be detected is prepared into solution, unit: mL;
m: sample weighing and weighing for the sample to be detected, unit: and (5) mg.
Compared with the prior art, the invention has the beneficial effects that:
(1) because D-folic acid is a chiral isomer of folic acid, and the chiral isomer is difficult to be effectively separated by a conventional alkylsilane bonded silica gel chromatographic column, the method adopts a weak anion exchange chiral column suitable for chiral separation of an acidic compound to carry out detection methodology verification on the residual quantity of D-folic acid in folic acid.
(2) A blank solution prepared by diluting 2mL of 28.6g/L sodium carbonate solution with water to 100mL does not interfere with the D-folic acid peak, which indicates that the system applicability of the detection method is good.
(3) The sensitivity is higher, and the content of D-folic acid with the content of 48ng/mL can be detected.
(4) The linear range of the measured D-folic acid is 95.7-1910 ng/mL, and the correlation coefficient R 2 Not less than 0.9998, simple, fast, accurate, stable and reliable method, and various technical indexes meet the detection requirements.
(5) The detection method of the invention can ensure that the chromatographic peak separation degree of folic acid and D-folic acid is more than 5.
(6) The operation method provided by the invention is simple, strong in operability, high in precision, good in reproducibility, and less than or equal to 2% of RSD (relative standard deviation), and less than or equal to 0.5% of RSD in the preferred embodiment, so that the accuracy of data is effectively guaranteed, the cost is low, and the method is suitable for various types of laboratory detection and industrial application.
Drawings
FIG. 1 HPLC chromatogram for detecting the content of D-folic acid in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the following will explain in detail the separation and detection method of folic acid and its impurity D-folic acid with reference to the examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1) Chromatographic conditions
The instrument comprises the following steps: japanese Shimadzu LC-20AT high performance liquid chromatograph;
a chromatographic column: weak anion exchange chiral column CHIRLPAK QD-AX for chiral separation of acidic compound, wherein o-9- (tert-butyl ester carbamoyl) quinidine is covalently bonded on the surface of silica gel, and the thickness is 4.6mm, 150mm and 5 μm;
column temperature: 30 ℃;
mobile phase: the component A is methanol, and the component B is 0.1mol/L acetic acid solution (the PH is adjusted to 4.0 by ammonia water); a: b is 90: 10;
flow rate: 1.2 mL/min;
detection wavelength: 280 nm;
a detector: a PDA detector.
2) Sample configuration
Test solution (i.e., sample solution to be tested): taking 20mg of folic acid test product, precisely weighing, placing in a 100mL measuring flask, adding 2mL of 28.6g/L sodium carbonate solution to dissolve folic acid, adding water to constant volume to scale, making 0.2mg/mL folic acid solution, and shaking up to obtain test solution.
Control solution: a proper amount of D-folic acid standard substance is precisely weighed, firstly dissolved quantitatively by 2mL of 28.6g/L sodium carbonate solution, and then diluted by water to prepare 1 mu g/mL of D-folic acid solution as a reference substance solution.
3) Calculation method
Respectively taking 20 μ L of reference solution and sample solution, injecting into HPLC for analysis, and calculating according to external standard method and peak area.
Calculating the formula:
remarking:
x: the mass content of the D-folic acid in the test sample is as follows: percent;
a: peak area of D-folic acid in the test solution;
A 0 : peak area of D-folic acid in the control solution;
C 0 : control solution concentration, unit: mu g/mL;
v: volume of the test sample with constant volume, unit: mL;
m: sample weighing and measuring unit: mg;
the limit requirements are as follows: the folic acid contains no more than 0.15% of D-folic acid.
4) Results of the experiment
The chromatogram of this example is shown in FIG. 1, and the retention time of the chromatographic peak and the degree of separation of folic acid and D-folic acid are shown in Table 1 below.
Detection example 1
The detection method in the embodiment 1 is examined according to the conventional test mode in the field, the detection limit and the quantification limit of the detection method are mainly examined, the linear interval of the content determination of the folic acid impurity D-folic acid is determined, a quantitative standard curve of the D-folic acid is prepared, and the repeatability, the accuracy and the solution stability of the method are examined.
(1) Investigation test of detection limit and quantification limit: weighing a proper amount of D-folic acid, dissolving the D-folic acid with 2mL of diluent (28.6g/L of sodium carbonate) respectively, diluting the D-folic acid with water into a solution of 1 mu g/mL of D-folic acid, then continuously diluting the D-folic acid with water to obtain solutions to be detected with different concentrations, detecting the solutions to be detected according to the chromatographic conditions in the embodiment 1, diluting the solutions according to the signal-to-noise ratio of each peak in a detection map, wherein the concentration of the solutions to be detected is the detection limit when the signal-to-noise ratio is 3, the concentration of the solutions to be detected is the quantification limit when the signal-to-noise ratio is 10, and the detection results are shown in the following table 3.
