CN117388404A - Method for detecting impurity tromethamine residue in pregabalin - Google Patents
Method for detecting impurity tromethamine residue in pregabalin Download PDFInfo
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- CN117388404A CN117388404A CN202311454821.8A CN202311454821A CN117388404A CN 117388404 A CN117388404 A CN 117388404A CN 202311454821 A CN202311454821 A CN 202311454821A CN 117388404 A CN117388404 A CN 117388404A
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 30
- 229960001233 pregabalin Drugs 0.000 title claims abstract description 22
- AYXYPKUFHZROOJ-ZETCQYMHSA-N pregabalin Chemical compound CC(C)C[C@H](CN)CC(O)=O AYXYPKUFHZROOJ-ZETCQYMHSA-N 0.000 title claims abstract description 22
- 239000012535 impurity Substances 0.000 title abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 229960000281 trometamol Drugs 0.000 claims abstract description 51
- 238000001212 derivatisation Methods 0.000 claims abstract description 28
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 62
- 238000012360 testing method Methods 0.000 claims description 39
- 239000000523 sample Substances 0.000 claims description 27
- 239000013558 reference substance Substances 0.000 claims description 25
- 238000005303 weighing Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000012488 sample solution Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 14
- 239000000543 intermediate Substances 0.000 abstract description 13
- 239000007788 liquid Substances 0.000 abstract description 12
- 229940079593 drug Drugs 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 3
- 238000000105 evaporative light scattering detection Methods 0.000 abstract description 3
- 238000005251 capillar electrophoresis Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 abstract description 2
- -1 amino compound Chemical class 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 239000002904 solvent Substances 0.000 description 29
- 238000002156 mixing Methods 0.000 description 26
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 24
- 239000007983 Tris buffer Substances 0.000 description 19
- 238000007865 diluting Methods 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000012071 phase Substances 0.000 description 14
- 239000011550 stock solution Substances 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 10
- 238000001816 cooling Methods 0.000 description 10
- 238000010438 heat treatment Methods 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 9
- UGJMXCAKCUNAIE-UHFFFAOYSA-N Gabapentin Chemical compound OC(=O)CC1(CN)CCCCC1 UGJMXCAKCUNAIE-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000007789 sealing Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 229960002870 gabapentin Drugs 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- AMDBBAQNWSUWGN-UHFFFAOYSA-N Ioversol Chemical compound OCCN(C(=O)CO)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I AMDBBAQNWSUWGN-UHFFFAOYSA-N 0.000 description 3
- 239000001961 anticonvulsive agent Substances 0.000 description 3
- 229960004537 ioversol Drugs 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229960003965 antiepileptics Drugs 0.000 description 2
- 239000006177 biological buffer Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 208000001640 Fibromyalgia Diseases 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010036376 Postherpetic Neuralgia Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001773 anti-convulsant effect Effects 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000000949 anxiolytic effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 230000001037 epileptic effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000004868 gas analysis Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 238000001474 liquid chromatography-evaporative light scattering detection Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012088 reference solution Substances 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
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
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- 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
-
- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
-
- 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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention provides a method for detecting the content of trace impurity tromethamine in pregabalin finished products and intermediates by combining a high performance liquid chromatography-ultraviolet detector with pre-column derivatization. The fluorescent group is added on the amino group of the tromethamine by adopting a low-cost derivatization reagent 2, 4-dinitrofluorobenzene, and the modified tromethamine derivative has ultraviolet absorption and is quantitatively analyzed by utilizing high performance liquid chromatography. The invention overcomes the interference of amino groups on the structure of pregabalin finished products and intermediates on the derivative reaction, and realizes the accurate quantitative detection of the tromethamine residue in the amino compound medicines. The method provided by the invention has the advantages of strong specificity, low sensitivity and good durability, and the high performance liquid chromatograph-ultraviolet detector detection equipment used by the method is easy to obtain, enriches the detection method of the tromethamine content, and breaks through the limitations of the conventional quantitative detection methods such as ion chromatograph, capillary electrophoresis apparatus and evaporative light scattering detector equipment.
Description
Technical Field
The invention provides a method for detecting the content of tromethamine, and belongs to the technical field of drug detection.
