CN114166975B - Method for analyzing photodecomposition degree of medicine by adjuvant tracing - Google Patents
Method for analyzing photodecomposition degree of medicine by adjuvant tracing Download PDFInfo
- Publication number
- CN114166975B CN114166975B CN202111493639.4A CN202111493639A CN114166975B CN 114166975 B CN114166975 B CN 114166975B CN 202111493639 A CN202111493639 A CN 202111493639A CN 114166975 B CN114166975 B CN 114166975B
- Authority
- CN
- China
- Prior art keywords
- medicine
- acetaldehyde
- chlorpromazine hydrochloride
- photodecomposition
- liquid chromatography
- 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.)
- Active
Links
- 239000003814 drug Substances 0.000 title claims abstract description 108
- 238000006303 photolysis reaction Methods 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 47
- 239000002671 adjuvant Substances 0.000 title abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 51
- 238000001514 detection method Methods 0.000 claims abstract description 44
- 238000001212 derivatisation Methods 0.000 claims abstract description 39
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 38
- 239000011159 matrix material Substances 0.000 claims abstract description 20
- 230000015843 photosynthesis, light reaction Effects 0.000 claims abstract description 15
- 238000004454 trace mineral analysis Methods 0.000 claims abstract description 3
- AKUVRZKNLXYTJX-UHFFFAOYSA-N 3-benzylazetidine Chemical group C=1C=CC=CC=1CC1CNC1 AKUVRZKNLXYTJX-UHFFFAOYSA-N 0.000 claims description 131
- 229960001657 chlorpromazine hydrochloride Drugs 0.000 claims description 131
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims description 126
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 65
- 239000000243 solution Substances 0.000 claims description 59
- 238000012417 linear regression Methods 0.000 claims description 35
- -1 hydroxyl free radical Chemical class 0.000 claims description 30
- 150000003254 radicals Chemical class 0.000 claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- 238000000354 decomposition reaction Methods 0.000 claims description 22
- 230000008569 process Effects 0.000 claims description 17
- 239000012752 auxiliary agent Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 239000012086 standard solution Substances 0.000 claims description 15
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical group [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 claims description 13
- 239000012483 derivatization solution Substances 0.000 claims description 13
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 10
- 238000002474 experimental method Methods 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 229960001076 chlorpromazine Drugs 0.000 claims 1
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 9
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000005286 illumination Methods 0.000 description 29
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 24
- MNUOZFHYBCRUOD-UHFFFAOYSA-N hydroxyphthalic acid Natural products OC(=O)C1=CC=CC(O)=C1C(O)=O MNUOZFHYBCRUOD-UHFFFAOYSA-N 0.000 description 13
- 239000012535 impurity Substances 0.000 description 13
- 238000001819 mass spectrum Methods 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- YMVFQWULFRMLRA-UHFFFAOYSA-N 10-[3-(dimethylamino)propyl]phenothiazin-2-ol Chemical compound C1=C(O)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 YMVFQWULFRMLRA-UHFFFAOYSA-N 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000000546 pharmaceutical excipient Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229950000688 phenothiazine Drugs 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 3
- 150000001793 charged compounds Chemical class 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 229940001470 psychoactive drug Drugs 0.000 description 2
- 239000004089 psychotropic agent Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000007348 radical reaction Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- AXDJCCTWPBKUKL-UHFFFAOYSA-N 4-[(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]aniline;hydron;chloride Chemical compound Cl.C1=CC(=N)C(C)=CC1=C(C=1C=CC(N)=CC=1)C1=CC=C(N)C=C1 AXDJCCTWPBKUKL-UHFFFAOYSA-N 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000012445 acidic reagent Substances 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000382 dechlorinating effect Effects 0.000 description 1
- 238000006298 dechlorination reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 239000003684 drug solvent Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002990 phenothiazines Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- HGBOYTHUEUWSSQ-UHFFFAOYSA-N valeric aldehyde Natural products CCCCC=O HGBOYTHUEUWSSQ-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
-
- 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
- G01N30/32—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
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)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A method for adjuvant-trace analysis of the photodecomposition of a drug, comprising the steps of derivatizing: taking a quantity of a drug comprising a drug matrix capable of undergoing a photolysis reaction and generating free radicals and a drug adjuvant capable of capturing the free radicals generated by the drug matrix during photolysis and generating a first component, adding a quantity of a derivatizing agent to the drug to obtain a derivatizing solution, the derivatizing agent being capable of derivatizing with the first component to form a derivatization product; and (3) detection: determining the photodecomposition degree of the drug by liquid chromatography on the derivatized solution, wherein the liquid chromatography is performed at a specific wavelength, the specific wavelength being the wavelength at the chromatographic peak of the derivatized product. The method has the advantages of simple and easy operation, high sensitivity and quick detection, and is suitable for quick analysis of the photodecomposition degree of the medicine.
Description
Technical Field
The invention relates to the field of analysis and detection of drug impurities, in particular to a method for analyzing the photodecomposition degree of a drug by tracing an auxiliary agent.
Background
Drug quality control is a basic element for ensuring safe drug administration, and some impurities in the drug can cause potential toxicity and side effects, and have an influence on the health of patients. The impurities in the medicine are mainly introduced from the production and storage processes, such as impurity generated by degradation of raw material medicines under the influence of external conditions or microorganisms in the storage process.
In the prior art, detection methods commonly used in the field of drug impurity detection mainly include chromatography, spectrometry, liquid phase or gas chromatography-mass spectrometry (LC/GC-MS) combination, and the like, and most of detection objects of these methods are impurities generated based on drug photodecomposition.
However, since the impurities generated by photodecomposition of the drug and the drug are similar in structure and physical and chemical properties, the traditional separation method has the defects of low separation degree, poor selectivity and the like, and is difficult to effectively identify the slight difference of the physical and chemical properties of the drug and the drug, and the separation of the drug and the drug into a core technical problem, the solution of the core technical problem often depends on the adoption of a high-performance chromatographic separation column, the increase of chromatographic separation time or complex sample pretreatment, and the measures are not only based on expensive equipment, but also require the application of a pretreatment method with a plurality of steps, and are time-consuming and labor-consuming, so that the rapid detection of the impurities in the drug is not facilitated; in addition, the matrix is complex, the impurity trace amount is easy and quick, the sensitivity is low, and the trace analysis standard is difficult to reach in some quick detection technologies, so that the development of the impurity quick detection method which is high in sensitivity, good in selectivity, simple and easy to operate is one of the challenges in the field of the traditional medicine analysis.