(2) Linear investigation test: preparation of standard stock solution: taking 10mg of a D-folic acid reference substance (with the purity of 93.3 percent), precisely weighing 10.26mg, placing the D-folic acid reference substance into a 100mL measuring flask, adding about 2mL of 28.6g/L sodium carbonate solution to dissolve the D-folic acid reference substance, diluting with water to a constant volume, shaking up to obtain a standard stock solution, wherein the concentration of D-folic acid in the standard stock solution is 95.73 mu g/mL.
Diluting the standard stock solution with water to be 0.1-2 times of the limit concentration (95ng/mL) obtained in the limit concentration test in the step (1) to obtain the following 7 solutions to be detected with different concentrations, namely 95.73ng/mL (10% limit concentration), 191.46ng/mL (20% limit concentration), 382.92ng/mL (40% limit concentration), 765.84ng/mL (80% limit concentration), 957.3ng/mL (100% limit concentration), 1531.68ng/mL (160% limit concentration) and 1914.6ng/mL (200% limit concentration), according to the chromatographic conditions of the example 1, feeding 1 needle into each solution to be detected with each concentration, recording the peak area of the chromatogram, and making a linear equation of the concentration and the peak area, wherein the obtained linear equation and the related coefficient are shown in the following table 3.
(3) And (3) repeatability inspection:
the reproducibility was investigated by sample recovery tests.
Preparation of standard stock solution: taking about 10mg of the D-folic acid reference substance, precisely weighing, placing in a 100mL measuring flask, adding about 2mL of 28.6g/L sodium carbonate solution, dissolving, diluting with water to a constant volume, and shaking up.
Weighing about 20mg of a test sample (folic acid), precisely weighing, placing in a 100mL measuring flask, precisely adding 1mL of the standard stock solution, fully mixing, adding 2mL of 28.6g/L sodium carbonate solution for dissolving, adding water for diluting to a scale, shaking uniformly, preparing 6 parts of the solution in parallel, taking 20 μ L of each solution, and detecting according to the detection method in example 1, wherein the 20 μ L of each solution is marked as 100% tagger-1, 100% tagger-2, 100% tagger-3, 100% tagger-4, 100% tagger-5 and 100% tagger-6, and the detection results are shown in the following table 2. The calculation method of the recovery rate comprises the following steps: (the content of the D-folic acid in the test sample is subtracted from the measured amount of the D-folic acid)/the added amount is multiplied by 100 percent.
TABLE 2
(4) And (3) solution stability investigation: and (3) sampling 7 solutions to be detected with different concentrations, which are prepared in the linear investigation step, at 0 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours and 24 hours according to the chromatographic conditions in the example 1, and recording a chromatogram.
Specific results are shown in Table 3 below
TABLE 3
Comparative example 1
This comparative example was identical to example 1 except that the flow rate was different from example 1, and the process conditions and operation were the same, and the flow rates of this comparative example were: 1.0 ml/min. However, in comparative example 1, there were disadvantages that the peak-forming time was slow, and that the peak width was large, the peak shape was not sharp enough, and the appearance was not beautiful.
Comparative example 2
This comparative example was the same as example 1 except that the mobile phase was different from example 1, and the process conditions and operation were the same as in example 1, and the mobile phase of this comparative example was: the component A is methanol, the component B is 0.1mol/L acetic acid solution (the PH is adjusted to 5.0 by ammonia water); a: and B is 90: 10. However, under the condition, folic acid and D-folic acid can not be well separated, and the separation degree is low.
Comparative example 3
The process conditions and operation of this comparative example were the same as in example 1 except that the diluent used in the sample preparation step was not water, the diluent used in the sample preparation step of this comparative example was a 50% aqueous methanol solution, and the specific sample preparation steps were as follows:
test solution (i.e., sample solution to be tested): taking 20mg of folic acid test product, precisely weighing, placing in a 100mL measuring flask, adding 2mL of 28.6g/L sodium carbonate solution to dissolve folic acid, adding 50% methanol water solution to constant volume to scale, making 0.2mg/mL folic acid solution, and shaking up to obtain test solution.
Control solution: a proper amount of D-folic acid standard substance is precisely weighed, firstly dissolved quantitatively by 2mL of 28.6g/L sodium carbonate solution, and then diluted by 50% methanol water solution to prepare 1 mu g/mL of D-folic acid solution as a reference substance solution.
In this comparative example, since the diluent contained methanol and affected the dissolution of folic acid, the recovery rate was low, all of which were 95% or less, when the detection was performed with accuracy, and was not as high as that in example 1.