Background
Pregabalin (II), an antiepileptic drug, is clinically used to treat postherpetic neuralgia and fibromyalgia. In recent years, medicines for treating epilepsy are more and more, wherein gabapentin medicines have certain advantages in treating the diseases, and pregabalin has similar structure and function to gabapentin and has antiepileptic, analgesic and anxiolytic activities. In laboratory studies, pregabalin has anticonvulsant activity against various epileptic models; the activity spectrum of the animal model is similar to that of gabapentin, but the activity of pregabalin is 3-10 times that of gabapentin.
Tromethamine (Tris for short in English), which is a weak base, is commonly used as a biological buffer system in the production process of biological products. Tris can cause hypoglycemia, hypotension, nausea, vomiting, and can also inhibit or even stop breathing. Excessive intake or renal insufficiency can cause alkaline symptoms.
Since Tris-HCl buffer solution is used in many drug preparation processes, to determine the Tris removal effect in the production process, the sample solution before and after purification needs to be detected, and the maximum residual amount of Tris in the drug is required to be not higher than 100ppm (0.1 mg/mL) by FDA. Currently, the method for detecting Tris in the chinese pharmacopoeia (2020 edition) is: about 0.25g of the product is taken, precisely weighed, 80ml of water is added for dissolution, 2-3 drops of methyl red indicator liquid are added, and hydrochloric acid titration solution (0.1 mol/L) is used for titration, thus obtaining the product. Each 1ml of hydrochloric acid titration (0.1 mol/L) corresponds to 12.11mg of C4H11NO3. When the acid-base titration method is adopted to measure the content of the Tris raw material, the interference of the main medicine and other auxiliary materials may cause larger difference between the measurement result and the prescription amount of a manufacturer, and the measurement result is inaccurate. And the biological product contains a large amount of carboxyl, amino and anions and cations, so that the Tris in the product cannot be quantitatively detected by a titration method.
Tromethamine is a biological buffer (Tris-HCl buffer solution) added during the synthesis of pregabalin intermediates, which remains weak on conventional reverse phase chromatography columns without uv absorption; tris has a high boiling point (220 ℃) and is difficult to gasify in gas chromatography, and is not suitable for gas analysis after formation of Tris-HCl salt, so that the control in production is not easy. As Tris has no ultraviolet absorption and large polarity, an ultraviolet detector cannot effectively detect by adopting a high performance liquid chromatography, and the existing literature reports that an ion chromatograph, a differential refraction detector, an evaporative light scattering detector, an electrospray detector and a capillary electrophoresis apparatus are adopted, the methods have the characteristics of poor precision, complex operation, expensive detection equipment and the like. There are also reports of pre-column derivatization of Tris with 6-aminoquinolinyl-N-hydroxysuccinimidyl formate (AQC), but the derivatization reagent is expensive, about 100 times of the 2, 4-dinitrofluorobenzene as the derivatization reagent used in the present invention, and for drugs containing amino groups in structure such as pregabalin and its intermediates, in order to ensure complete derivatization of Tris, additional derivatization reagents are required to ensure complete derivatization of both the drug itself and Tris, so that accurate detection of Tris content in the amino drug can be achieved.
Further, as a result of the search, "determination of tromethamine in ioversol injection by HPLC" Zhang Mian, journal of western medicine, 2012,27 (6): 722-723 discloses a method for detecting Tris in ioversol injection: the ammonium dihydrogen phosphate buffer is used as a mobile phase, and a differential refraction detector is used as a detector to detect Tris in the ioversol injection. But the detection sensitivity is low, and the detection limit height cannot meet the existing detection requirement.
Patent CN112946123 a discloses a method for detecting tromethamine, which can effectively separate other substances in a detection sample from tris by combining high performance liquid chromatography-evaporative light scattering method, but the sensitivity of the evaporative light scattering detector is poor, the detection limit of the method is 14.5ppm, and the quantitative limit is 49.6ppm. In addition, the detection equipment of the evaporation light detector is expensive, the operation is complex, and the detection cost is high.
Therefore, providing a rapid, simple, economical and widely applicable Tris liquid phase detection method is a technical problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention aims to provide a method for simply, economically and efficiently detecting the Tris content in pregabalin or an intermediate thereof.
The invention provides a method for detecting trace Tris in pregabalin or an intermediate thereof, which comprises the following steps:
(1) Preparing a reference substance solution:
precisely weighing a proper amount of tromethamine reference substance to prepare a reference substance solution with a certain concentration. Precisely measuring a proper amount of the solution, adding a derivatization reagent 2, 4-dinitrofluorobenzene for derivatization, and preparing a reference substance solution;
(2) Preparing a sample solution of a test sample:
adding a derivatization reagent 2, 4-dinitrofluorobenzene into pregabalin or an intermediate thereof to be detected for derivatization treatment, and preparing a sample solution;
(3) And (3) sample injection detection:
detecting and analyzing the reference substance solution and the sample solution in the step (1) and the step (2) by adopting a high performance liquid chromatography ultraviolet detector; and precisely measuring 10 mu l of a derivative test sample, injecting the sample into a liquid chromatograph after the chromatographic column is balanced according to preset chromatographic conditions, and collecting a chromatogram.
(4) Calculating the content of tromethamine:
wherein:
w pairs: weighing the reference substance; p pairs: the content of the reference substance;
v pairs: dilution of the reference substance; and A pair: peak area of tromethamine derivative in control solution;
sample a: peak area of tromethamine derivative in the sample solution; v sample: dilution of the test sample;
w sample: sample weighing is carried out on the test sample.
Further, the concentration range of the reference substance solution prepared in the step (1) is 0.05 mug/ml to 0.3 mug/ml.
Further, the chromatographic conditions at the time of detection in the step (3) are:
a detector: an ultraviolet detector;
chromatographic column: octadecylsilane chemically bonded silica column;
the detection wavelength is as follows: 360nm;
mobile phase: water-acetonitrile 42:58-38:62;
the flow rate of the mobile phase is as follows: 0.8-1.2 ml/min
Column temperature: 27-33 ℃;
sample injection volume: 10 μl.
Still further, the mobile phase: the volume ratio of water to acetonitrile is in the range of 40:60.
further, the detection limit of the tromethamine obtained in the step (4) is 3ppm, and the quantitative limit is 10ppm.
The analysis method of trace impurity tromethamine in pregabalin or intermediate sample adopts the derivatization reagent 2, 4-dinitrofluorobenzene to conduct pre-column derivatization on tromethamine before detection, and then uses high performance liquid chromatography-ultraviolet method to detect. The method has strong anti-interference capability, even if pregabalin and the intermediate thereof contain amino, the complete derivatization of the tromethamine in the amino medicine can be realized through the developed derivatization conditions, and the sample adding recovery rate is good. The derivatization reagent 2, 4-dinitrofluorobenzene adopted by the method is low in price and easy to obtain, and the detection cost is effectively saved. The method is simple to operate, high in sensitivity and wide in linear range, and can be used for rapidly and effectively detecting the content of the trace impurity tromethamine in the pregabalin or intermediate samples, so that the impurity residue in the pregabalin medicine can be effectively controlled, and the medication safety of patients can be protected.
Drawings
FIG. 1 is a graph of the hollow white solution of example 1;
FIG. 2 is a graph of a specific solution for tromethamine assay in example 1;
FIG. 3 is a graph of the limit of detection solution (3 ppm) for tromethamine assay in example 1;
FIG. 4 is a graph of the quantitative limit solution (10 ppm) of the tromethamine assay of example 1;
FIG. 5 is a graph of a control solution (0.2. Mu.g/ml) for tromethamine assay in example 1;
FIG. 6 is a graph of the accuracy of the tromethamine assay in example 1;
FIG. 7 is a graph showing the detection of tromethamine (2 mg/ml) in pregabalin intermediate to be tested in example 1;
fig. 8 is a linear regression graph of test example 2.
Detailed Description
Examples
Chromatographic conditions
Instrument: high performance liquid chromatograph equipped with ultraviolet detector
Chromatographic column: siemens flight Hypersil GOLD C18,250mm 4.6mm,5 μm
Mobile phase: water-acetonitrile (40:60); flow rate: 1ml/min; sample injection volume: 10 μl of
Column temperature: 30 ℃; run time: for 40min; detection wavelength: 360nm; solvent: methanol
Derivatizing reagent solution: weighing a proper amount of 2, 4-dinitrofluorobenzene, precisely weighing, adding a proper amount of solvent, dissolving and quantitatively diluting to prepare a solution containing 300mg per 1 ml.
Blank solution: precisely weighing 1ml of methanol, placing into a 50ml measuring flask, respectively adding 128mg of sodium hydroxide and 1ml of derivative reagent solution, sealing, and mixing. Heating in water bath at 60deg.C for 2 hr, taking out, cooling to room temperature, adding appropriate amount of solvent, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing.
Tromethamine stock solution: taking a proper amount of tromethamine reference substance, precisely weighing, adding a proper amount of solvent for dissolving and quantitatively diluting to prepare a solution containing about 10 mug per 1 ml.
Control solution: precisely measuring 1ml of tromethamine stock solution, placing into a 50ml measuring flask, adding 128mg of sodium hydroxide and 1ml of derivatization agent solution, sealing, and mixing uniformly. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing.
Quantitative limiting solution: precisely measuring 1ml of tromethamine stock solution, placing into a 10ml measuring flask, diluting to scale with solvent, mixing well, and taking as quantitative limit stock solution. Precisely measuring 1ml of quantitative limit stock solution, placing into a 50ml measuring flask, adding 128mg of sodium hydroxide and 1ml of derivatization agent solution, sealing, and mixing. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing.
Accuracy solution: about 100mg of pregabalin intermediate is taken, precisely weighed, placed in a 50ml measuring flask, and 1ml of a storage solution is quantitatively limited by adding tromethamine to dissolve. 128mg of sodium hydroxide and 1ml of derivatizing agent solution are added, sealed and uniformly mixed. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing.
Test solution: about 100mg of pregabalin intermediate is taken, precisely weighed, placed in a 50ml measuring flask, and 1ml of solvent is added to dissolve. 128mg of sodium hydroxide and 1ml of derivatizing agent solution are added, sealed and uniformly mixed. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing.
After the derivatization reaction was completed, each solution was transferred to a liquid-phase sample vial. Precisely measuring 10 μl, injecting into a liquid chromatograph, and recording the chromatogram. The peak area calculated by the external standard method should not exceed 100ppm.
Test example 1 System applicability test
Taking a proper amount of tromethamine reference substance, precisely weighing, adding a proper amount of solvent for dissolving and quantitatively diluting to prepare a solution containing about 10 mug per 1 ml. Precisely weighing 1ml of the solution, placing into a 50ml measuring flask, adding 128mg of sodium hydroxide and 1ml of derivatization agent solution, sealing, and mixing uniformly. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing. Precisely measuring 10 μl of the solution, injecting into a liquid chromatograph, continuously sampling for 6 times, and recording the chromatogram. The results showed that the tromethamine derivative had a retention time with an RSD value of 0.03%, a peak area with an RSD value of 0.74% and less than 2.0%, a theoretical plate number and a tailing factor all meeting the requirements, and the test results are shown in table 1 below.
Table 1 results of System suitability test
Test example 2 Linear Range test
Taking a proper amount of tromethamine reference substance, precisely weighing, adding a proper amount of solvent, dissolving and quantitatively diluting to prepare a solution with the concentration of about 50 mug per 1ml, and taking the solution as a stock solution. The stock solution was precisely measured and diluted with methanol to prepare a series of reference solutions having concentrations of 1. Mu.g/ml, 5. Mu.g/ml, 8. Mu.g/ml, 10. Mu.g/ml, and 15. Mu.g/ml. Precisely weighing 1ml of each solution, placing into 50ml measuring flask, adding 128mg of sodium hydroxide and 1ml of derivatization agent solution, sealing, and mixing. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing. 10 μl of the solution was measured precisely, and the solution was poured into a liquid chromatograph to record a chromatogram. The concentration of the derivative tromethamine reference substance is respectively 0.02 mug/ml, 0.1 mug/ml, 0.16 mug/ml, 0.2 mug/ml and 0.3 mug/ml, and linear regression is carried out by taking the concentration (mug/ml) of the tromethamine reference substance as an x axis and the peak area as a y axis, so as to obtain a linear regression equation: y= 1409.3744x-0.0029, r=0.9993. The results show that when the concentration of tromethamine in the solution is in the range of 0.02-0.3 mug/ml, the linear relation between the concentration and the peak area is good. The test results are shown in Table 2, FIG. 8.
TABLE 2 results of Linear and Range experiments
Test example 3 limit of detection test
3ml of the control solution of 1. Mu.g/ml in test example 2 was precisely measured, placed in a 10ml measuring flask, and diluted with methanol to prepare a detection limit solution having a concentration of 0.3. Mu.g/ml. Precisely measuring 1ml of detection limit solution, placing into a 50ml measuring flask, adding 128mg of sodium hydroxide and 1ml of derivatization agent solution, sealing, and mixing. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing. 10 μl of the solution was measured precisely, and the solution was poured into a liquid chromatograph to record a chromatogram. The concentration of the detection limit solution of the tromethamine after the derivatization is 0.024 mug/ml, and the result shows that the signal to noise ratio of the tromethamine derivative is 5.2 and is more than 3. The test results are shown in Table 3, FIG. 3.
TABLE 3 detection limit test results
Test example 4 quantitative limit test
0.0213. Mu.g/ml of the control solution in test example 2 was taken. Precisely measuring 10 μl of the solution, injecting into a liquid chromatograph, continuously sampling for 6 times, and recording the chromatogram. The test results are shown in Table 4, FIG. 4.
TABLE 4 quantitative limit test results
Test example 5 accuracy test
Taking a proper amount of tromethamine reference substance, precisely weighing, adding a proper amount of solvent, dissolving and quantitatively diluting to prepare a solution with the concentration of about 50 mug per 1ml, and taking the solution as a stock solution. The stock solution was precisely measured and diluted with methanol to prepare accurate solutions of 5. Mu.g/ml, 10. Mu.g/ml and 15. Mu.g/ml, respectively, in the order of 50%,100% and 150% in level. About 100mg of the product is taken, precisely weighed, placed in a 50ml measuring flask, and 1ml of each concentration accuracy solution is added respectively to dissolve. 128mg of sodium hydroxide and 1ml of derivatizing agent solution are added, sealed and uniformly mixed. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing. Test solutions at each concentration were prepared in triplicate. Precisely measuring 10 μl of each solution, injecting into a liquid chromatograph, recording the chromatogram, and calculating recovery rate of tromethamine. The experimental results show that the recovery rate of the tromethamine with each concentration is 93-102%, the RSD of the recovery rate is 2.7%, the experimental results are shown in Table 5, and the results show that the method is good in accuracy and reliable in experimental results.
TABLE 5 sample recovery test results
Test example 6 durability test
Table 6 durability inspection project
6.1 Effect of column temperature
Tromethamine stock solution: taking a proper amount of tromethamine reference substance, precisely weighing, adding a proper amount of solvent for dissolving and quantitatively diluting to prepare a solution containing about 10 mug per 1 ml.
Durable solution: about 100mg of the product is taken, precisely weighed, placed in a 50ml measuring flask, and 1ml of tromethamine stock solution is added to dissolve the product. 128mg of sodium hydroxide and 1ml of derivatizing agent solution are added, sealed and uniformly mixed. Heating in water bath at 60deg.C for 2 hr, cooling to room temperature, adding appropriate amount of solvent, mixing, adding 1mol/L hydrochloric acid aqueous solution 5ml, diluting with solvent to scale, and mixing. Duplicate was prepared in duplicate.
Under the condition that the rest detection conditions are unchanged, the column temperature of the chromatographic column is changed, the influence on the detection result is examined, and the test result is shown in Table 7. The results show that when the column temperature is 28-32 ℃, no obvious influence is caused on the detection of tromethamine, and the method disclosed by the invention has good durability on the change of the column temperature.
