CN119165093B - High-sensitivity and low-limit tert-butylamine residual trace analysis method - Google Patents
High-sensitivity and low-limit tert-butylamine residual trace analysis method Download PDFInfo
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- YBRBMKDOPFTVDT-UHFFFAOYSA-N tert-butylamine Chemical compound CC(C)(C)N YBRBMKDOPFTVDT-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000004454 trace mineral analysis Methods 0.000 title claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 45
- 238000001212 derivatisation Methods 0.000 claims abstract description 36
- 230000035945 sensitivity Effects 0.000 claims abstract description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 118
- 239000000243 solution Substances 0.000 claims description 78
- 239000013558 reference substance Substances 0.000 claims description 48
- 239000000523 sample Substances 0.000 claims description 34
- 239000002253 acid Substances 0.000 claims description 33
- 239000011230 binding agent Substances 0.000 claims description 31
- 239000012488 sample solution Substances 0.000 claims description 30
- 239000003814 drug Substances 0.000 claims description 29
- 238000012360 testing method Methods 0.000 claims description 29
- 239000006184 cosolvent Substances 0.000 claims description 28
- PASDCCFISLVPSO-UHFFFAOYSA-N benzoyl chloride Chemical group ClC(=O)C1=CC=CC=C1 PASDCCFISLVPSO-UHFFFAOYSA-N 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 25
- 239000003795 chemical substances by application Substances 0.000 claims description 25
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical group CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 23
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical group CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 239000013064 chemical raw material Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 229960001399 clenbuterol hydrochloride Drugs 0.000 claims description 9
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000010812 external standard method Methods 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 7
- -1 amide compound Chemical class 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- 238000004364 calculation method Methods 0.000 claims description 5
- DDUHZTYCFQRHIY-UHFFFAOYSA-N 7-chloro-3',4,6-trimethoxy-5'-methylspiro[1-benzofuran-2,4'-cyclohex-2-ene]-1',3-dione Chemical compound COC1=CC(=O)CC(C)C11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 DDUHZTYCFQRHIY-UHFFFAOYSA-N 0.000 claims 1
- 229960001117 clenbuterol Drugs 0.000 claims 1
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 claims 1
- 238000005185 salting out Methods 0.000 abstract description 5
- 238000005220 pharmaceutical analysis Methods 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 20
- 229940079593 drug Drugs 0.000 description 19
- 239000012071 phase Substances 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 239000012490 blank solution Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 11
- 239000012895 dilution Substances 0.000 description 11
- 239000002904 solvent Substances 0.000 description 11
- 239000012088 reference solution Substances 0.000 description 9
- 239000008213 purified water Substances 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000012085 test solution Substances 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 150000002466 imines Chemical class 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000033962 Fontaine progeroid syndrome Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 150000008043 acidic salts Chemical group 0.000 description 1
- 150000001263 acyl chlorides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 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
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011165 process development Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 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
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- 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
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Abstract
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a high-sensitivity and low-limit trace analysis method for tert-butylamine residue. The invention creatively develops a derivatization reverse detection method with high sensitivity, which can obviously reduce the detection limit and the detection quantitative limit of the tert-butylamine content, can avoid salting-out risks and improve the stability of derivative products, and has extremely high commercial application value.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a high-sensitivity and low-limit trace analysis method for tert-butylamine residue.
Background
Tert-butylamine is a colorless transparent liquid, and is soluble in water, absolute ethyl alcohol, benzene, chloroform, diethyl ether and the like, and is commonly used as a chemical agent such as a rubber accelerator, a synthetic drug, a dye, an insecticide and the like. Tert-butylamine is toxic and there is a risk of mortality, for example by inhalation, oral or transdermal absorption of tert-butylamine. Moreover, tert-butylamine has a strong stimulating effect on the eyes, skin, mucous membranes and respiratory tract. Therefore, the residual quantity of tert-butylamine in the crude drug needs to be strictly detected and controlled.