The above embodiments are merely examples for clearly illustrating the present invention and do not limit the present invention. Other variants and modifications of the invention, which are obvious to those skilled in the art and can be made on the basis of the above description, are not necessary or exhaustive for all embodiments, and are therefore within the scope of the invention.
Claims (8)
1. An HPLC detection method of folic acid and an impurity D-folic acid thereof comprises the following steps:
preparing a sample solution to be detected;
preparing a reference substance solution by using a D-folic acid standard substance;
respectively carrying out HPLC detection on the prepared sample solution to be detected and the prepared reference substance solution; wherein: the HPLC detection conditions were as follows: the chromatographic column adopts a weak anion exchange chiral column; the mobile phase comprises a component A and a component B; the component A is methanol, and the component B is acetic acid solution; the volume ratio of the methanol to the acetic acid solution is (88: 12) - (92: 8);
calculating the content of the D-folic acid in the sample to be detected: and calculating the content of the D-folic acid in the sample to be detected by using the peak area of the D-folic acid in the sample solution to be detected and the peak area of the D-folic acid in the reference solution by adopting an external standard method.
2. The detection method according to claim 1, wherein the volume ratio of the methanol to the acetic acid solution is 90: 10.
3. The detection method of claim 1, wherein the weak anion exchange chiral column is CHIRLPAK QD-AX,4.6mm x150mm,5 μm.
4. The detection method according to claim 1, wherein the concentration of the acetic acid solution is 0.1 mol/L; preferably, the acetic acid solution is prepared by diluting glacial acetic acid to 0.1mol/L with water; preferably, the pH of the acetic acid solution is 4.0; further preferably, the pH of the acetic acid solution is adjusted to 4.0 using ammonia water.
5. The assay of claim 1, wherein the HPLC assay conditions are at a column temperature of 25-35 ℃; preferably, the flow rate of the mobile phase in the HPLC detection condition is 1.0-1.2 mL/min; preferably, the detection wavelength in the HPLC detection condition is 254 nm-280 nm; preferably, the detector in the HPLC detection condition is a PDA detector; preferably, the sample solution to be detected and the control solution are added in an amount of 20 μ L under the HPLC detection condition.
6. The assay of claim 1, wherein the step of formulating the sample solution to be assayed comprises: weighing a sample to be detected with the weight m, dissolving the sample to be detected by using a solvent, and diluting the sample to be detected to a constant volume V by using a diluent; preferably, the solvent is a 28.6g/L sodium carbonate solution; preferably, the diluent is water.
7. The assay of claim 6, wherein the control solution is formulated by a method comprising: precisely weighing a proper amount of D-folic acid standard substance, firstly dissolving the D-folic acid standard substance by using a solvent used in the preparation of a sample solution to be detected, and then diluting by using a diluent used in the preparation of the sample solution to be detected to obtain a solution with the concentration of C 0 The control solution of (4); preferably, the concentration of D-folic acid in the control solution is 1 mug/mL.
8. The detection method according to claim 7, wherein the calculation formula for calculating the content of D-folic acid in the sample to be detected is:
in the formula, the first step is that,
x: the mass content of the D-folic acid in the sample to be detected is as follows: percent;
a: peak area of D-folic acid in the sample solution to be detected;
A 0 : peak area of D-folic acid in the control solution;
C 0 : control solution concentration, unit: mu g/mL;
v: the final volume of constant volume after the sample to be detected is prepared into solution, unit: mL;
m: sample weighing and weighing for the sample to be detected, unit: and (5) mg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210590278.3A CN115078613A (en) | 2022-05-26 | 2022-05-26 | HPLC detection method for folic acid and D-folic acid as impurity thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210590278.3A CN115078613A (en) | 2022-05-26 | 2022-05-26 | HPLC detection method for folic acid and D-folic acid as impurity thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115078613A true CN115078613A (en) | 2022-09-20 |
Family
ID=83250241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210590278.3A Pending CN115078613A (en) | 2022-05-26 | 2022-05-26 | HPLC detection method for folic acid and D-folic acid as impurity thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115078613A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110190305A1 (en) * | 2010-02-02 | 2011-08-04 | Allos Therapeutics, Inc. | Optically Pure Diastereomers of 10-Propargyl-10-Deazaaminopterin and Methods of Using Same |