TABLE 7 column temperature tolerance investigation results
6.2 influence of flow Rate
Under the condition that the rest detection conditions are unchanged, the flow rate of the mobile phase is changed, the influence on the detection result is examined, and the test result is shown in Table 8. The result shows that when the flow rate of the mobile phase is 0.8-1.2 ml/min, no obvious influence is caused on the detection of tromethamine, and the method provided by the invention has good durability on the change of the flow rate of the mobile phase.
TABLE 8 flow resistance investigation results
6.3 Effect of mobile phase ratio
Under the condition that the rest detection conditions are unchanged, the proportion of the mobile phase is changed, the influence on the detection result is examined, and the test result is shown in Table 9.9, the results show that when the mobile phase ratio is water-acetonitrile (42:58) - (38:62), no obvious influence is caused on tromethamine detection, and the method provided by the invention has good durability on the change of the mobile phase ratio.
TABLE 9 Mobile phase proportional tolerance test results
6.4 influence of wavelength
Under the condition that the rest detection conditions are unchanged, the wavelength is changed, the influence on the detection result is examined, and the test result is shown in table 10. The result shows that when the detection wavelength is 358-365 nm, no obvious influence is caused on the detection of tromethamine, and the method disclosed by the invention has good durability on wavelength change.
Table 10 wavelength tolerance investigation results
6.5 Effect of different chromatographic columns
Under the condition that the rest detection conditions are unchanged, different octadecylsilane chemically bonded silica columns are used, the influence on the detection result is examined, and the test result is shown in table 11. The results show that there is no significant effect on tromethamine detection when different chromatographic columns are used.
Table 11 column tolerance test results
The results of the tests for each examined tolerating condition are summarized in Table 12, with an RSD of 2.2%. The results show that the small changes of column temperature, flow rate, mobile phase proportion, wavelength and different chromatographic column test conditions have no obvious influence on sample detection.
Table 12 summary of the results of the tolerance test investigation
The above description is merely illustrative of the preferred embodiments of the present invention, and the present invention is not limited to the above embodiments, and any changes and modifications of the present invention fall within the scope of the present invention.
Claims (6)
1. A method for detecting the content of trace tromethamine in pregabalin or an intermediate thereof comprises the following steps:
preparing a reference substance solution:
precisely weighing tromethamine reference substance to prepare a reference substance solution with a certain concentration, precisely weighing a proper amount of the reference substance solution, adding a derivatization reagent 2, 4-dinitrofluorobenzene for derivatization, and preparing the reference substance solution;
preparing a sample solution of a test sample:
adding a derivatization reagent 2, 4-dinitrofluorobenzene into pregabalin or an intermediate thereof to be detected for derivatization treatment, and preparing a sample solution;
and (3) sample injection detection:
detecting and analyzing the reference substance solution and the sample solution in the step (1) and the step (2) by adopting a high performance liquid chromatography ultraviolet detector;
calculating the content of tromethamine:
wherein:
W for a pair of : weighing the reference substance;P control : the content of the reference substance;
V for a pair of : dilution of the reference substance;A for a pair of : peak area of tromethamine derivative in control solution;
A sample : peak area of tromethamine derivative in the sample solution;V sample : dilution of the test sample;
W sample : sample weighing is carried out on the test sample.
2. The method for detecting the content of tromethamine according to claim 1, wherein the concentration of the control solution prepared in the step (1) is in the range of 0.05 μg/ml to 0.3 μg/ml.
3. The method for detecting the content of tromethamine according to claim 1, wherein the concentration of the sample solution prepared in the step (2) is 2mg/ml.
4. The method for detecting the content of tromethamine according to claim 1, wherein the detection conditions in the step (3) are as follows:
chromatographic column: octadecylsilane chemically bonded silica column; the detection wavelength is as follows: 360nm; mobile phase: the volume ratio of water to acetonitrile is 42:58-38:62; the flow rate of the mobile phase is as follows: 0.8-1.2 ml/min; column temperature: 27-33 ℃; sample injection volume: 10 μl.
5. The method for detecting the content of tromethamine according to claim 4, wherein the detection conditions in the step (3) are as follows: mobile phase: water-acetonitrile 40:60.
6. the method for detecting the content of tromethamine according to claim 4, wherein the detection conditions in the step (3) are as follows: the flow rate was 1.0ml/min.
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