Disclosure of Invention
Based on the above, the invention aims to provide a method for analyzing the photodecomposition degree of a drug by tracing with auxiliary agents, which has the advantages of simple and easy operation, high sensitivity and rapid detection, and is suitable for rapid analysis of the photodecomposition degree of the drug.
A method for adjuvant tracking analysis of the degree of photodecomposition of a drug comprising the steps of:
and (3) derivatization treatment: taking a quantity of a drug comprising a drug matrix capable of undergoing a photolysis reaction and generating free radicals and a drug adjuvant capable of capturing the free radicals generated by the drug matrix during photolysis and generating a first component, adding a quantity of a derivatizing agent to the drug to obtain a derivatizing solution, the derivatizing agent being capable of derivatizing with the first component to form a derivatization product;
and (3) detection: determining the photodecomposition degree of the drug by liquid chromatography on the derivatized solution, wherein the liquid chromatography is performed at a specific wavelength, the specific wavelength being the wavelength at the chromatographic peak of the derivatized product.
The invention utilizes the auxiliary agent of the medicine to capture the free radical generated by the medicine matrix in the photolysis process to generate a first component, then adds the derivatization reagent and the derivatization product generated by the derivatization of the first component, and adopts the liquid chromatography to determine the photodecomposition degree of the medicine. The adjuvant tracing concept disclosed by the invention is firstly proposed in the field of medicine analysis, the traditional concept that medicine quality monitoring mainly starts from quantification is changed, the time required by sample pretreatment is shortened, the detection and analysis process of medicine impurities is greatly simplified, the steps are simple, and the sensitivity is high.
Further, the medicine is a solution, and the volume fraction of the medicine auxiliary agent is 5% -50% of the medicine solution. The volume fraction of the pharmaceutical adjuvant is too low to be detected, too high to cause pain or other side effects when the drug acts on the human body.
Further, the pharmaceutical adjuvant is an alcohol compound. The alcohol compound has small toxic and side effects and does not influence the curative effect of the medicine, the first component generated after the alcohol compound captures free radicals is an aldehyde compound, and the first component has large chemical property difference with a medicine matrix and is active in chemical property.
Further, the alcohol compound is one of ethanol, propanol, isopropanol and butanol. The ethanol, propanol, isopropanol and butanol can further generate corresponding aldehyde compounds, and have large chemical property difference and active chemical property with the drug matrix.
Further, the derivatizing reagent is one of 2, 4-dinitrophenylhydrazine, a phenol reagent and a Schiff reagent. And selecting a corresponding derivatization reagent according to the generated aldehyde compound to obtain a derivatization product.
Further, the step of determining the photodecomposition degree of the drug by liquid chromatography is:
under the same conditions, preparing a series of standard solutions of a first component with concentration gradients, respectively carrying out derivatization treatment, respectively carrying out liquid chromatography detection under the same specific wavelength, and drawing a standard curve of the peak area of a concentration-derivatization product chromatographic peak to obtain a linear regression equation;
substituting the peak area of the chromatographic peak of the derivatization product obtained by carrying out liquid chromatography detection on the derivatization solution into the linear regression equation to determine the concentration of the first component in the medicine, thereby determining the photodecomposition degree of the medicine.
Further, the medicine matrix is chlorpromazine hydrochloride, the medicine auxiliary agent is ethanol, the free radical is hydroxyl free radical, the first component is acetaldehyde, the photodecomposition degree of the medicine is expressed by the decomposition rate omega% of the chlorpromazine hydrochloride, and the photodecomposition degree of the medicine accords with the following equation:
wherein: c (acetaldehyde) is the concentration of acetaldehyde in the drug; c (original chlorpromazine hydrochloride) is the original concentration of chlorpromazine hydrochloride in the medicine; m (chlorpromazine hydrochloride) is the relative average molecular mass of chlorpromazine hydrochloride; m (acetaldehyde) is the relative average molecular mass of acetaldehyde;conversion of chlorpromazine hydrochloride to acetaldehyde by free radical; said->The determination method of (1) is as follows: through designing high performance liquid chromatography experiments to detect respectivelyMeasuring the decomposition amount of chlorpromazine hydrochloride and the generation amount of acetaldehyde in the photodecomposition process of the medicine, drawing a standard curve of the generation amount of acetaldehyde-the decomposition amount of chlorpromazine hydrochloride, and obtaining a second linear regression equation, wherein the slope of the second linear regression equation is->
The decomposition amount of chlorpromazine hydrochloride is obtained by making a difference between the original chlorpromazine hydrochloride in the medicine and the amount of chlorpromazine hydrochloride after photodecomposition, and the amount of chlorpromazine hydrochloride after photodecomposition is obtained by the following steps: preparing a series of chlorpromazine hydrochloride solutions with concentration, performing liquid chromatography detection under the same wavelength, drawing a standard curve of the concentration-chromatographic peak area of the chlorpromazine hydrochloride, obtaining a third linear regression equation, respectively performing liquid chromatography detection on the photo-decomposed medicine, obtaining chromatographic peak areas, substituting the chromatographic peak areas into the third linear regression equation, respectively obtaining the concentration of the chlorpromazine hydrochloride in the photo-decomposed medicine, and respectively determining the decomposition amount of the chlorpromazine hydrochloride;
the acetaldehyde generation amount is obtained by: preparing a series of concentration acetaldehyde standard solutions, adding the same amount of derivatization reagent, performing liquid chromatography detection at the same wavelength, drawing a concentration acetaldehyde-chromatographic peak area standard curve, obtaining a fourth linear regression equation, adding the equivalent derivatization reagent into the photo-decomposed medicine, and substituting the peak area obtained by liquid chromatography detection into the fourth linear regression equation to obtain the concentration of acetaldehyde generated in the medicine, thereby determining the generation amount of acetaldehyde.
The invention adopts liquid chromatography to determine the photodecomposition degree of the medicine, does not need expensive instruments and equipment and complicated sample pretreatment technology to separate the medicine from photodecomposition products thereof, and is suitable for detecting impurities with low content, complex matrix and similar structural properties to main components.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 is a diagram of a mechanism of photodissociation of phenothiazines;
FIG. 2 is a diagram of the molecular photolysis mechanism of chlorpromazine hydrochloride;
FIG. 3 is a chromatogram of an illuminated solution detected by the HPLC-SPD-FL method, wherein FIG. a is a fluorescence chromatogram, and FIG. b is an ultraviolet chromatogram, 1 represents an illuminated mixed standard solution of TPA and HTA, 2 represents an illuminated aqueous solution of chlorpromazine hydrochloride containing TPA, and 3 represents an illuminated solution of chlorpromazine hydrochloride containing TPA and 10% volume fraction ethanol;
FIG. 4 is a mass spectrum of 2-hydroxy promazine in a solution after LC-MS detection and illumination, wherein FIG. a is a mass spectrum of 2-hydroxy promazine in chlorpromazine hydrochloride solution without ethanol, and FIG. b is a mass spectrum of 2-hydroxy promazine in chlorpromazine hydrochloride solution with 10% ethanol by volume fraction;
FIG. 5 is a graph of the mass spectrum of 2-ethoxypromazine in a solution after LC-MS detection and illumination, wherein FIG. a is a graph of the mass spectrum of 2-ethoxypromazine in a chlorpromazine hydrochloride solution without ethanol, and FIG. b is a graph of the mass spectrum of 2-ethoxypromazine in a chlorpromazine hydrochloride solution with 10% ethanol by volume fraction;
FIG. 6 is a liquid chromatogram of a derivatization solution of chlorpromazine hydrochloride solution, wherein FIG. a is a DNPH-acetaldehyde peak area diagram and FIG. b is a DNPH-acetaldehyde peak area diagram; 1 represents a chlorpromazine hydrochloride derivatization solution with indoor illumination of 30s, and 2 represents a chlorpromazine hydrochloride derivatization solution with indoor illumination of 0 s;
FIG. 7 is a liquid chromatogram of a derivatization solution of an acetaldehyde standard solution, wherein FIG. a is a DNPH-acetaldehyde peak plot and FIG. b is a linear regression fit of acetaldehyde standard concentration versus DNPH-acetaldehyde peak area;
FIG. 8 is a linear regression fit of the decomposition amount of chlorpromazine hydrochloride to the formation amount of acetaldehyde;
FIG. 9 is a graph showing the comparison of chlorpromazine hydrochloride liquid chromatography after 30s of non-illumination and indoor natural illumination;
FIG. 10 is a graph showing the effect of different alcohols as radical scavengers during the photodecomposition of chlorpromazine hydrochloride.
Detailed Description
In order to further illustrate the invention, the embodiment takes phenothiazine psychotropic drugs as an example, and the method for tracing and analyzing the photodecomposition degree of the drugs by auxiliary agents is described in detail. However, it will be appreciated by those skilled in the art that the specific examples are intended to be illustrative of the principles of the present invention and are not intended to limit the invention to alternative embodiments, and that the determination of the degree of photodecomposition by those skilled in the art based on actual drug may be similarly performed using the methods of the present invention.
Specifically, in the embodiment, chlorpromazine hydrochloride which is a common phenothiazine psychotropic drug is taken as an example, namely, the drug matrix is chlorpromazine hydrochloride, ethanol which is a common drug solvent is taken as a drug auxiliary agent, and the ethanol can capture hydroxyl radicals generated after the chlorpromazine hydrochloride is photolyzed to generate acetaldehyde. 2, 4-Dinitrophenylhydrazine (DNPH) is adopted as a derivatization reagent to carry out derivatization reaction with acetaldehyde to generate a derivatization product DNPH-acetaldehyde.
In other embodiments, in addition to ethanol as a pharmaceutical adjuvant, one of the alcohol compounds propanol, isopropanol, butanol, etc. may be selected as a pharmaceutical adjuvant. In addition to using 2, 4-Dinitrophenylhydrazine (DNPH) as the derivatizing agent, a phenol reagent, a Schiff reagent (fuchsin sulfurous acid reagent) can be selected as the derivatizing agent. According to different application scenes and requirements, proper pharmaceutical auxiliary agents and derivatization reagents are selected.
The beneficial effects of the invention are further illustrated by simulating the photodecomposition process of the medicine, and the specific steps are as follows:
(1) Sample liquid preparation:
preparing a chlorpromazine hydrochloride solution with the concentration of 10mg/mL, wherein the chlorpromazine hydrochloride solution contains 10% ethanol by volume fraction, and shading after illumination for 30s under indoor natural light.
(2) And (3) derivatization treatment:
taking 200 mu L of chlorpromazine hydrochloride solution subjected to illumination for 30s in the step (1) and adding 2000 mu L of 2, 4-dinitrophenylhydrazine solution with the concentration of 2mg/mL into a 20mL brown bottle, adding 2800 mu L of phosphate buffer solution with the pH of 5.00, placing the solution in a constant-temperature oscillating water bath kettle at the temperature of 60 ℃ for reaction for 40min, placing the solution at the room temperature to the normal temperature, and filtering the solution through a microporous filter membrane to obtain chlorpromazine hydrochloride derivatization solution.
(3) And (3) detection:
and (3) introducing the derivatization solution after the derivatization treatment in the step (2) into a liquid chromatograph for analysis and detection to obtain a chromatogram and the peak area of a derivatization product DNPH-acetaldehyde chromatographic peak. Specifically, the liquid chromatography conditions were: using a column of Shim-pack GIST C18 (2.1X100 mm,3 μm), mobile phase A:0.1% trifluoroacetic acid; mobile phase B: acetonitrile, gradient elution (0-0.50 min,70.0% A;0.50-5.00min,70.0% A-20.0% A;5.00-6.00min,20.0% A-20.0% A;6.00-6.50min,20.0% A-70.0% A;6.50-9.00min,70.0% A-70.0% A), flow rate: 0.500mL/min, column temperature: 40 ℃, sample injection amount: 10. Mu.L, the detection wavelength was 355nm, respectively.
In order to facilitate determination of the photodecomposition degree of the chlorpromazine hydrochloride solution by liquid chromatography, the steps of this embodiment further include:
(4) Derivatization treatment and detection of an acetaldehyde standard solution:
to brown bottles of 50. Mu.g/mL of acetaldehyde standard solution 10, 20, 30, 40, 50, 100, 150, 200, 250, 400, 600, 800, 1000. Mu.L to 20mL, 2000. Mu.L of 2mg/mL of 2, 4-dinitrophenylhydrazine solution was added, and then buffer solution of pH=5.00 was added to 5mL, respectively, at which time the concentration of acetaldehyde in the derivative reagent was 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 6.0, 8.0, 10.0. Mu.g/mL, respectively. And (3) respectively placing the materials in a constant-temperature oscillating water bath kettle at 60 ℃ for reaction for 40min, taking out the materials, placing the materials at room temperature, passing the materials through a microporous filter membrane, respectively entering a liquid chromatograph in the step (3) for analysis and detection, and respectively obtaining a liquid chromatogram and the peak area of the chromatographic peak of the derivative DNPH-acetaldehyde. And drawing an acetaldehyde standard regression curve by taking the peak area of the DNPH-acetaldehyde chromatographic peak as an ordinate and the acetaldehyde concentration as an abscissa to obtain a linear regression equation.
Specifically, in this example, the principle of measurement of the photodecomposition degree of chlorpromazine hydrochloride is as follows:
please refer to fig. 1, which is a graph of a photodissociation mechanism of a phenothiazine drug, wherein the phenothiazine drug is easy to generate a photodissociation reaction under an illumination condition to generate free radicals; the free radicals react with water to form hydroxyl free radicals, and the hydroxyl free radicals are captured by ethanol serving as a pharmaceutical adjuvant to generate acetaldehyde; and then derivatizing the mixture with 2, 4-Dinitrophenylhydrazine (DNPH) and acetaldehyde to generate a derivatization product DNPH-acetaldehyde. In this embodiment, please refer to fig. 2, which is a diagram of a mechanism of chlorpromazine hydrochloride molecule photolysis, wherein the chlorpromazine hydrochloride molecule becomes a high-excitation molecule and decomposes to generate free radicals under the condition of illumination, and a series of cascade reactions occur; the free radical reacts with water in the solvent to form a hydroxyl radical, which is captured by ethanol as a pharmaceutical adjuvant to form acetaldehyde.
Further, in order to verify that chlorpromazine hydrochloride generates hydroxyl radicals in the photolysis process, experimental verification is also performed on the photolysis mechanism process of chlorpromazine hydrochloride in the embodiment. In this example, the presence of hydroxyl radicals in chlorpromazine hydrochloride solution was demonstrated by high performance liquid chromatography-diode array-fluorescence detector (HPLC-SPD-FL) method, and further confirmed by liquid chromatography-mass spectrometry (LC-MS) method that the chlorpromazine hydrochloride molecule generated hydroxyl radicals during photolysis, and also confirmed ethoxy (C) 2 H 5 O.cndot.) free radical reaction process, specifically as follows:
terephthalic acid (TPA) is a commonly used fluorescent probe molecule for detecting hydroxyl radicals, TPA is non-fluorescent per se, but the generated hydroxyphthalic acid (HTA) after reacting with the hydroxyl radicals has fluorescence, and the product is single and stable, and has been widely used in the catalysis field to prove that the hydroxyl radicals are generated in the reaction process. Since other photolytic products of chlorpromazine hydrochloride also have fluorescence, HTA needs to be detected after separation from other fluorescent products, and thus high performance liquid chromatography-diode array-fluorescence detector (HPLC-SPD-FL) combination method with good definite ability is used to detect HTA in chlorpromazine hydrochloride.
After adding TPA into chlorpromazine hydrochloride solution, light is irradiated, and high performance liquid chromatography-diode array-fluorescence detector (HPLC-SPD-FL) combined method is used for detecting whether HTA is generated or not to prove the generation of hydroxyl free radicals. As a result, referring to fig. 3, which is a chromatogram of an illuminated solution detected by the HPLC-SPD-FL method, referring to fig. a of fig. 3, which is a fluorescence chromatogram, the illuminated mixed standard solution 1 of TPA and HTA, the illuminated aqueous solution 2 of chlorpromazine hydrochloride containing TPA, and the illuminated solution 3 of chlorpromazine hydrochloride containing TPA and 10% volume fraction ethanol all have fluorescence chromatographic peak responses, that is, HTA is generated, which can prove that hydroxyl radicals are generated during the chlorpromazine hydrochloride photolysis process. Further comparing, the peak height of 3 is lower than that of 2, that is to say, the content of HTA generated by the chlorpromazine hydrochloride alcohol solution after illumination is lower than that of the chlorpromazine hydrochloride aqueous solution, because ethanol can be used as a hydroxyl radical capturing agent and can compete with TPA to capture hydroxyl radicals, thereby reducing the yield of HTA; thus, this side demonstrates that ethanol can capture hydroxyl radicals generated during chlorpromazine hydrochloride illumination, ultimately forming acetaldehyde. Referring to fig. 3 b, which shows a uv chromatogram, the uv detector does not detect the uv response peaks of HTA of 2 and 3, which is due to the weak uv signal and strong fluorescence signal of HTA itself; TPA has only an ultraviolet absorbance signal and no fluorescence signal; making it a common reagent for proving the presence of hydroxyl radicals.
To gain insight into the mechanism of chlorpromazine hydrochloride photolysis and acetaldehyde generation upon addition of ethanol, we identified two chlorpromazine hydrochloride photolysis products associated with the free radical process using liquid chromatography-mass spectrometry (LC-MS). Referring to fig. 4, a mass spectrum of 2-hydroxy promazine in the solution after LC-MS detection and illumination is shown, wherein fig. a is a mass spectrum of 2-hydroxy promazine in chlorpromazine hydrochloride solution without ethanol, and fig. b is a mass spectrum of 2-hydroxy promazine in chlorpromazine hydrochloride solution with 10% ethanol by volume fraction; the result shows that no matter whether ethanol auxiliary agent is added, an m/z= 301.14 molecular ion peak is detected, and the abundance ratio of m/z= 302.06 to m/z= 301.13 is close to 18.4 percent, which is completely matched with the isotope theoretical distribution ratio of 2-hydroxy promazine, so that the substance can be estimated to be the product of the chlorpromazine hydrochloride after dechlorination and being replaced by hydroxyl radicals, namely the 2-hydroxy promazine according to the isotope mass spectrometry identification method; this demonstrates from the side that free radicals are generated during chlorpromazine hydrochloride photolysis.
Referring to fig. 5, a mass spectrum of 2-ethoxypromazine in a solution after LC-MS detection and illumination is shown, wherein fig. a is a mass spectrum of 2-ethoxypromazine in a chlorpromazine hydrochloride solution without ethanol, and fig. b is a mass spectrum of 2-ethoxypromazine in a chlorpromazine hydrochloride solution with 10% ethanol by volume fraction; the results indicate that in the absence of ethanol, noAn m/z= 329.22 molecular ion peak is generated, whereas in the presence of ethanol; m/z= 330.22 molecular ion peak to abundance ratio close to 1:5, matching with the isotope theory distribution proportion of the 2-ethoxypromazine, so that the substance can be presumed to be a product obtained by dechlorinating chlorpromazine hydrochloride and replacing the chlorpromazine hydrochloride by ethoxy, namely the 2-ethoxypromazine according to the isotope mass spectrometry identification method; in this context, however, ethoxy groups can only be derived from ethanol. Thus, it can be presumed that ethanol participates in the radical reaction process. The formation of acetaldehyde may be the reaction of hydroxyl radicals with ethanol to form ethoxy (C 2 H 5 O.radical, ethoxy (C) 2 H 5 O.cndot.) the radicals react with hydroxyl radicals to form acetaldehyde.
In the embodiment, ethanol is used for capturing hydroxyl radicals generated in the photolysis process of chlorpromazine hydrochloride to generate acetaldehyde, and 2, 4-Dinitrophenylhydrazine (DNPH) is used as a derivatization reagent to react with the generated acetaldehyde to generate a derivatization product DNPH-acetaldehyde.
Referring to FIG. 6, a liquid chromatogram of a derivatization solution of chlorpromazine hydrochloride solution is shown, wherein FIG. a shows a DNPH-acetaldehyde peak area diagram, and FIG. b shows a DNPH-acetaldehyde peak area diagram; 1 represents a chlorpromazine hydrochloride derivatization solution with indoor illumination of 30s, and 2 represents a chlorpromazine hydrochloride derivatization solution with indoor illumination of 0 s; the chlorpromazine hydrochloride solution after illumination generates acetaldehyde, the acetaldehyde and DNPH further generate derivatization reaction to generate DNPH-acetaldehyde, and the chromatographic peak intensity of the DNPH-acetaldehyde is enhanced.
Referring to FIG. 7, a liquid chromatogram of a derivatization solution of an acetaldehyde standard solution is shown, wherein FIG. a is a DNPH-acetaldehyde peak diagram, and FIG. b is a linear regression fit of the standard concentration of acetaldehyde to the DNPH-acetaldehyde peak area; in the range of 0-10.0 mug/mL of standard concentration of acetaldehyde, the peak area of DNPH-acetaldehyde chromatographic peak gradually increases with the increase of standard concentration of acetaldehyde, and the linear regression equation of the standard curve is y=479889x+7540.9, wherein x represents the standard concentration of acetaldehyde, y represents the peak area of DNPH-acetaldehyde, and the correlation coefficient R 2 ＝0.9998。
And (3) detecting by an ultraviolet light intensity value measuring method: after the DNPH-acetaldehyde chromatographic peak area a after light irradiation for 30s in fig. 6 was removed from the blank, the area was substituted into a linear regression equation y=479889x+7540.9 of the acetaldehyde standard solution shown in fig. 7, and the concentration c of acetaldehyde generated in the chlorpromazine hydrochloride solution was calculated, thereby determining the photodecomposition degree of the chlorpromazine hydrochloride solution.
In this example, the concentration of acetaldehyde in chlorpromazine hydrochloride solution was measured to be 2.415 μg/mL by substituting the DNPH-acetaldehyde chromatographic peak area of fig. 6 after light irradiation for 30s into y=479889x+7540.9, and the t-test result (P < 0.05) showed that there was a significant difference in the acetaldehyde content generated in chlorpromazine hydrochloride solution after light irradiation.
Specifically, the procedure for determining the photodecomposition of chlorpromazine hydrochloride solutions is as follows:
according to the reaction mechanism diagram of chlorpromazine hydrochloride in fig. 2, the relationship between the decomposed chlorpromazine hydrochloride and the hydroxyl radical is:
n (decomposed chlorpromazine hydrochloride) =n (hydroxyl radical)
The relationship between acetaldehyde and the hydroxyl radicals reacted (called available hydroxyl radicals) is:
2n (acetaldehyde) =n (available hydroxyl radicals)
Although not all of the hydroxyl radicals generated by chlorpromazine hydrochloride can be reacted with ethanol to convert into acetaldehyde, we hypothesize that in a certain concentration range, the yield of acetaldehyde is in direct proportion to the decomposition amount of chlorpromazine hydrochloride, so the following formula is given:
since the reactions are in the same solution and the volumes are equal, there are also:
decomposition rate of chlorpromazine hydrochloride:
wherein:
c (acetaldehyde) is the concentration of acetaldehyde in chlorpromazine hydrochloride solution, and the unit is mug/mL;
c (original chlorpromazine hydrochloride) is the original concentration of chlorpromazine hydrochloride solution, and the unit is mg/mL;
m (chlorpromazine hydrochloride) is the relative average molecular weight of chlorpromazine hydrochloride, namely 355.32g/mol;
m (acetaldehyde) is the relative average molecular mass of acetaldehyde, i.e., 44.05g/mol;
the conversion rate of chlorpromazine hydrochloride into acetaldehyde by free radical is generally 15% -25%, the decomposition amount of chlorpromazine hydrochloride and the generation amount of acetaldehyde are detected by designing related high performance liquid chromatography experiments, the generation amount of acetaldehyde is X-axis, the decomposition amount of chlorpromazine hydrochloride is Y-axis, and a linear regression curve is drawn to obtain a linear regression equation, wherein the slope is the conversion rate of chlorpromazine hydrochloride into acetaldehyde by free radical.
Specifically, in this example, a correlation experiment was designed by detecting the relationship between the decomposition amount of chlorpromazine hydrochloride and the generation amount of acetaldehyde, and a linear regression equation was drawn.
The relevant experiments were as follows:
firstly, according to the decomposition amount of chlorpromazine hydrochloride, the quantification is realized through a working curve, namely chlorpromazine hydrochloride standard solutions with different concentrations are prepared, the solution is detected by a liquid chromatography, and a concentration-peak area linear regression curve (namely the working curve) is drawn, wherein the specific steps are as follows:
a1. preparing a standard solution: precisely weighing 0.0500g of chlorpromazine hydrochloride, dissolving and transferring to a 100mL volumetric flask with a proper amount of ultrapure water, fixing the volume to the scale with ultrapure water, and shaking to obtain chlorpromazine hydrochloride mother liquor. And preparing the chlorpromazine hydrochloride mother liquor into chlorpromazine hydrochloride solutions of 10, 20, 40, 60, 80, 100 and 120 mug/mL by using ultrapure water respectively, then passing through a microporous filter membrane, performing liquid chromatography detection, and drawing a standard chlorpromazine hydrochloride concentration-peak area linear regression curve to obtain a linear regression equation.
b1. Sample solution pretreatment: diluting chlorpromazine hydrochloride solution with the original concentration of 10mg/mL after natural illumination for 0, 5, 30, 60 and 90min by 10 times, passing through a microporous filter membrane, and performing liquid chromatography detection to obtain chromatographic peak areas respectively.
The liquid chromatography detection conditions of a1 and b1 are as follows: the chromatographic conditions were a column using a Shim-pack GIST C18 (2.1X100 mm,3 μm), mobile phase A:0.1% trifluoroacetic acid, mobile phase B: acetonitrile, gradient elution (0-1.00 min,80.0% A;1.00-10.00min,80.0% A-20.0% A;10.00-10.50min,20.0% A-80.0% A;10.50-13.00min,80.0% A-80.0% A), flow rate: 0.500mL/min, column temperature: 40 ℃, sample injection amount: 10 mu L, and the detection wavelength was 265nm.
c1. And c, substituting the chromatographic peak areas obtained in the step b1 into the linear regression equation of the step a1 respectively to obtain the concentration of chlorpromazine hydrochloride in the chlorpromazine hydrochloride solution after illumination, so as to make a difference with the original concentration of the chlorpromazine hydrochloride, and respectively calculating the decomposition amount of the chlorpromazine hydrochloride.
Secondly, the generation amount of acetaldehyde is also tested by an acetaldehyde working curve, and the specific steps are as follows:
a2. preparing a standard solution: respectively taking 50 mug/mL of acetaldehyde standard solution 10, 20, 30, 40, 50, 100, 150, 200, 250, 400, 600, 800 and 1000 mug to 20mL of brown bottle, adding water with corresponding volume, and supplementing the volume to 1000 mug; then, 2000. Mu.L of 2mg/mL 2, 4-dinitrophenylhydrazine solution was added in sequence, and then, buffer solution with pH=5.00 was added to 5mL, at which time the concentration of acetaldehyde in the derivatization reagent was 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 6.0, 8.0, 10.0. Mu.g/mL, respectively. Placing the mixture in a constant-temperature oscillating water bath kettle at 60 ℃ for reaction for 40min, taking out the mixture, placing the mixture at room temperature, passing the mixture through a microporous filter membrane, performing liquid chromatography detection, and drawing a standard acetaldehyde concentration-peak area linear regression curve to obtain a linear regression equation.
b2. Sample pretreatment: taking 200 mu L of chlorpromazine hydrochloride solution with the concentration of 10mg/mL after natural illumination for 0, 5, 30, 60 and 90min, adding 800 mu L of water, sequentially adding 2000 mu L of 2mg/mL of 2, 4-dinitrophenylhydrazine solution, then adding buffer solution with the pH value of=5.00 to 5mL, standing at the constant temperature of 60 ℃ for 40min, taking out, standing at the room temperature, passing through a microporous filter membrane, and carrying out liquid chromatography detection to obtain chromatographic peak areas respectively.
The liquid chromatography detection conditions of a2 and b2 are as follows: the chromatographic conditions were a column using a Shim-pack GIST C18 (2.1X100 mm,3 μm), mobile phase A:0.1% trifluoroacetic acid; mobile phase B: acetonitrile, gradient elution (0-0.50 min,70.0% A;0.50-5.00min,70.0% A-20.0% A;5.00-6.00min,20.0% A-20.0% A;6.00-6.50min,20.0% A-70.0% A;6.50-9.00min,70.0% A-70.0% A), flow rate: 0.500mL/min, column temperature: 40 ℃, sample injection amount: 10. Mu.L, the detection wavelength was 355nm, respectively.
c2. Substituting the chromatographic peak areas obtained in the step b2 into the linear regression equation of the step a2 respectively to obtain the concentration of the generated acetaldehyde, namely the generated amount of the acetaldehyde, in the chlorpromazine hydrochloride solution after illumination.