| CN105806966A (en) * | 2014-12-30 | 2016-07-27 | 北京斯利安药业有限公司 | Quality control method for high optical purity folic acid preparation process |
| CN111239301A (en) * | 2020-02-14 | 2020-06-05 | 济南康和医药科技有限公司 | Method for detecting content of folic acid impurity D |
| CN112649524A (en) * | 2020-12-02 | 2021-04-13 | 重庆华邦胜凯制药有限公司 | Method for separating and detecting folic acid and folic acid optical isomer in folic acid |
| CN113063889A (en) * | 2021-03-12 | 2021-07-02 | 北京斯利安药业有限公司 | Method for detecting content of folic acid enantiomer |
-
2022
- 2022-05-26 CN CN202210590278.3A patent/CN115078613A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110190305A1 (en) * | 2010-02-02 | 2011-08-04 | Allos Therapeutics, Inc. | Optically Pure Diastereomers of 10-Propargyl-10-Deazaaminopterin and Methods of Using Same |
| CN105806966A (en) * | 2014-12-30 | 2016-07-27 | 北京斯利安药业有限公司 | Quality control method for high optical purity folic acid preparation process |
| CN111239301A (en) * | 2020-02-14 | 2020-06-05 | 济南康和医药科技有限公司 | Method for detecting content of folic acid impurity D |
| CN112649524A (en) * | 2020-12-02 | 2021-04-13 | 重庆华邦胜凯制药有限公司 | Method for separating and detecting folic acid and folic acid optical isomer in folic acid |
| CN113063889A (en) * | 2021-03-12 | 2021-07-02 | 北京斯利安药业有限公司 | Method for detecting content of folic acid enantiomer |
Non-Patent Citations (4)
| Title |
|---|
| DANCEL GROUP: "INSTRUCTION MANUAL FOR CHIRALPAK QN-AX and CHIRALPAK QD-AX", pages 1, Retrieved from the Internet <URL:https://mz-at.de/fileadmin/user_upload/Brochures/CHIRALPAK-QD-AX_QN-AX.pdf> * |
| 刘育坚 等: "反相手性液相色谱法在对映体拆分中的应用研究", 化学试剂, no. 07 * |
| 赵琦 等: "HPLC法测定叶酸原料中光学异构体的含量", 药学研究, vol. 41 * |
| 郑彦云 等: "化学检验技术", 暨南大学出版社, pages: 104 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111855856B (en) | Method for simultaneously detecting contents of sodium azide, sodium nitrite and sodium nitrate in sodium azide synthesis reaction liquid | |
| CN111443151B (en) | Method for detecting content of trace cysteine in compound amino acid injection | |
| CN110940745A (en) | Method for detecting related substances in S-2-amino-2- (2-chlorphenyl) methyl acetate or salts thereof | |
| CN111537637A (en) | Method for directly measuring 3 degradation products of tryptophan in compound amino acid injection | |
| CN107870209B (en) | Method for determining impurity content in linagliptin bulk drug | |
| CN107271592B (en) | Liquid chromatographic purity detection method for completely separating tipiraxib hydrochloride from related impurities | |
| CN112763629A (en) | Method for simultaneously detecting 6 acidifiers in compound acidifier | |
| CN110220989B (en) | Method for detecting fasudil hydrochloride and 9 related substances thereof | |
| CN115078613A (en) | HPLC detection method for folic acid and D-folic acid as impurity thereof | |
| CN108645925B (en) | Method for detecting related substances in cinnamyl octane related products through high performance liquid chromatography | |
| CN113820409B (en) | Method for detecting related substances in mother nucleus of moxifloxacin | |
| CN115616133A (en) | Method for detecting cysteine in compound amino acid injection and application thereof | |
| CN113484450B (en) | Derivatization treatment method for detecting drug enantiomer, determination method and application | |
| CN112129847A (en) | Method for detecting content of D-p-hydroxyphenylglycine methyl ester | |
| CN111443150A (en) | Method for detecting content of acetylcysteine and acetyltyrosine in compound amino acid injection | |
| CN107091895B (en) | Method for separating and measuring related substances in riociguat raw material medicine by adopting HPLC (high performance liquid chromatography) | |
| CN112881538A (en) | Detection method for impurities and enantiomers in fudosteine and fudosteine tablets | |
| CN110824059A (en) | Detection method of formyl impurities in febuxostat | |
| CN113514582B (en) | Impurity detection method for p-nitro-alpha-acetamido-beta-hydroxy propiophenone | |
| CN112379018B (en) | Method for detecting 3-methylxanthine in linagliptin starting material A | |
| CN117310021B (en) | Method for separating and detecting related substances of ubenimex by high performance liquid chromatography | |
| CN117310022B (en) | Method for separating and detecting related substances of quinuclidine benzhydrol as intermediate of ubenimex | |
| CN116879428B (en) | High performance liquid analysis method for residual content of phosphorylcholine in L-alpha-phosphorylcholine | |
| CN115266963B (en) | Method for detecting cysteine and cystine in compound amino acid injection | |
| CN113917043B (en) | Detection method of afatinib maleate genotoxic impurities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |