CN113834885A - HPLC (high Performance liquid chromatography) detection method and methodology verification for clotrimazole raw material genotoxic impurities - Google Patents
HPLC (high Performance liquid chromatography) detection method and methodology verification for clotrimazole raw material genotoxic impurities Download PDFInfo
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- CN113834885A CN113834885A CN202110941523.6A CN202110941523A CN113834885A CN 113834885 A CN113834885 A CN 113834885A CN 202110941523 A CN202110941523 A CN 202110941523A CN 113834885 A CN113834885 A CN 113834885A
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- clotrimazole
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- glyoxal
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- VNFPBHJOKIVQEB-UHFFFAOYSA-N clotrimazole Chemical compound ClC1=CC=CC=C1C(N1C=NC=C1)(C=1C=CC=CC=1)C1=CC=CC=C1 VNFPBHJOKIVQEB-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229960004022 clotrimazole Drugs 0.000 title claims abstract description 63
- 239000002994 raw material Substances 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 32
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 24
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 24
- 239000012535 impurity Substances 0.000 title claims abstract description 24
- 238000001514 detection method Methods 0.000 title claims description 67
- 238000012795 verification Methods 0.000 title claims description 29
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 195
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims abstract description 132
- 239000000243 solution Substances 0.000 claims abstract description 111
- 239000013558 reference substance Substances 0.000 claims abstract description 75
- 229940015043 glyoxal Drugs 0.000 claims abstract description 66
- 239000012085 test solution Substances 0.000 claims abstract description 59
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000003085 diluting agent Substances 0.000 claims abstract description 38
- 239000011550 stock solution Substances 0.000 claims abstract description 37
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000001212 derivatisation Methods 0.000 claims abstract description 33
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 230000035945 sensitivity Effects 0.000 claims abstract description 7
- 238000012360 testing method Methods 0.000 claims description 50
- 239000000523 sample Substances 0.000 claims description 36
- 239000013641 positive control Substances 0.000 claims description 30
- 239000012088 reference solution Substances 0.000 claims description 24
- 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 23
- 239000000706 filtrate Substances 0.000 claims description 20
- 239000012488 sample solution Substances 0.000 claims description 17
- 238000011084 recovery Methods 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 12
- 230000014759 maintenance of location Effects 0.000 claims description 12
- 238000011002 quantification Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000012490 blank solution Substances 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 230000004044 response Effects 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 6
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- 238000010200 validation analysis Methods 0.000 claims description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 230000003044 adaptive effect Effects 0.000 claims description 3
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims 1
- 239000007858 starting material Substances 0.000 claims 1
- 230000009897 systematic effect Effects 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 7
- 238000001914 filtration Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 238000005259 measurement Methods 0.000 description 10
- -1 ketone compounds Chemical class 0.000 description 9
- 238000001816 cooling Methods 0.000 description 8
- 238000012417 linear regression Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
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- 239000012071 phase Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000012483 derivatization solution Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 150000001299 aldehydes Chemical class 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 150000007857 hydrazones Chemical class 0.000 description 3
- 238000005935 nucleophilic addition reaction Methods 0.000 description 3
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 description 3
- 229940067157 phenylhydrazine Drugs 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- YEIYMELXENLJSN-SOSXVSKCSA-N (e)-[(2z)-2-hydrazinylideneethylidene]hydrazine Chemical compound N\N=C\C=N/N YEIYMELXENLJSN-SOSXVSKCSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- QZUDBNBUXVUHMW-UHFFFAOYSA-N clozapine Chemical compound C1CN(C)CCN1C1=NC2=CC(Cl)=CC=C2NC2=CC=CC=C12 QZUDBNBUXVUHMW-UHFFFAOYSA-N 0.000 description 1
- 229960004170 clozapine Drugs 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
- G01N2030/042—Standards
Abstract
A method for detecting genotoxic impurity in clotrimazole raw material by HPLC and its methodology are verified, taking formaldehyde reference substance or glyoxal reference substance to prepare corresponding positive reference substance stock solution quantitatively with diluent, taking clotrimazole raw material and diluent or corresponding positive reference substance stock solution, phosphoric acid solution, derivative solution to conduct derivatization reaction, preparing the clotrimazole raw material and diluent into test solution and positive test solution, injecting the test solution and positive test solution into a liquid chromatograph, detecting and recording chromatogram under the conditions that the column temperature is 30-35 ℃, the flow phase is acetonitrile and water, and the flow rate is 0.5-1ml/min, and calculating the content of formaldehyde or glyoxal according to the following formula:
proceeding according to specificity, sensitivity, linearity and range, precision, accuracy, durabilityThe legislation verifies that the potential genotoxic impurities, namely formaldehyde and glyoxal, in the clotrimazole bulk drug can be quickly and effectively detected so as to control the quality of the clotrimazole bulk drug.
Description
Technical Field
The invention relates to an HPLC (high performance liquid chromatography) detection method and a methodology verification for clotrimazole raw material genotoxic impurities, belonging to the technical field of clotrimazole raw material detection.
Background
Clotrimazole is a broad-spectrum antifungal drug, has a good antibacterial effect on various fungi, particularly candida albicans, has an action mechanism of inhibiting the synthesis of fungal cell membranes and influencing the metabolic process of the fungal cell membranes, and is used for systemic fungal infection and topical fungal infection. The synthesis process of clotrimazole comprises the following steps:
the method comprises the following steps: imidazole is synthesized according to the following synthesis process flow I:
step two: synthesizing chloro (o-chlorophenyl) diphenylmethane according to the following synthesis process flow:
step three: synthesizing clotrimazole according to the following synthesis process flow:
therefore, the clotrimazole has the risk of introducing genotoxic impurities of formaldehyde and glyoxal in the imidazole synthesis process, so that a method for quickly and effectively detecting and analyzing the contents of the potential genotoxic impurities of the clozapine bulk drug, namely the formaldehyde and the glyoxal, needs to be developed, and the method is verified by methodology to evaluate the detection method so as to control the quality of clotrimazole raw material drugs.
Disclosure of Invention
The invention aims to provide an HPLC (high performance liquid chromatography) detection method and a methodology verification for clotrimazole raw material genotoxic impurities, which aim to quickly and effectively detect potential genotoxic impurities, namely formaldehyde and glyoxal, in clotrimazole bulk drugs, and the result of the methodology verification meets the requirement so as to control the quality of clotrimazole raw materials.
The invention is realized by the following technical scheme:
an HPLC detection method for clotrimazole raw material genotoxic impurities comprises the following steps: quantitatively preparing a corresponding positive control stock solution by using a formaldehyde reference substance or a glyoxal reference substance with a diluent, preparing a specimen solution and a positive specimen solution by using a clotrimazole raw material and the diluent or the corresponding positive control stock solution, a phosphoric acid solution and a derivative solution through derivatization reaction, preparing the specimen solution and the positive specimen solution with the diluent, respectively injecting the corresponding positive control stock solution, the specimen solution and the positive specimen solution into a liquid chromatograph, detecting and recording chromatograms at a column temperature of 30-35 ℃, a circulation phase of acetonitrile and water and a flow rate of 0.5-1ml/min, and calculating the content of formaldehyde or glyoxal according to the following formula:
in the formula ATest solutionIs the peak area of formaldehyde or glyoxal in the chromatogram of the test solution; a. thePositive test solutionThe peak area of formaldehyde or glyoxal in the chromatogram of the positive test solution is shown; cPositive control stock solutionConcentration of formaldehyde or glyoxal (μ g/ml) in the stock solution for the positive control; w is the weight (g) of the test solution clotrimazole;
when the content of formaldehyde is detected, the diluent is prepared from acetonitrile, the derivative solution is prepared from 2, 4-dinitrophenylhydrazine and acetonitrile, and the chromatographic conditions of a liquid chromatograph are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as filler, the column temperature is 30 ℃, the volume ratio of acetonitrile to water is 75:25 as mobile phase, and the running time is 40 min; the flow rate is 0.5ml/min, the detection wavelength is 355nm, and the sample injection volume is 10-20 mul;
when detecting the content of glyoxal, the diluent adopts absolute ethyl alcohol, the derivative solution adopts 2, 4-dinitrophenylhydrazine and the absolute ethyl alcohol to prepare, and the chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as filler, the column temperature is 35 ℃, acetonitrile-water volume ratio is 70:30 as mobile phase, and the running time is 25 min; the flow rate is 1ml/min, the detection wavelength is 400nm, and the sample injection volume is 10-20 mul;
further, when preparing a test sample solution and a positive test sample solution, the clotrimazole raw material and a diluent or a corresponding positive control sample storage solution are shaken for 30min at 150 revolutions per minute, then the mixture is filtered, the filtrate is added with a phosphoric acid solution, the derivative solution is shaken for 1h at 70 ℃ at 150 revolutions per minute, then the diluent is added, the mixture is mixed evenly, and then the filtrate is filtered and the continuous filtrate is obtained.
Further, when detecting the formaldehyde content, the formaldehyde content in the positive control stock solution is 0.15 mug/ml, and the dosage-mass ratio of the clotrimazole raw material to the 2, 4-dinitrophenylhydrazine in the derivative solution when preparing the test solution and the positive test solution is 2500: 1, when detecting glyoxal, the content of glyoxal in the positive control storage solution is 0.3 mug/ml, and the dosage mass ratio of the clotrimazole raw material to the 2, 4-dinitrophenylhydrazine in the derivative solution when preparing the test solution and the positive test solution is 5000: 1.
the methodology of the HPLC detection method for the genotoxic impurities in the clotrimazole raw material is verified, wherein the verification items comprise specificity, sensitivity, linearity and range, precision, accuracy and durability, and a reference solution is prepared by taking a corresponding positive reference stock solution, adding a phosphoric acid solution, shaking a derivative solution and adding a diluent;
when the specificity is verified, a diluent, a phosphoric acid solution and a derivative solution are shaken and then added to prepare a blank solution, and the mass ratio of the amount of the 2, 4-dinitrophenylhydrazine in the methanol reference substance to the amount of the 2, 4-dinitrophenylhydrazine in the derivative solution is 0.75:100 when the reference substance solution is prepared during formaldehyde detection; verifying that the mass ratio of the amount of the 2, 4-dinitrophenylhydrazine in the methanol reference substance and the derivative solution is 1.5:100 when the reference substance solution is prepared during glyoxal detection, respectively injecting a blank solution and the reference substance solution into a liquid chromatograph for detection, wherein the interference peak area in the blank solution chromatogram is less than or equal to 10% of the main peak area of the reference substance solution;
when the sensitivity is verified, respectively performing derivatization reaction on the positive control stock solution and the positive control stock solution, performing HPLC detection, and determining a detection limit and a quantification limit by adopting a signal-to-noise ratio (S/N), wherein the S/N is more than or equal to 3; the quantitative limit S/N is more than or equal to 10, the quantitative limit concentration is less than 30% of the limit concentration, the parallel 6 detection peak area RSD is less than or equal to 15%, the retention time RSD is less than or equal to 2%, when the linearity and range are verified, reference substance solutions with multiple concentrations are prepared within 200% of the limit concentration and subjected to derivatization and HPLC detection, the measured response value peak area A is used as a function drawing of the Y axis to the concentration C of the measured object as the X axis, the correlation coefficient r is more than or equal to 0.999, and the I Y axis intercept I/100% response value is less than or equal to 20%;
and (3) sample injection precision during precision verification: multiple reference substance solutions have continuous sample injection retention time RSD less than or equal to 1, peak area RSD less than or equal to 2%, and repeatability is as follows: the absolute deviation of the formaldehyde content is not more than 5% of the quality standard, the absolute deviation of the formaldehyde content in the intermediate precision is not more than 5% of the quality standard, the verification accuracy yield range is 75-120%, and the RSD is not more than 10%;
compared with 0 hour when the durability is verified, the maximum change rate of the main peak area of the reference substance solution is less than or equal to 10 percent, the relative standard deviation of the main peak area obtained by continuously injecting the reference substance solution for 6 times in a system adaptability manner is less than or equal to 5 percent, the content RSD of the formaldehyde or glyoxal in the test substance solution is less than or equal to 20 percent, and the recovery rate of the formaldehyde or glyoxal in the test substance solution is between 75 and 120 percent.
The invention has the beneficial effects that:
the HPLC detection method is adopted to carry out medicine detection on the clotrimazole raw material, the methodological verification result meets the requirement, carbonyl groups in aldehyde and ketone compounds can carry out nucleophilic addition reaction with amino groups in phenylhydrazine to generate aldehyde hydrazone or ketone hydrazone products, larger pi-pi conjugated systems exist in the molecular structure of hydrazone, characteristic absorption is realized in an ultraviolet-visible light region, and formaldehyde and 2, 4-dinitrophenylhydrazine in a derivative solution react to generate 2, 4-dinitrophenylhydrazone, glyoxal and 2, 4-dinitrophenylhydrazine to generate glyoxaldihydrazone.
Drawings
FIG. 1 is a linear regression equation for formaldehyde detection.
FIG. 2 is a chromatogram of a sample solution.
FIG. 3 is a graph of a linear regression equation for glyoxal testing.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings.
An HPLC detection method for genotoxic impurities in clotrimazole raw materials is used for detecting the content of formaldehyde:
the diluent is acetonitrile; the formaldehyde reference substance is purchased from Aladdin K1822057, and the content is 100 mg/L;
control stock solutions: precisely measuring a proper amount of formaldehyde reference substances, and quantitatively diluting with a diluent to prepare a solution containing 5 mu g/ml of formaldehyde in each 1 ml;
positive control stock solution: precisely measuring 3ml of reference stock solution, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking;
test solution and positive test solution: respectively and precisely weighing 0.5g of each of two parts of clotrimazole raw materials, adding 10ml of diluent into one part of the clotrimazole raw material, adding 10ml of positive reference substance stock solution into the other part of the clotrimazole raw material, placing the clotrimazole raw material in a constant temperature oscillator to shake for 30 minutes at 150 revolutions per minute, filtering, taking continuous filtrate as test sample working solution and positive test sample working solution, precisely measuring 5ml of each of the test sample working solution and the positive test sample working solution, adding 2.0ml of 5% phosphoric acid solution and 1.0ml of derivative solution, mixing uniformly, immediately placing the mixture in a 70 ℃ water bath constant temperature oscillator to shake at 150 revolutions per minute, taking out the mixture after 1 hour, cooling the mixture to room temperature, adding 2.0ml of diluent, mixing uniformly, filtering, and taking continuous filtrate;
blank solution: precisely measuring 5ml of diluent, adding 1.0ml of 1% phosphoric acid solution, adding 1.0ml of derivative solution (10 mg of 2, 4-dinitrophenylhydrazine is taken and placed in a 100ml measuring flask, and is dissolved and diluted to scale with acetonitrile), immediately placing the mixture in a 70 ℃ water bath constant temperature oscillator, shaking at 150 revolutions per minute, taking out the mixture after 1 hour, cooling to room temperature, adding 3.0ml of diluent, uniformly mixing, filtering, and taking the subsequent filtrate.
Control solution: precisely measuring 5ml of positive control stock solution, adding 1.0ml of 1% phosphoric acid solution and 1.0ml of derivative solution, immediately placing in a 70 ℃ water bath constant temperature oscillator, shaking at 150 rpm, taking out after 1 hour, cooling to room temperature, adding 3.0ml of diluent, mixing uniformly, filtering, and taking the subsequent filtrate.
Precisely measuring 10 mul of each of the test solution and the positive test solution, and respectively injecting into a liquid chromatograph under the chromatographic conditions: octadecylsilane bonded silica gel as filler (Waters Symmetry C18, 250 × 4.6mm, 5 μm, or equivalent performance column); the column temperature is 30 ℃; acetonitrile-water (volume ratio is 75:25) is used as a mobile phase; the running time is 40 min; the flow rate is 0.5 ml/min; the detection wavelength is 355 nm; the sample injection volume is 10 mul; detecting and recording a chromatogram, and calculating the content of formaldehyde according to the following formula;
in the formula ATest solutionIs the peak area of formaldehyde in the chromatogram of the test solution; a. thePositive test solutionThe peak area of formaldehyde in the chromatogram of the positive test solution is shown; cPositive control stock solutionConcentration of formaldehyde in stock solution (μ g/ml) for positive control; w is the weight (g) of the test solution clotrimazole;
the carbonyl in the aldehyde and ketone compound can carry out nucleophilic addition reaction with the amino in the phenylhydrazine to generate aldehyde hydrazone or ketone hydrazone products. The molecular structure of hydrazone has a larger pi-pi conjugated system, and has characteristic absorption in an ultraviolet-visible light region. Formaldehyde reacts with 2, 4-dinitrophenylhydrazine to generate 2, 4-dinitrophenylhydrazone, so that the HPLC method is adopted for measurement, and the maximum absorption wavelength of a main component is about 355nm and is determined to be 355nm as can be seen from an HPLC spectrogram of a reference substance solution;
weighing 0.5g of the product, precisely weighing, placing in 4 centrifuge tubes, adding 10.0ml of formaldehyde reference substance solution of 0.15 μ g/ml, shaking for 20 min, 30min, 45 min and 60 min, and filtering. Precisely taking 5ml of subsequent filtrate, placing the subsequent filtrate in a centrifuge tube, adding 1.0ml of derivative solution, adding 2.0ml of 5% phosphoric acid, shaking for 1 hour in a water bath constant temperature oscillator at 70 ℃, taking out the subsequent filtrate, cooling to room temperature, adding acetonitrile to 10ml, filtering, taking 10 mu l of the subsequent filtrate, injecting the subsequent filtrate into a chromatograph, and recording a chromatogram, wherein the results are shown in the following table 1:
TABLE 1 extraction time and Peak area
| Extraction time | Peak area |
| 20min | 74953 |
| 30min | 71641 |
| 45min | 68060 |
| 60min | 70712 |
As can be seen from the table above, the extraction time is 30 minutes, and the peak area of the derivative product is basically stable at 20 minutes, which indicates that the sample is completely extracted;
2, 4-dinitrophenylhydrazine is used as a derivatization reagent, in order to ensure that the derivatization reaction is complete, different amounts of derivatization solutions are respectively added, and simultaneously, 5ml of a reference solution is subjected to derivatization reaction, and the result is shown in the following table 2:
TABLE 2 addition of derivatization solution and peak area
| Addition of derivatizing solution | Peak area |
| 0.5ml | 64064 |
| 0.8ml | 66430 |
| 1.0ml | 70712 |
| 1.5ml | 71123 |
As can be seen from the above table, when the derivatization is performed by using an excessive amount of the derivatization solution, the peak area of the derivatization product does not change with the change of the amount of the 2, 4-dinitrophenylhydrazine, and the addition amount of 1.0ml of the derivatization solution is selected;
according to the characteristics of the solvent acetonitrile, 70 ℃ is selected as the derivatization temperature, and the reference substance solution is respectively derivatized for 1 hour, 1.5 hours, 2 hours and 3 hours, and the results are shown in the following table 3:
TABLE 3 derivatization time and peak area
| Time of derivation | Crest planeProduct of large quantities |
| 0.5h | 69055 |
| 1h | 70712 |
| 1.5h | 70123 |
| 2h | 70465 |
As can be seen from the above table, when the derivatization time is 1h, the sample is basically completely derivatized, so the derivatization time is 1 h;
verification of a methodology for detecting formaldehyde content:
blank test: respectively measuring a blank solution and a reference solution, and inspecting the interference condition of the blank solution, wherein the peak area of an interference peak in a chromatogram of the blank solution is less than 10% of the main peak area of the reference solution according to the test result, so that the requirements are met;
detection limit and quantitation limit: determining a detection limit and a quantification limit by adopting a signal-to-noise ratio, diluting stock solutions of a formaldehyde reference substance, performing derivatization respectively, adjusting detection sensitivity, and determining the detection limit by using corresponding concentration when the signal-to-noise ratio (S/N) is not less than 3; determining a quantification limit according to the corresponding concentration when the signal-to-noise ratio (S/N) is not less than 10, repeatedly investigating 6 test solutions at the concentration level, and requiring that the relative standard deviation of each solvent peak area in the chromatogram obtained 6 times is not more than 15% to confirm that the quantification limit determination result has certain precision and require that the detection limit S/N is more than or equal to 3; the quantitative limit S/N is more than or equal to 10, the quantitative limit concentration is less than 30% of the limit concentration, the RSD of the parallel 6 peak areas is less than or equal to 15%, the RSD of the retention time is less than or equal to 2%, and the test results of the quantitative limit and the detection limit of the formaldehyde are shown in the following table 4 and table 5:
TABLE 4 Formaldehyde quantitation limit and detection limit test results
| Item | Concentration (μ g/ml) | S/N | Corresponding to the limit concentration |
| Detection limit | 0.0005 | 4 | 0.3％ |
| Limit of quantification | 0.0023 | 40 | 1.5％ |
TABLE 5 Formaldehyde quantitative limit precision test results
| Quantitative limiting solution | Retention time (min) | Peak area | S/N |
| Limit of quantitation 1 | 7.348 | 4395 | 35 |
| Limit of quantitation 2 | 7.358 | 4541 | 25 |
| Limit of quantitation 3 | 7.350 | 4146 | 28 |
| Limit of quantitation 4 | 7.352 | 4723 | 39 |
| Limit of quantitation 5 | 7.351 | 4564 | 31 |
| Limit of quantitation 6 | 7.348 | 4696 | 40 |
| RSD(％) | 0.1 | 4.8 | N/A |
As can be seen from the table above, the detection limit concentration of formaldehyde is 0.0005 μ g/ml, and S/N is 4, which corresponds to the limit concentration of 0.3%; the limit concentration of quantification is 0.0023 mug/ml, S/N is 40, which is equivalent to the limit concentration of 1.5%, the RSD of the parallel 6 peak areas is 4.8%, and the RSD of the retention time is 0.1%, which all meet the verification requirements.
Linearity and range: preparing 6 concentrations of reference solutions within a range from LOQ concentration to a certain concentration (limit concentration is 200%) and performing derivatization, drawing a linear relation by using a function of a measured response value A peak area to a measured substance concentration C, performing linear regression, and using a correlation coefficient r to confirm a good linear relation, wherein the value of the linear regression coefficient r is required to be not less than 0.999, the linear solution of each concentration is prepared as shown in Table 6, the linear test result is shown in Table 7, and the linear regression equation is shown in figure 1:
TABLE 6 Formaldehyde Linear solution preparation
TABLE 7 results of Formaldehyde Linear test
As can be seen from the graph, the derivatization of formaldehyde in the range of 0.002 to 0.300. mu.g/ml (equivalent to a limit concentration of 2 to 200%) has a good linear relationship, and meets the detection requirements.
Precision:
sample introduction precision: continuously injecting the reference solution for 6 times respectively, wherein the retention time RSD is less than or equal to 1%; the peak area RSD should be less than or equal to 2% (n-6), with the results shown in Table 8 below:
TABLE 8 sample introduction precision test results
As can be seen from the above table, the reference solution was continuously injected for 6 times, the peak area RSD was 0.4%, the retention time RSD was 0.1%, and the validation requirements were met; the sample injection precision of the measuring method is proved to be good.
Repeatability: one analyst takes the same clotrimazole sample (batch number: 0102-:
TABLE 9 results of the repeatability tests
Test results show that 6 parts of test solution is prepared under the same conditions, the maximum absolute deviation of the formaldehyde content is 0.03ppm, and the test solution meets the verification requirements; the determination method is proved to have better repeatability.
Intermediate precision: taking the same batch of clotrimazole samples (batch number: 0102-:
TABLE 10 results of intermediate precision test
Test results show that the maximum absolute deviation of the formaldehyde content of 12 parts of test sample solutions of two analysts is 0.08ppm, and the maximum absolute deviation meets the verification requirements; the determination method is proved to have better intermediate precision.
Accuracy: preparing 9 parts of test solution in the range of 50-150% of the limit concentration, wherein the preparation process is shown in Table 11, and the recovery rate is 75-120%; RSD is less than or equal to 10%, and the detection results are shown in Table 12:
TABLE 11 Formaldehyde recovery ratio solution preparation
TABLE 12 results of the Formaldehyde recovery test
Note:
the peak area of the test solution is 2025.
As can be seen from the table, the average recovery rate of 96.0% and the RSD of 2.5% were found to meet the validation requirements in 9 samples of 50%, 100% and 150%.
Durability: the sample solution and the reference solution are placed at room temperature for 30 hours, sample injection measurement is carried out for 0h, 4h, 8h, 12h, 18h, 24h and 30h respectively, the stability condition of the solutions is investigated, a basis is provided for the placing time of the sample solution and the reference solution, the RSD value of the formaldehyde content in the sample solution is less than or equal to 20%, compared with 0h, the change rate of the main peak area is less than or equal to 10%, and the results are shown in a table 13.14.
TABLE 13 stability test results of test solutions
| Time | 0h | 4h | 8h | 12h | 18h | 24h | 30h | RSD(％) |
| Formaldehyde content (%) | 0.11 | 0.10 | 0.08 | 0.11 | 0.13 | 0.12 | 0.13 | 16.0 |
TABLE 14 stability test results for control solutions
| Time | 0h | 4h | 8h | 12h | 18h | 24h | 30h |
| Peak area | 54946 | 54957 | 55392 | 55679 | 55135 | 55413 | 55445 |
| Rate of change (%) | N.A | 0.0 | 0.8 | 1.3 | 0.3 | 0.8 | 0.9 |
The sample solution is placed for 30 hours, the content of the formaldehyde RSD is 16.0 percent, the sample solution meets the verification requirement, and the sample solution is stable within 24 hours; and (3) standing the reference solution for 30 hours, comparing the peak area with 0 hour, wherein the maximum change rate is 1.3 percent, and the reference solution is stable within 30 hours according with the verification requirement.
Chromatographic condition parameter variation effects: the wavelength (+ -5 nm), the flow rate (+ -0.05 ml/min), the organic phase proportion (+ -5%) in the mobile phase and the chromatographic columns of different batches are properly changed, the adaptive solution of the system and the solution of the test sample are measured, and the bearing degree of the measurement result which is not influenced is evaluated when the measurement condition parameters have slight changes, and the chromatographic condition parameters are shown in a table 15.
TABLE 15 chromatographic Condition parameters
The peak area RSD of the main component of the reference solution is less than or equal to 5 percent; the separation degree of the main component and the adjacent chromatographic peak is not less than 1.5, the recovery rate of formaldehyde in the test solution is 75-120%, and the results are shown in the following table 16:
TABLE 16 durability test results
a: a chromatographic column 1: waters Symmetry C18 (250X 4.6mm, 5 μm), SN: 03293009913886
b: and (3) chromatographic column 2: waters Symmetry C18 (250X 4.6mm, 5 μm), SN: 03253004813858
Note: "N/A" means not applicable.
The chromatographic conditions are slightly changed, the reference solution is continuously injected for 6 times, the RSD of the peak area is less than 5%, the separation degrees of main components and adjacent chromatographic peaks in the test solution are respectively greater than 1.5, the recovery rate of formaldehyde is respectively in the range of 75-120%, and the result shows that when the measurement condition parameters are slightly changed, the method has good durability.
Sample detection: three batches of clotrimazole samples are taken for detection (batch numbers: 0102-200258, 0102-.
An HPLC detection method for clotrimazole raw material genotoxic impurities is used for detecting the content of glyoxal:
diluent agent: absolute ethyl alcohol; the glyoxal reference substance is purchased from national drug group batch No. 20200506, and the content is 40.8%;
control stock solutions: taking a proper amount of glyoxal reference substance, precisely weighing, and quantitatively diluting with a diluent to prepare a solution containing 3mg of glyoxal in each 1 ml;
positive control stock solution: precisely measuring 1ml of reference stock solution, placing in a 100ml measuring flask, diluting to scale with diluent, and shaking; precisely measuring 1ml, placing in a 100ml measuring flask, diluting to scale with diluent, and shaking;
blank solution: precisely measuring 5ml of diluent, adding 1.0ml of 10% phosphoric acid solution, adding 1.0ml of derivative solution (10 mg of 2, 4-dinitrophenylhydrazine is taken and placed in a 100ml measuring flask, and is dissolved and diluted to scale by absolute ethyl alcohol), immediately placing the solution in a 70 ℃ water bath constant temperature oscillator, shaking at 150 revolutions per minute, taking out the solution after 1 hour, cooling to room temperature, adding 3.0ml of diluent, uniformly mixing, and filtering to obtain subsequent filtrate;
control solution: precisely measuring 5ml of positive control stock solution, adding 2.0ml of 10% phosphoric acid solution, adding 1.0ml of derivatization test solution, immediately placing the positive control stock solution in a 70 ℃ water bath constant temperature oscillator, shaking at 150 rpm, taking out after 1 hour, cooling to room temperature, adding 2.0ml of diluent, uniformly mixing, filtering, and taking a subsequent filtrate;
test solution and positive test solution: accurately weighing two parts (1 g each) of clotrimazole, adding 10ml of diluent into one part, adding 10ml of positive control stock solution into the other part, placing in a constant temperature oscillator, shaking at 150 rpm for 30min, filtering, and collecting the filtrate as test sample working solution and positive test sample working solution. Precisely measuring the sample working solution and the positive sample working solution by 5ml respectively, adding 2.0ml of 20% phosphoric acid solution and 1.0ml of derivatization test solution respectively, uniformly mixing, immediately placing in a 70 ℃ water bath constant temperature oscillator to shake at 150 rpm, taking out after 1 hour, cooling to room temperature, adding 2.0ml of diluent, uniformly mixing, filtering, and taking the subsequent filtrate.
Precisely measuring 10 mul of each of the test solution and the positive test solution, and respectively injecting into a liquid chromatograph under the chromatographic conditions: octadecylsilane bonded silica gel as filler (Waters Symmetry C18, 250 × 4.6mm, 5 μm, or equivalent performance column); the column temperature was 35 ℃; acetonitrile-water (70:30) is used as a mobile phase; the flow rate is 1.0 ml/min; the running time is 25 min; the detection wavelength is 440 nm; the sample injection volume is 20 mu l, the chromatogram is recorded, and the quantity of glyoxal is calculated according to the following formula;
in the formula ATest solutionThe peak area of glyoxal in the chromatogram of the test solution is shown; a. thePositive test solutionThe peak area of glyoxal in the chromatogram of the positive test solution is shown; cPositive control stock solutionThe concentration of glyoxal in the stock solution for the positive control (μ g/ml); w is the weight (g) of the test solution clotrimazole;
the carbonyl in aldehyde and ketone compounds can perform nucleophilic addition reaction with amino in phenylhydrazine to generate aldehyde hydrazone or ketone hydrazone products, a large pi-pi conjugated system exists in the molecular structure of the hydrazone, characteristic absorption exists in an ultraviolet-visible light region, glyoxal and 2, 4-dinitrophenylhydrazine react to generate glyoxal dihydrazone, so that the measurement is performed by adopting an HPLC method, the maximum absorption wavelength of a main component is 440nm according to an HPLC spectrogram of a reference solution, the detection wavelength is determined to be 440nm, a chromatogram of a sample solution is shown in figure 2, a main peak and an adjacent chromatographic peak are separated from each other by a baseline, and the separation degree is 6.3.
Clotrimazole is dissolved in absolute ethyl alcohol, acetone and chloroform, and hardly dissolved in water, and glyoxal is dissolved in ethyl alcohol, ether and water, so that absolute ethyl alcohol is preferably used as an extraction solvent of the product, 1g of the product is precisely weighed and placed into 4 centrifuge tubes, and 10.0ml of glyoxal reference substance solution with the concentration of 0.3 mu g/ml is added. Shake for 20 min, 30min, 45 min, 60 min, respectively, and filter. Precisely measuring 5ml of subsequent filtrate, placing the subsequent filtrate in a centrifuge tube, adding 1.0ml of derivative solution, adding 1.5ml of 10% phosphoric acid, shaking for 2 hours in a water bath constant temperature oscillator at 70 ℃, cooling to room temperature after taking out, adding acetonitrile to 10ml, filtering, injecting into a chromatograph, recording a chromatogram, wherein the peak area of a derivative product is basically stable at 20 minutes, which indicates that the sample is completely extracted, and comprehensively considering, selecting 30 minutes as extraction time; using 2, 4-dinitrophenylhydrazine as a derivatization reagent, using an excessive derivatization solution for derivatization, wherein the peak area of a derivatization product does not change along with the change of the amount of the 2, 4-dinitrophenylhydrazine, and selecting the addition amount of 1.0ml derivatization solution; according to the characteristics of the product extraction solvent absolute ethyl alcohol, 70 ℃ is selected as a derivatization temperature, a reference substance solution is used for derivatization for 1 hour, 1.5 hours, 2 hours and 3 hours respectively, and when the derivatization is 1 hour, a sample is basically completely derivatized, so that the derivatization time is 1 hour and is selected as the derivatization time;
the specificity is as follows: and respectively measuring a blank derivative solution and a reference solution, and inspecting the interference condition of the blank derivative solution, wherein the blank derivative solution has no obvious interference at a main peak, and the blank derivative solution does not have an interference peak at the main peak position in a blank test result, does not interfere with measurement and meets the verification requirement.
Detection limit and quantitation limit: determining a detection limit and a quantification limit by adopting a signal-to-noise ratio, diluting stock solutions of glyoxal reference substances, performing derivatization respectively, adjusting detection sensitivity, determining the detection limit by using corresponding concentrations when a signal-to-noise ratio (S/N) is not less than 3, determining the quantification limit by using corresponding concentrations when the signal-to-noise ratio (S/N) is not less than 10, repeatedly inspecting 6 test solutions at the concentration level, and verifying that the determination result of the quantification limit has certain precision by requiring that the relative standard deviation of each solvent peak area in a chromatogram obtained for 6 times is not more than 15%, wherein the S/N is not less than 3; the quantitative limit S/N is more than or equal to 10, the quantitative limit concentration is less than 30 percent of the limit concentration, the RSD of the parallel 6 peak areas is less than or equal to 15 percent, the RSD of the retention time is less than or equal to 2 percent, and the results are shown in the following table 17.18;
TABLE 17 glyoxal quantitation limit and detection limit test results
| Item | Concentration (μ g/ml) | S/N | Corresponding to the limit concentration |
| Detection limit | 0.005 | 4 | 1.7％ |
| Limit of quantification | 0.015 | 40 | 5.0％ |
TABLE 18 results of glyoxal quantitation limit precision test
The detection limit concentration of glyoxal is 0.005 mu g/ml, which corresponds to the limit concentration of 1.7%, and S/N is 4; the limit concentration of the quantification is 0.015 mu g/ml, which corresponds to the limit concentration of 5.0 percent, and S/N is 40; the quantitative limit is equal to 6 parts, the peak area RSD is less than or equal to 15 percent, the retention time RSD is less than or equal to 2 percent, and the requirements of verification are met.
Linearity and range: the control solutions of 6 concentrations were used to perform derivatization from LOQ to a range of concentrations (limit concentration 200%). The linear relation is plotted by the measured response value A (peak area) to the function of the concentration C of the measured object, linear regression is carried out, a good linear relation is verified by using a correlation coefficient r, the numerical value of the linear regression coefficient r is required to be not lower than 0.999, the preparation of linear solutions with various concentrations is shown in a table 19, the correlation coefficient r is not less than 0.999, | Y-axis intercept |/100% response value should be not more than 20%, the experimental result is shown in a table 20, and the equation of linear regression for glyoxal inspection is shown in an attached figure 3.
TABLE 19 Linear glyoxal solution formulation
TABLE 20 examination of the Linear test results with glyoxal
As can be seen from the graph, the derivatization of glyoxal in the concentration range of 0.02 mug/ml to 0.60 mug/ml (equivalent to the limit concentration of 5% to 200%) has good linear relation and meets the detection requirements.
Precision: sample introduction precision: taking a reference substance solution, continuously injecting samples for 6 times respectively, wherein the retention time RSD is less than or equal to 1%; the peak area RSD should be 2% or less (n 6), and the results are shown in table 21:
TABLE 21 sample introduction precision test results
Test results show that the reference solution is continuously injected for 6 times, the peak area RSD is 0.3 percent, and the verification requirements are met; the sample injection precision of the measuring method is proved to be good.
Repeatability: the same person was tested, and the same lot of clotrimazole samples (lot number: 0102-:
TABLE 22 results of the repeatability tests
Test results show that 6 parts of test solution is prepared by one analyst under the same conditions, the maximum absolute deviation of the glyoxal content is 0.03ppm, and the verification requirements are met; the determination method is proved to have better repeatability.
Intermediate precision: taking the same batch of clotrimazole samples (batch number: 0102-:
TABLE 23 results of intermediate precision test
Test results show that the maximum absolute deviation of the glyoxal content of 12 test sample solutions of two analysts is 0.05ppm, and the maximum absolute deviation meets the verification requirements; the determination method is proved to have better intermediate precision.
Accuracy: preparing 9 parts of test solution in the range of 50-150% of the limit concentration, wherein the preparation process is shown in Table 24, and the recovery rate is 75-120%; RSD should be less than or equal to 10%, and the results of glyoxal recovery test are shown in Table 25:
TABLE 24 glyoxal recovery ratio solution preparation
TABLE 25 glyoxal recovery test results
Note:
the peak area of the sample solution was 2917.
As can be seen from the table, the average recovery rate of 9 samples of 50%, 100% and 150% was 99.8%, and the RSD was 1.9%, which all met the validation requirements.
Durability: solution stability: the test solution and the reference solution are placed at room temperature for 24 hours, sample introduction is carried out for determination at 0h, 4h, 8h, 16h and 24h respectively, the stability condition of the solutions is investigated, a basis is provided for the placing time of the test solution and the reference solution, the RSD value of the glyoxal content in the test solution is less than or equal to 20%, the change rate of the main peak area is less than or equal to 10% when the reference solution is compared with 0h, and the experimental results are shown in a table 26.27.
TABLE 26 stability test results for control solutions
| Time | 0h | 4h | 8h | 16h | 24h |
| Peak area | 122851 | 127676 | 129982 | 132677 | 133385 |
| Rate of change (%) | N.A | 3.9 | 5.8 | 8.0 | 8.6 |
Note: N.A. does not apply
TABLE 27 test results of solution stability of test article
| Time | 0h | 4h | 8h | 16h | 24h | RSD(％) |
| Glyoxal content (ppm) | 0.12 | 0.14 | 0.14 | 0.14 | 0.14 | 6.6 |
The sample solution is placed for 24 hours, the content of the glyoxal RSD is 6.6 percent, the verification requirement is met, and the sample solution is stable within 24 hours; and (3) standing the reference solution for 24 hours, comparing the peak area with 0 hour, wherein the maximum change rate is 8.6 percent, and the reference solution is stable within 24 hours according with the verification requirement.
Chromatographic condition parameter variation effects: properly changing the wavelength (+ -5 nm), the flow rate (+ -0.1 ml/min), the organic phase proportion (+ -3%) in the mobile phase, replacing chromatographic columns of different batches, measuring the adaptive solution of the system and the solution of a test sample, and evaluating the bearing degree of the measurement result which is not influenced when the measurement condition parameters have slight changes, wherein the chromatographic condition parameters are shown in the table 28, and the RSD (reference substance) peak area of the main component of the solution is not more than 5%; the separation degree of the main component and the adjacent chromatographic peak is not less than 1.5, the recovery rate of the glyoxal is 75-120%, and the experimental results are shown in Table 29.
TABLE 28 chromatographic Condition parameters
TABLE 29 durability test results
a: a chromatographic column 1: waters Symmetry C18 (250X 4.6mm, 5 μm), SN: 03293009913886
b: and (3) chromatographic column 2: waters Symmetry C18 (250X 4.6mm, 5 μm), SN: 03253004813858
Note: "N/A" means not applicable.
The chromatographic condition is slightly changed, the reference solution is continuously injected for 6 times, the RSD of the peak area is less than 5 percent, the separation degrees of the main component and the adjacent chromatographic peak in the test solution are more than 1.5, and the recovery rate of the glyoxal is within the range of 75-120 percent. The results show that the method is excellent in durability when the measurement condition parameters slightly vary.
Sample detection: three batches of clotrimazole samples were tested (batch Nos. 0102-200258, 0102-200259 and 0102-200124), and the content (ppm) of glyoxal was undetected, undetected and 0.1, respectively.
Test results show that the content of glyoxal in three batches of samples is less than 3ppm, and detection meets the requirements, so that the method can be used for measuring glyoxal of clotrimazole.
In summary, the verification results of the methodology for detecting the formaldehyde content in clotrimazole are shown in table 30 below, and the verification results of the methodology for detecting the glyoxal content are shown in table 31 below:
TABLE 30 methodological test results for detecting Formaldehyde content in Clotrimazole
TABLE 31 methodological validation results for testing glyoxal content in clotrimazole
The verification items all meet the requirements, and the method can be used for measuring the contents of formaldehyde and glyoxal in the clotrimazole, so that the HPLC detection method is suitable for quickly and effectively detecting potential genotoxic impurities, namely formaldehyde and glyoxal, in the clotrimazole bulk drug, so as to control the quality of the clotrimazole raw material.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. An HPLC detection method for clotrimazole raw material genotoxic impurities is characterized by comprising the following steps: quantitatively preparing a corresponding positive control stock solution by using a formaldehyde reference substance or a glyoxal reference substance with a diluent, preparing a specimen solution and a positive specimen solution by using a clotrimazole raw material and the diluent or the corresponding positive control stock solution, a phosphoric acid solution and a derivative solution through derivatization reaction, preparing the specimen solution and the positive specimen solution with the diluent, respectively injecting the corresponding positive control stock solution, the specimen solution and the positive specimen solution into a liquid chromatograph, detecting and recording chromatograms at a column temperature of 30-35 ℃, a circulation phase of acetonitrile and water and a flow rate of 0.5-1ml/min, and calculating the content of formaldehyde or glyoxal according to the following formula:
in the formula ATest solutionIs the peak area of formaldehyde or glyoxal in the chromatogram of the test solution; a. thePositive test solutionThe peak area of formaldehyde or glyoxal in the chromatogram of the positive test solution is shown; cPositive control stock solutionConcentration of formaldehyde or glyoxal (μ g/ml) in the stock solution for the positive control; w is the test solution clotrimazole weight (g).
2. The HPLC detection method for clotrimazole raw material genotoxic impurities as claimed in claim 1, wherein in the detection of formaldehyde content, the diluent is acetonitrile, the derivative solution is prepared from 2, 4-dinitrophenylhydrazine and acetonitrile, and the chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as filler, the column temperature is 30 ℃, the volume ratio of acetonitrile to water is 75:25 as mobile phase, and the running time is 40 min; the flow rate is 0.5ml/min, the detection wavelength is 355nm, and the sample injection volume is 10-20 μ l.
3. The HPLC detection method for clotrimazole raw material genotoxic impurities as claimed in claim 1, wherein when detecting the glyoxal content, the diluent is absolute ethanol, the derivative solution is prepared from 2, 4-dinitrophenylhydrazine and absolute ethanol, and the chromatographic conditions of the liquid chromatograph are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica as filler, the column temperature is 35 ℃, acetonitrile-water volume ratio is 70:30 as mobile phase, and the running time is 25 min; the flow rate is 1ml/min, the detection wavelength is 400nm, and the sample injection volume is 10-20 mul.
4. The HPLC method for detecting clotrimazole raw material genotoxic impurities as claimed in claim 1, wherein when preparing the test sample solution and the positive test sample solution, the clotrimazole raw material and the diluent or the corresponding positive control sample stock solution are shaken at 150 rpm for 30min and then filtered, the filtrate is added with the phosphoric acid solution, the derivative solution is shaken at 150 rpm for 1h at 70 ℃, the diluent is added and mixed evenly, and then the filtrate is filtered and continued.
5. The HPLC method for detecting the genotoxic impurities in the clotrimazole raw material as claimed in claim 1, wherein the content of formaldehyde in the positive control stock solution is 0.15 μ g/ml when detecting the content of formaldehyde, and the mass ratio of the clotrimazole raw material to the 2, 4-dinitrophenylhydrazine in the derivative solution when preparing the test solution and the positive test solution is 2500: 1, when detecting glyoxal, the content of glyoxal in the positive control storage solution is 0.3 mug/ml, and the dosage mass ratio of the clotrimazole raw material to the 2, 4-dinitrophenylhydrazine in the derivative solution when preparing the test solution and the positive test solution is 5000: 1.
6. the methodological validation of the HPLC detection method of clotrimazole starting material genotoxic impurities according to any of claims 1 to 5, wherein validation items include specificity, sensitivity, linearity and range, precision, accuracy, durability, and the control solution is prepared by taking the stock solution of the corresponding positive control, adding phosphoric acid solution, shaking the derivative solution, and adding diluent.
7. The methodological verification of the clotrimazole raw material genotoxic impurity HPLC detection method according to claim 6, characterized in that, when verifying specificity, the diluent, the phosphoric acid solution and the derivative solution are shaken and then added to prepare a blank solution, and when verifying formaldehyde detection, the mass ratio of the methanol reference substance to the 2, 4-dinitrophenylhydrazine in the derivative solution is 0.75: 100; verifying that the mass ratio of the amount of the 2, 4-dinitrophenylhydrazine in the methanol reference substance and the derivative solution is 1.5:100 when the reference substance solution is prepared during glyoxal detection, respectively injecting a blank solution and the reference substance solution into a liquid chromatograph for detection, wherein the interference peak area in the blank solution chromatogram is less than or equal to 10% of the main peak area of the reference substance solution.
8. The methodological verification of the clotrimazole raw material genotoxic impurity HPLC detection method according to claim 6, characterized in that, when the sensitivity is verified, the positive control stock solution is respectively subjected to derivatization reaction and HPLC detection, the detection limit and the quantification limit are determined by adopting the signal-to-noise ratio S/N, and the detection limit S/N is more than or equal to 3; the quantitative limit S/N is more than or equal to 10, the quantitative limit concentration is less than 30% of the limit concentration, the parallel 6 detection peak area RSD is less than or equal to 15%, the retention time RSD is less than or equal to 2%, when the linearity and range are verified, reference substance solutions with multiple concentrations are prepared within 200% of the limit concentration and subjected to derivatization and HPLC detection, the measured response value peak area A is used as a function of the Y axis to the concentration C of the measured object as the X axis to be plotted, the correlation coefficient r is more than or equal to 0.999, and the I Y axis intercept is less than or equal to 20% of the response value 100%.
9. The methodological verification of the clotrimazole raw material genotoxic impurity HPLC detection method according to claim 6, characterized in that, the precision of the injection is verified as follows: multiple reference substance solutions have continuous sample injection retention time RSD less than or equal to 1, peak area RSD less than or equal to 2%, and repeatability is as follows: the absolute deviation of the formaldehyde content is not more than 5% of the quality standard, the absolute deviation of the formaldehyde content in the intermediate precision is not more than 5% of the quality standard, the verification accuracy yield range is 75-120%, and the RSD is not more than 10%.
10. The methodological test of the method for detecting the genotoxic clotrimazole impurity in the claim 6, wherein the maximum change rate of the main peak area of the reference solution is less than or equal to 10% compared with 0 time when the durability is tested, the relative standard deviation of the main peak area obtained by continuously feeding 6 times of the systematic adaptive reference solution is less than or equal to 5%, the content of the formaldehyde or the glyoxal in the test solution is less than or equal to 20%, and the recovery rate of the formaldehyde or the glyoxal in the test solution is between 75% and 120%.
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Application publication date: 20211224 |