Tert-butylamine is a volatile basic organic compound having a molecular weight of 73.14 and a boiling point of 46 ℃. In the field of pharmaceutical analysis, gas chromatography (Gas Chromatography, GC) and hydrogen flame ionization detectors (Flame Ionization Detector, FID) are generally preferred for the process development of drug molecules. The tert-butylamine has strong alkalinity, is easy to remain on the chromatographic column and has poor peak shape, so that the alkaline compound chromatographic column can be adopted for detection. However, the detection limit of the tert-butylamine analyzed by the conventional gas chromatography method can only reach 100ppm at the minimum at present, and the requirement of lower and more accurate detection limit is difficult to realize. In addition, the molecular weight of tert-butylamine is very close to the molecular weight of the column loss peak of a gas chromatography-mass spectrometer (Gas Chromatography Mass Spectrometer, GCMS) and is easily interfered by the column loss peak, so that the detection by the gas chromatography-mass spectrometer is not suitable. In addition, tert-butylamine is organic amine, exists in the form of tert-butylamine cation in aqueous solution, but is limited by poor solubility of chemical bulk drugs of a test product in a large proportion of aqueous phase, and the detection limit of tert-butylamine only reaches 100ppm when the method is developed by an ion chromatography-cation mode, so that the lower detection requirement cannot be realized.
Disclosure of Invention
The prior art has the problems of low sensitivity or poor popularity in detecting the residual amine compounds in chemical raw material medicines. Aiming at the problem, the invention creatively develops a derivatization reverse phase detection method with high sensitivity, which can obviously reduce the detection limit and the detection quantitative limit of the tert-butylamine content, can avoid salting-out risk and improve the stability of the derivative product, and has extremely high commercial application value.
The method for analyzing the trace amount of the residual tert-butylamine with high sensitivity and low limit comprises the following steps:
Dissolving a chemical raw material medicinal cosolvent and acetonitrile, adding an acid binding agent and a derivatization agent for derivatization reaction, and quenching the reaction with water after the reaction is finished to obtain a sample solution;
dissolving tert-butylamine reference substance with cosolvent and acetonitrile, adding acid-binding agent and derivatizing agent for derivatization reaction, and quenching with water after the reaction is finished to obtain reference substance solution;
thirdly, measuring a proper amount of sample solution and reference substance solution, injecting the sample solution and the reference substance solution into a high performance liquid chromatograph, and recording the peak area of the tert-butylamine derivative in the chromatogram;
the fourth step is to calculate the residual quantity of tert-butylamine in the chemical raw material medicine by an external standard method, wherein the calculation formula is as follows:
;
Wherein:
A S peak area of tert-butylamine derivative in sample solution;
w S the sample weighing amount (mg) of the test sample;
w STD the sample weighing amount (mg) of the reference substance;
V STD dilution volume (ml) of control solution;
V S dilution volume (ml) of the test solution;
A STD peak area of tert-butylamine derivative in control solution;
pw is the content of tert-butylamine reference substance.
Preferably, the chemical raw material medicine is acidic salt, and preferably, the chemical raw material medicine is clenbuterol hydrochloride.
Preferably, the cosolvent is N-methyl pyrrolidone and/or N, N-dimethylformamide, wherein the volume ratio of the cosolvent to acetonitrile is 1:1-1:20.
Preferably, the acid-binding agent is selected from one or more of triethylamine, diethanolamine, N-diisopropylethylamine.
Preferably, the mol ratio of the acid binding agent to the tert-butylamine reference substance is 10000:1-200000:1.
Preferably, the derivatizing agent is benzoyl chloride.
Preferably, the molar ratio of the derivatizing agent to the tert-butylamine reference substance is 100:1-1000:1.
Preferably, the tert-butylamine derivative is an amide compound.
Preferably, the reaction temperature is room temperature to 60 ℃.
Preferably, the concentration of the chemical raw material medicine in the sample solution is 36+/-2 mg/ml, and the concentration of the tert-butylamine reference substance in the reference substance solution is 0.18+/-0.01 mug/ml.
Preferably, octadecyl bonded silica gel is used as filler, the column temperature is 35+/-3 ℃, the mobile phase A is 0.1+/-0.01% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the flow rate is 1.0+/-0.2 ml/min.
Preferably, the detection wavelength of the high performance liquid chromatograph is 224+ -5 nm or 205+ -5 nm. Preferably, the detection wavelength of the high performance liquid chromatograph is 224nm.
Compared with the prior art, the invention has the following beneficial effects:
1. Aiming at the problem of low detection sensitivity of the residual quantity of the tert-butylamine, the invention develops a derivatization reversed-phase liquid chromatography method for analyzing the content of the tert-butylamine in the bulk drug, can quantitatively and accurately detect the residual quantity of the tert-butylamine with the content of more than 1.5ppm, and remarkably enhances the sensitivity of the detection method.
2. According to the invention, benzoyl chloride is used as a derivatization reagent, so that the rapid derivatization at room temperature is realized, the derivatization stability is improved, and the detection time is saved.
3. The invention optimizes and verifies the detection method, has the advantages of simplicity, convenience, rapidness, high accuracy and the like, and reduces the salting-out risk.
Drawings
FIG. 1 is a diagram showing the specificity of a blank solution, a positioning solution, a reference solution and a test sample solution by HPLC, wherein the reference solution is 1-reference solution, the blank solution is 2-solution, the positioning solution is 3-solution, and the test sample solution is 4-solution;
FIG. 2 is a plot showing the sensitivity profiles of blank solution, detection limit solution, quantitative limit solution and control solution by HPLC, wherein 1-control solution, 2-quantitative limit solution, 3-detection limit solution, 4-blank solution;
FIG. 3 is a linear graph of peak area of tert-butylamine derivatives versus tert-butylamine concentration for linear regression;
FIG. 4 is an accuracy representative graph of characterization of blank solution, control solution, test solution, and AC2-100% solution (accuracy limit plus standard solution) by HPLC, wherein 1-AC2-100% solution, 2-test solution, 3-control solution, 4-blank solution.
Detailed Description
The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof. Specific experimental methods not mentioned in the following examples are generally carried out according to conventional experimental methods.
How to obtain a detection method with good specificity, so that tert-butylamine derivatives and crude drug impurities can be effectively separated, and the sensitivity and accuracy of the detection method are improved by improving the derivatization process, which is the technical problem to be solved by the invention. The trace analysis method of the residual amount of tert-butylamine in the chemical raw material drug according to the present invention is exemplified below.
After the chemical raw material medicinal cosolvent and solvent acetonitrile are dissolved, an acid binding agent and a derivatization agent are added for derivatization reaction, and after the reaction is finished, water is used for reaction quenching to obtain a sample solution.
The chemical raw material medicine is acid salt. Preferably, the chemical bulk drug is clenbuterol hydrochloride. The chemical raw material medicine contains impurities. The impurity is a relatively basic fatty amine including, but not limited to, t-butylamine, n-butylamine, diethylamine, isopropylamine, and the like.
And (3) selecting a derivatizing agent. Tert-butylamine itself has no uv absorption. The tert-butylamine derivative can be provided with ultraviolet absorption by selecting a proper derivatizing agent with ultraviolet absorption. Preferably, the derivatizing agent is benzoyl chloride. The derivative formed by the reaction of benzoyl chloride and tert-butylamine is an amide compound, the derivative has ultraviolet absorption, is a neutral compound with stable property, has rapid derivatization reaction speed, and can shorten derivatization time. If a salicylaldehyde-methanol derivative system is adopted, the formed derivative is an imine compound, and the potential decomposition risk exists, so that alkali environment is formed by adding alkali such as sodium hydroxide and the like to increase the stability of the derivative product imine compound.
The solubility of clenbuterol hydrochloride as a chemical raw material in acetonitrile is limited, and an acetonitrile solution with expected concentration is difficult to obtain. Water and methanol react with the derivatizing agent benzoyl chloride and are therefore not suitable as co-solvents. For example, the co-solvent is selected from N-methylpyrrolidone (NMP) and/or N, N-Dimethylformamide (DMF). Preferably, NMP is selected as the co-solvent.
The volume ratio of the cosolvent to the acetonitrile can be 1:1-1:20. As an example, the volume ratio of the co-solvent to acetonitrile is 1:10.
And (3) selecting an acid binding agent. The acid binding agent is selected from Triethylamine (TEA), diethanolamine (DEA), N-Diisopropylethylamine (DIEA). Preferably, the acid binding agent is DIEA. The acid binding agent can adsorb hydrochloric acid which is a byproduct of the derivatization reaction and is contained in the raw material medicine, so that the derivatization reaction is promoted to reach the end point. Strong bases such as sodium hydroxide react with the derivative reagent acyl chloride and are not suitable for the derivative system of the invention.
The derivatization temperature is room temperature to 60 ℃. For example, the reaction temperatures are room temperature, 40 ℃ and 60 ℃. The water bath may be heated to maintain the desired derivatization reaction temperature. Unless otherwise stated, room temperature refers to 15 to 25 ℃.
The derivatization time can be adjusted as desired. The derivatization time may be within 2 hours. For example, the derivatization time is 0.5 minutes to 2 hours. Preferably, the derivatization time is 1 minute to 1 hour. For example, the derivatization times are shaking (around 1 min), 10 minutes and 1 hour.
The mol ratio of the acid binding agent to the tert-butylamine reference substance is 10000:1-200000:1. For example, the molar ratio of the acid binding agent to the tert-butylamine control is 10000:1, 120000:1 and 200000:1. Preferably, the molar ratio of the acid binding agent to the tert-butylamine control is 120000:1.
The mole ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100:1-1000:1. For example, the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine control is 100:1, 250:1, 500:1, and 1000:1. Preferably, the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine control is 250:1.
To eliminate the effect of excess derivatizing agent benzoyl chloride on the chromatographic system, the benzoyl chloride was quenched with purified water and the reaction stopped.
The preparation process of the reference substance solution is basically the same as that of the test substance solution. For example, after the tert-butylamine reference substance is dissolved by using a cosolvent and a solvent acetonitrile, an acid binding agent and a derivatizing agent are added for derivatization reaction, and after the reaction is finished, water is used for quenching the reaction to obtain a reference substance solution.
And respectively precisely measuring a proper amount of the sample solution and the reference solution, injecting into a high performance liquid chromatograph, and recording the peak area of the tert-butylamine derivative in the chromatogram. The detection wavelength of the high performance liquid chromatograph is 224+/-5 nm or 205+/-5 nm. For example, the detection wavelength of the high performance liquid phase condition is 224nm or 205nm. Preferably, the detection wavelength of the high performance liquid phase condition is 224nm.
Optimization of chromatographic parameters. The mobile phase, chromatographic column and elution procedure are optimized, and finally a conventional C18 chromatographic column and phosphoric acid aqueous solution-acetonitrile are adopted as a mobile phase system. Specifically, octadecyl bonded silica gel is used as a filler, the column temperature is 35+/-3 ℃, the mobile phase A is phosphoric acid aqueous solution with the mass concentration of 0.1+/-0.01%, the mobile phase B is acetonitrile, and the flow rate is 1.0+/-0.2 ml/min. For example, octadecyl bonded silica gel is used as a filler, wherein the octadecyl bonded silica gel is Waters XBiridge ® C18, 4.6 x 150mm x 3.5 mu m, the column temperature is 35 ℃, the detection wavelength is 224nm, the mass concentration of 0.1% phosphoric acid aqueous solution is a mobile phase A, acetonitrile is a mobile phase B, and gradient elution is carried out, and the flow rate is 1.0 ml/min. Buffer salt solutions present a potential salting-out risk if an aqueous mobile phase of potassium dihydrogen phosphate solution is employed.
And calculating the residue of tert-butylamine in the test sample according to an external standard method. The calculation formula is as follows:
。
Wherein A S is the peak area of tert-butylamine derivative in the sample solution, W S is the sample weighing amount (mg) of the sample, W STD is the sample weighing amount (mg) of the reference substance, V STD is the product (ml) of the reference substance solution, V S is the volume (ml) of the sample solution, A STD is the peak area of tert-butylamine derivative in the reference substance solution, and Pw is the content of tert-butylamine reference substance.
In some embodiments, the invention provides an analysis method of the residual quantity of tert-butylamine in clenbuterol hydrochloride bulk drug, comprising the following steps:
Firstly, taking a proper amount of clenbuterol hydrochloride crude drug sample, adding NMP for ultrasonic dissolution, adding acetonitrile for dilution, adding DIEA for shaking, adding benzoyl chloride solution for shaking, adding purified water for volume fixing to a scale, and taking the sample solution as a sample solution.
And secondly, adding a proper amount of NMP, adding acetonitrile for dilution, adding a proper amount of tert-butylamine reference substance solution, adding DIEA, shaking uniformly, adding benzoyl chloride, shaking uniformly, and adding purified water to fix the volume to a scale to obtain the reference substance solution.
And thirdly, precisely measuring a proper amount of the two solutions obtained in the first step and the second step respectively, injecting the two solutions into a high performance liquid chromatograph, and recording the peak area of the tert-butylamine derivative in the chromatogram. Wherein octadecyl bonded silica gel is used as a filler, the column temperature is 35 ℃, the detection wavelength is 224nm, a phosphoric acid aqueous solution with the mass concentration of 0.1% is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is carried out at the flow rate of 1.0 ml/min.
And fourthly, calculating the residue of the tert-butylamine derivative in the test sample according to an external standard method.
In conclusion, the derivatization reverse phase detection method can accurately determine the residual quantity of the tert-butylamine in the chemical bulk drug by optimizing the cosolvent, the acid-binding agent and the derivatization agent, realizes the detection limit and the detection quantitative limit of the content of the tert-butylamine to be obviously reduced, can quantitatively detect the residual quantity of the tert-butylamine of more than 1.5ppm in the chemical bulk drug, and has the advantages of high sensitivity and good reproducibility. In addition, the derivatization process of the method does not need heating, is convenient and quick, can avoid salting-out risks, improves the stability of the derivative products, is favorable for ensuring the quality of chemical raw materials, and has extremely high commercial application value.
The present invention will be described in more detail by way of examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.
The following are the instruments, controls, reagents and chromatographic conditions related to examples and comparative examples.
TABLE 1
TABLE 2
Example 1
In the first step, the sample is derived. The method comprises the steps of placing 720mg of clenbuterol hydrochloride crude drug sample in a volumetric flask, adding a cosolvent NMP for ultrasonic dissolution, adding a solvent acetonitrile 12.8 ml for dilution, adding an acid binding agent DIEA for shaking uniformly, adding a benzoyl chloride solution (1.7 mg/ml, acetonitrile) for shaking uniformly, and adding purified water for volume fixing to a scale to obtain a sample solution.
And the second step, derivatizing the reference substance. The method is basically the same as the first step, and only differs in that the clenbuterol hydrochloride crude drug sample is replaced by a tert-butylamine reference substance solution. Placing cosolvent NMP into a volumetric flask, adding 2.0ml of tert-butylamine reference substance solution (1.825 μg/ml, acetonitrile), adding acetonitrile 10.8 ml for dilution, adding acid-binding agent DIEA, shaking uniformly, adding benzoyl chloride (1.7 mg/ml, acetonitrile) for shaking uniformly, adding purified water to fix volume to scale, and taking the reference substance solution.
And thirdly, sampling procedure and method parameters. Taking the two solutions obtained in the first step and the second step, precisely measuring 10 mu l of each solution, injecting the solutions into a high performance liquid chromatograph, and recording the peak area of the tert-butylamine derivative in the chromatogram. Octadecyl bonded silica gel is used as filler. The octadecyl bonded silica gel is Waters XBiridge ® C18, 4.6X105 mm 3.5 μm, the column temperature is 35 ℃, the detection wavelength is 224nm,0.1% phosphoric acid aqueous solution is mobile phase A, acetonitrile is mobile phase B, gradient elution is carried out, and the flow rate is 1.0 ml/min.
TABLE 3 Table 3
And fourthly, calculating the residual quantity of the tert-butylamine derivative in the test sample according to an external standard method. The calculation formula is as follows:
。
Wherein A S is the peak area of tert-butylamine derivative in the sample solution, W S is the sample weighing amount (mg) of the sample, W STD is the sample weighing amount (mg) of the reference substance, V STD is the dilution volume (ml) of the reference substance solution, V S is the dilution volume (ml) of the sample solution, A STD is the peak area of tert-butylamine derivative in the reference substance solution, and Pw is the content of tert-butylamine reference substance.
In the first step and the second step of the embodiment, the cosolvent is NMP, the acid binding agent DIEA is used in a molar ratio of 200000:1 with respect to the tert-butylamine reference substance, and the derivatizing agent benzoyl chloride is used in a molar ratio of 250:1 with respect to the tert-butylamine reference substance. The quantitative determination results using different amounts of the cosolvent NMP are shown in Table 4.
TABLE 4 Table 4
As can be seen from Table 4, when the volume ratio of the cosolvent NMP to the solvent acetonitrile was 1:10, the peak area of the tert-butylamine derivative was large and the test sample was completely dissolved. This means that the larger the volume of the co-solvent, the greater the inhibition of the derivatization reaction, but that too low a volume of the co-solvent results in precipitation of the test sample. Thus, the volume ratio of the cosolvent NMP to the solvent acetonitrile was chosen to be 1:10 for subsequent experiments.
Example 2
Substantially the same as in example 1, except that in the first and second steps, the volume ratio of the cosolvent NMP to the solvent acetonitrile was 1:10, the molar ratio of the acid-binding agent (TEA or DIEA) to the tert-butylamine control was 40000:1, and the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine control was 100:1. The quantitative detection results using different kinds of acid binding agents are shown in Table 5.
TABLE 5
As can be seen from Table 5, the acid scavenger was DIEA substantially non-interfering. Thus, the acid-binding agent was chosen for DIEA for subsequent experiments.
Example 3
Substantially the same as in example 1, except that in the first and second steps, the volume ratio of the cosolvent NMP to the solvent acetonitrile was 1:10, the acid-binding agent was DIEA, and the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine control was 100:1. The quantitative detection results using different amounts of acid binding agent are shown in Table 6.
TABLE 6
Recovery = (tert-butylamine content of sample after labeling-tert-butylamine content of sample itself)/tert-butylamine addition amount
As can be seen from Table 6, the molar ratio of acid-binding agent DIEA to tert-butylamine control was 120000:1, which gave the best results, high recovery and no interference. If the amount of DIEA added is small, the recovery rate is low, and if the amount of DIEA added is high, the interference becomes large.
Example 4
Substantially the same as in example 1, except that in the first and second steps, the volume ratio of the cosolvent NMP to the solvent acetonitrile was 1:10, the acid-binding agent was DIEA, and the molar ratio of the acid-binding agent to the tert-butylamine control was 120000:1. The quantitative determination results using the mole ratio of derivatizing agent to tert-butylamine control are shown in Table 7.
TABLE 7
As can be seen from Table 7, the molar ratio of benzoyl chloride to tert-butylamine control was 250:1, which works best. If the amount of benzoyl chloride added is small, the recovery rate is low, and if the amount of benzoyl chloride added is high, the interference is large and detection cannot be performed.
The conclusion is that the volume ratio of cosolvent NMP to solvent acetonitrile is 1:10, the acid binding agent is DIEA, the mol ratio of the acid binding agent to the tert-butylamine reference substance is 120000:1, and the mol ratio of benzoyl chloride to the tert-butylamine reference substance is 250:1, which are used as the final sketching conditions. The specific derivatization conditions are as follows:
The sample is derived by placing about 720mg of clenbuterol hydrochloride crude drug sample in a volumetric flask with the volume of 20ml, adding 1.2ml of cosolvent NMP for ultrasonic dissolution, adding 12.8 ml ml of solvent acetonitrile for dilution, adding 1ml of DIEA for shaking uniformly, adding 1.0ml of benzoyl chloride solution (1.7 mg/ml of acetonitrile) for shaking uniformly, and adding purified water for constant volume to scale to obtain a sample solution;
The control is derived by placing 1.2ml of NMP in a volumetric flask with a volume of 20ml, adding 2.0ml of tert-butylamine control solution (1.825 mug/ml, acetonitrile), adding 10.8 ml of acetonitrile for dilution, adding 1ml of DIEA, shaking uniformly, adding 1.0 ml of benzoyl chloride (1.7 mg/ml, acetonitrile), shaking uniformly continuously, adding purified water to a scale, and taking the volume to be the control solution;
Preparing a blank solution, namely placing 1.2ml of NMP into a volumetric flask with a volume of 20ml, adding acetonitrile 12.8 ml to dilute, adding DIEA 0.5ml, shaking uniformly, adding benzoyl chloride (1.7 mg/ml, acetonitrile) 1.0 ml to continuously shake uniformly, and adding purified water to fix the volume to a scale to obtain the blank solution.
Example 5
5.1 Specificity test
Taking blank solution, positioning solution, reference substance solution and test sample solution, detecting after derivatization according to the established chromatographic conditions, and recording a chromatogram. The preparation of the positioning solution is similar to that of the reference substance solution, and the difference is that the addition amount of the tert-butylamine of the positioning solution is 5 times that of the reference substance solution.
As can be seen from FIG. 1, the chromatographic peak retention time of the tert-butylamine derivative is about 18.5min, and the blank solution and other components in the sample have no interference at the peak position of tert-butylamine, thus indicating good specificity.
5.2 Quantitative limit test
NMP is placed in a volumetric flask, tert-butylamine is added to prepare a solution with the required concentration, the solution is measured after being derived according to a formulated method, a chromatogram is recorded, and the concentration corresponding to the solution with the signal to noise ratio of more than 10:1 is taken as a quantitative limit. The preparation of the quantitative limiting solution is similar to that of the reference solution, except that the addition amount of tert-butylamine is 0.3 times of that of the reference solution. The quantitative limit test results are shown in Table 8.
TABLE 8
As shown by the test results, when the concentration of the crude drug sample solution is 36mg/mL and the sample injection amount is 10 mu L, the quantitative limit of tert-butylamine under the proposed method is 30 percent (1.5 ppm) of the limit (5 ppm), and the quantitative limit solution is continuously injected for 3 times, and the peak area RSD is smaller than 15 percent.
5.3 Limit of detection test
NMP is placed in a volumetric flask, tert-butylamine is added to prepare a solution with the required concentration, the solution is measured after being derived according to a formulated method, a chromatogram is recorded, and the concentration corresponding to the solution with the signal to noise ratio of more than 3:1 is taken as a detection limit. The preparation of the detection limit solution is similar to that of the reference solution, and the difference is that the addition amount of the tert-butylamine is 0.1 times of that of the reference solution. The detection limit test results are shown in Table 9.
TABLE 9
As is clear from the above test results, when the concentration of the crude drug sample solution was 36mg/mL and the sample injection amount was 10. Mu.L, the lower detection limit of the proposed method was 10% (0.5 ppm) of the limit (5 ppm).
As can be seen from fig. 2, the blank solution) is substantially free of interference, and the signal-to-noise ratio of the detection limit solution and the quantification limit solution can be satisfied.
5.4 Linearity of
30%, 50%, 100%, 150%, 250%, 500% Of the limiting concentration level of tert-butylamine (5 ppm) was prepared as a linear solution according to a proposed method. The preparation of each linear solution is similar to that of the control solution, and the addition amount of the tert-butylamine is only adjusted. The results of the linear test are shown in Table 10.
Table 10
FIG. 3 is a linear plot of peak area versus concentration for linear regression of tert-butylamine between 30% and 500% of the limit concentration for the linear test term.
As can be seen from FIG. 3, the linear test shows that the t-butylamine has a peak area of the derivative linearly regressing the concentration between the limit concentration of 30% (1.5 ppm) and the limit concentration of 500% (25 ppm), the linear regression coefficient R is 0.9999, and the absolute value of Y-axis intercept/peak area of 100% concentration is 6.10%. Indicating that under the proposed method, the concentration of tert-butylamine and the peak area are in good linear relationship.
5.5 Accuracy test
Preparing a test sample solution and a reference substance solution according to a formulated method, preparing standard test sample solutions with limit concentrations of 50%, 100% and 150%, preparing 2 parts of standard test sample solutions with each concentration in parallel, determining according to formulated chromatographic conditions, recording a chromatogram, calculating the content of tert-butylamine according to an external standard method by using peak area, and calculating the recovery rate of tert-butylamine in each standard test sample. The preparation of each accuracy test solution is similar to that of the test solution, and different amounts of tert-butylamine are added according to the requirements after the solution is dissolved. The accuracy test results are shown in Table 11.
TABLE 11
Test results show that in 6 parts of 3 horizontal standard-added bulk drug sample solutions, the recovery rate of tert-butylamine is in the range of 96.30% -100.50%, the average value is 98.5%, and the RSD is 1.6%. Experimental results prove that the method has good accuracy and precision.
As can be seen from fig. 4, the recovery at 100% of the spiked level was good.
Comparative example 1
Step A, derivatization of a sample, namely taking a 50 mg-25 mL volumetric flask of an arolol hydrochloride bulk drug sample, adding 10mL of methanol for dissolution, adding 0.5mL of 0.1mol/L sodium hydroxide and 1mL of derivatization agent (10% salicylaldehyde-methanol), derivatizing for 30min in a 60 ℃ water bath, taking out, cooling, and adding methanol for dilution to a scale;
step B, derivatizing the reference substance, namely taking a proper amount of tert-butylamine reference substance, adding methanol to dilute the tert-butylamine reference substance to prepare 50g/mL solution, taking 1mL of the solution, placing the solution into a 25mL volumetric flask, adding 10mL of methanol, 0.5mL of 0.1mol/L sodium hydroxide and 1mL of derivatizing agent (10% salicylaldehyde-methanol), derivatizing the solution in a 60 ℃ water bath for 30min, taking out, cooling, and adding methanol to dilute the solution to a scale;
And C, taking 20 mul of the two solutions, respectively precisely measuring, injecting into a liquid chromatograph, taking octadecyl bonded silica gel (WELCH Ultimate AQ-C18,5 mu m, 4.6X250 mm) as a filler, taking the column temperature as 30 ℃, taking the detection wavelength as 348nm, taking 0.02mol/L KH 2PO4 (pH 3.0+/-0.1) as a mobile phase A, taking acetonitrile as a mobile phase B, and carrying out gradient elution according to a table 12, wherein the flow rate is 1.0 ml/min.
Table 12
And D, recording the peak area of the tert-butylamine derivative in the chromatogram, and calculating the quantity of tert-butylamine in the sample according to an external standard method:
Calculation formula
。
Wherein:
A Sample -obtaining residual peak areas in a chromatogram of the sample solution;
Obtaining residual peak areas in a chromatogram of the A For a pair of -reference solution;
m For a pair of -control sample (mg);
m Sample -sample weight (mg).
As a result of the detection, the limit of detection of tert-butylamine in comparative example 1 was 1000ppm, and the limit of detection quantification was 10ppm.
Claims (3)
1. A method for analyzing trace amounts of residual tert-butylamine with high sensitivity and low limit, characterized in that it comprises the following steps:
Dissolving clenbuterol hydrochloride serving as a chemical raw material medicine by acetonitrile and a cosolvent, adding an acid binding agent and a derivatizing agent for derivatization reaction, and quenching the reaction with water after the reaction is finished to obtain a sample solution;
dissolving tert-butylamine reference substance with acetonitrile and cosolvent, adding acid-binding agent and derivatizing agent for derivatization reaction, and quenching with water after the reaction is finished to obtain reference substance solution;
thirdly, measuring a proper amount of sample solution and reference substance solution, injecting the sample solution and the reference substance solution into a high performance liquid chromatograph, and recording the peak area of the tert-butylamine derivative in the chromatogram;
the fourth step is to calculate the residual quantity of the clenbuterol Luo Zhongshu butylamine hydrochloride of the chemical raw material medicine by an external standard method, and the calculation formula is as follows:
;
Wherein:
A S peak area of tert-butylamine derivative in sample solution;
w S, weighing sample of the test sample, and mg;
W STD, weighing the reference substance, and mg;
v STD the volume of the reference substance solution, ml;
v S the volume of the sample solution, ml;
A STD peak area of tert-butylamine derivative in control solution;
pw, t-butylamine control;
Wherein:
The cosolvent is N-methyl pyrrolidone, and the volume ratio of the cosolvent to acetonitrile is 1:1-1:10;
The acid-binding agent is N, N-diisopropylethylamine, and the molar ratio of the acid-binding agent to the tert-butylamine reference substance is 120000:1;
The derivatization agent is benzoyl chloride, and the molar ratio of the benzoyl chloride of the derivatization agent to the tert-butylamine reference substance is 100:1-250:1;
octadecyl bonded silica gel is used as a filler, the column temperature is 35+/-3 ℃, the mobile phase A is 0.1+/-0.01% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the flow rate is 1.0+/-0.2 ml/min;
the detection wavelength of the high performance liquid chromatograph is 224+/-5 nm or 205+/-5 nm;
The gradient elution parameters of the high performance liquid chromatograph are as follows:
。
2. the method for analyzing trace amounts of residual tert-butylamine according to claim 1, wherein the reaction temperature is room temperature to 60 ℃.
3. The method for trace analysis of tert-butylamine residue according to claim 1, wherein the tert-butylamine derivative is an amide compound.
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