Referring to fig. 8, a linear regression curve is drawn according to the data of c1 and c2, wherein the linear regression equation of the linear regression fit of the decomposition amount of chlorpromazine hydrochloride and the generation amount of acetaldehyde in the present embodiment is y=0.15x, wherein y represents the decomposition amount of chlorpromazine hydrochloride, x represents the generation amount of acetaldehyde, and the correlation coefficient R 2 =0.9998, the slope is the conversion of chlorpromazine hydrochloride to acetaldehyde by free radical, that is to say in this example,
namely chlorpromazine hydrochloride with a decomposition rate of
To further demonstrate the beneficial effects of this example, the present example also diluted 100-fold with 100 mg/mL chlorpromazine hydrochloride solution with 30s of indoor illumination and 10.0mg/mL non-illumination, passed through a microporous filter membrane, detected by liquid chromatography, and three sets of parallel experiments were performed, respectively. Referring to fig. 9, a comparison chart of chlorpromazine hydrochloride liquid chromatography after non-illumination and indoor natural illumination for 30s shows that there is no obvious difference between the illumination for 30s and the non-illumination chlorpromazine hydrochloride signals, i.e. the chromatographic peak areas of chlorpromazine hydrochloride solution before and after illumination are not changed significantly, which indicates that no obvious decomposition of chlorpromazine hydrochloride is detected by adopting the traditional liquid chromatography. The method has high sensitivity, and can detect that chlorpromazine hydrochloride is decomposed after indoor natural light illumination for 30 s.
In other embodiments, in addition to ethanol, methanol, propanol, isopropanol, and butanol may also be used as radical traps for chlorpromazine hydrochloride, with the corresponding products being formaldehyde, propionaldehyde, acetone, and butyraldehyde, respectively; referring to FIG. 10, the corresponding DNPH-formaldehyde, DNPH-propanal, DNPH-acetone and DNPH-butanal signals were also detected by liquid chromatography after DNPH derivatization, but it is noted that methanol is toxic and generally not used as a pharmaceutical adjuvant.
The method of the invention utilizes a specific drug auxiliary agent (such as an alcohol compound) to be added into a drug matrix, free radicals generated by the drug matrix in the photolysis process of the drug matrix are captured by the drug auxiliary agent to generate substances (such as aldehyde compounds) with higher chemical activity and large difference between physical and chemical properties and the drug matrix, and then the substances are derivatized with a derivatization reagent to form a derivatization product, and the photodecomposition degree of the drug is determined by adopting a liquid chromatography.
Compared with the prior art, the method has the advantages of simple and quick treatment process, no need of complex separation and detection equipment, high sensitivity, and suitability for detecting impurities with low content, complex matrix and similar structural properties to main components
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention, and the invention is intended to encompass such modifications and improvements.
Claims (2)
1. The method for analyzing the photodecomposition degree of the medicine by tracing the auxiliary agent is characterized by comprising the following steps of: the method comprises the following steps:
and (3) derivatization treatment: taking a certain amount of medicine, wherein the medicine comprises a medicine matrix and a medicine auxiliary agent, the medicine matrix can generate photolysis reaction and generate free radicals, the medicine auxiliary agent can capture the free radicals generated by the medicine matrix in the photolysis process and generate a first component, the medicine is a solution, the volume fraction of the medicine auxiliary agent is 5% -50% of that of the medicine solution, and a certain amount of derivatization reagent is added into the medicine to obtain a derivatization solution, and the derivatization reagent can be derivatized with the first component to obtain a derivatization product;
and (3) detection: determining the photodecomposition degree of the drug by liquid chromatography on the derivatized solution, wherein the liquid chromatography is performed by liquid chromatography detection at a specific wavelength, and the specific wavelength is the wavelength at the chromatographic peak of the derivatized product;
the liquid chromatography method for determining the photodecomposition degree of the drug comprises the following steps: under the same conditions, preparing a series of standard solutions of a first component with concentration gradients, respectively carrying out derivatization treatment, respectively carrying out liquid chromatography detection under the same specific wavelength, and drawing a standard curve of the peak area of a concentration-derivatization product chromatographic peak to obtain a linear regression equation;
substituting the peak area of the chromatographic peak of the derivatization product obtained by carrying out liquid chromatography detection on the derivatization solution into the linear regression equation to determine the concentration of the first component in the medicine, thereby determining the photodecomposition degree of the medicine;
the medicine matrix is chlorpromazine hydrochloride, the medicine auxiliary agent is ethanol, the free radical is hydroxyl free radical, the first component is acetaldehyde, the photodecomposition degree of the medicine is represented by the decomposition rate omega% of chlorpromazine hydrochloride, and the photodecomposition degree of the medicine accords with the following equation:
wherein: c (acetaldehyde) is the concentration of acetaldehyde in the drug; c (original chlorpromazine hydrochloride) is the original concentration of chlorpromazine hydrochloride in the medicine; m (chlorpromazine hydrochloride) is chlorpromazine hydrochlorideIs a relative average molecular mass of (2); m (acetaldehyde) is the relative average molecular mass of acetaldehyde;conversion of chlorpromazine hydrochloride to acetaldehyde by free radical; said->The determination method of (1) is as follows: the method comprises the steps of respectively detecting the decomposition amount of chlorpromazine hydrochloride and the generation amount of acetaldehyde in the photodecomposition process of a medicine through a designed high performance liquid chromatography experiment, drawing a standard curve of the generation amount of acetaldehyde and the decomposition amount of chlorpromazine hydrochloride, and obtaining a second linear regression equation, wherein the slope of the second linear regression equation is ++ ->
The decomposition amount of chlorpromazine hydrochloride is obtained by making a difference between the original chlorpromazine hydrochloride in the medicine and the amount of chlorpromazine hydrochloride after photodecomposition, and the amount of chlorpromazine hydrochloride after photodecomposition is obtained by the following steps: preparing a series of chlorpromazine hydrochloride solutions with concentration, performing liquid chromatography detection under the same wavelength, drawing a standard curve of the concentration-chromatographic peak area of the chlorpromazine hydrochloride, obtaining a third linear regression equation, respectively performing liquid chromatography detection on the photo-decomposed medicine, obtaining chromatographic peak areas, substituting the chromatographic peak areas into the third linear regression equation, respectively obtaining the concentration of the chlorpromazine hydrochloride in the photo-decomposed medicine, and respectively determining the decomposition amount of the chlorpromazine hydrochloride;
the acetaldehyde generation amount is obtained by: preparing a series of concentration acetaldehyde standard solutions, adding the same amount of derivatization reagent, performing liquid chromatography detection at the same wavelength, drawing a concentration acetaldehyde-chromatographic peak area standard curve, obtaining a fourth linear regression equation, adding the equivalent derivatization reagent into the photo-decomposed medicine, and substituting the peak area obtained by liquid chromatography detection into the fourth linear regression equation to obtain the concentration of acetaldehyde generated in the medicine, thereby determining the generation amount of acetaldehyde.
2. The method for adjuvant-trace analysis of the degree of photodecomposition of a drug according to claim 1, wherein: the derivatization reagent is one of 2, 4-dinitrophenylhydrazine, phenol reagent and Schiff reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111493639.4A CN114166975B (en) | 2021-12-08 | 2021-12-08 | Method for analyzing photodecomposition degree of medicine by adjuvant tracing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111493639.4A CN114166975B (en) | 2021-12-08 | 2021-12-08 | Method for analyzing photodecomposition degree of medicine by adjuvant tracing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114166975A CN114166975A (en) | 2022-03-11 |
| CN114166975B true CN114166975B (en) | 2024-01-09 |
Family
ID=80484330
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111493639.4A Active CN114166975B (en) | 2021-12-08 | 2021-12-08 | Method for analyzing photodecomposition degree of medicine by adjuvant tracing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114166975B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363816A (en) * | 2008-08-22 | 2009-02-11 | 中国烟草总公司郑州烟草研究院 | Reagent for deriving small molecule aldehyde material and use thereof |
| CN101509903A (en) * | 2009-03-20 | 2009-08-19 | 南京工业大学 | High performance liquid chromatography analysis method for glyoxal and glyoxylic acid |
| CN109633041A (en) * | 2019-02-26 | 2019-04-16 | 广东食品药品职业学院 | The method that derivatization HPLC method measures protona in drug |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10556817B2 (en) * | 2017-01-30 | 2020-02-11 | Florida State University Research Foundation, Inc. | Gas-liquid plasma and bioreactor system and method for remediation of liquids and gases |
-
2021
- 2021-12-08 CN CN202111493639.4A patent/CN114166975B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363816A (en) * | 2008-08-22 | 2009-02-11 | 中国烟草总公司郑州烟草研究院 | Reagent for deriving small molecule aldehyde material and use thereof |
| CN101509903A (en) * | 2009-03-20 | 2009-08-19 | 南京工业大学 | High performance liquid chromatography analysis method for glyoxal and glyoxylic acid |
| CN109633041A (en) * | 2019-02-26 | 2019-04-16 | 广东食品药品职业学院 | The method that derivatization HPLC method measures protona in drug |
Non-Patent Citations (4)
| Title |
|---|
| Spectroscopic studies of cutaneous photosensitizing agentd viii a spin-trapping study of light induced free radicals from chlorpromazine and promazine;Ann G. Motten;《Phorochemistry and Photobiology》;第42卷(第1期);第9-15页 * |
| 光化学衍生-高效液相色谱法测定大米中黄曲霉毒素B_1含量;李梅;《粮食加工》(第06期);全文 * |
| 免疫亲和柱净化光化学衍生高效液相色谱-荧光检测法测定动物类药材中黄曲霉毒素;杨小丽 等;《中国药业》(第15期);全文 * |
| 气相色谱法测定Fenton反应产生的羟基自由基及其清除作用;王炳娟;《理化检验(化学分册)》(第01期);第88-90页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114166975A (en) | 2022-03-11 |
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 | |
| Possanzini et al. | Determination of formaldehyde and acetaldehyde in air by HPLC with fluorescence detection | |
| CN113009028B (en) | Method for detecting related substances of omeprazole sodium for injection | |
| CN114166975B (en) | Method for analyzing photodecomposition degree of medicine by adjuvant tracing | |
| CN113219089A (en) | Method for detecting urea by post-column derivatization-liquid chromatography | |
| Barakoti et al. | Formaldehyde analysis in non-aqueous methanol solutions by infrared spectroscopy and electrospray ionization | |
| Oliver et al. | Ethanol determination using pressure mobilization and free solution capillary electrophoresis by photo-oxidation assisted ultraviolet detection | |
| CN114166834B (en) | Method for analyzing photodecomposition degree of medicine by tracing auxiliary agent | |
| CN111855836A (en) | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography | |
| CN114166976B (en) | Method for analyzing drug content in health care product by tracing auxiliary agent | |
| CN114166824B (en) | Method for analyzing drug content in health care product by tracing auxiliary agent | |
| CN114839287A (en) | Method for detecting sodium tetradecanoate in miboplatin | |
| CN114166835B (en) | Method for analyzing drug content in health care product by tracing auxiliary agent | |
| Alzweiri et al. | Stability determination for cyclized 2, 4-dinitrophenyl hydrazone derivative of glucose | |
| Wang et al. | Analysis of spectinomycin by HPLC with evaporative light-scattering detection | |
| CN108982706B (en) | Method for detecting impurity cis-perhydroisoindole in mitiglinide calcium | |
| CN107085056B (en) | A kind of gaschromatographic mass spectrometry detection method of nitrofurazone biological marker 5- nitro -2- furfural | |
| Basheer | Extraction of Pregabalin in Urine Samples Using a Sulfonated Poly (ether ether ketone) Membrane | |
| CN110887930A (en) | Method for measuring oxalic acid content in workplace | |
| CN118566395B (en) | Method, application and system for determining impurity content in toluene sulfonic acid Lu Meipai protuberance orally disintegrating tablet | |
| Verma et al. | DETECTION OF METFORMIN IN BIOLOGICAL MATRICES EMPLOYING THIN LAYER CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY. | |
| CN113376294A (en) | Analysis method of ethyl cyanoacetate in tofacitinib citrate | |
| CN116953131A (en) | Glyceraldehyde detection method and application thereof | |
| CN117288860A (en) | Analysis method of genotoxic impurities in flurbiprofen | |
| Mohr et al. | Analysis of omarigliptin forced degradation products by ultra‐fast liquid chromatography, mass spectrometry, and in vitro toxicity assay |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |