CN115266983B - Method for measuring antifungal drugs in antibacterial agent of antibacterial product by using gas chromatography mass spectrometry - Google Patents
Method for measuring antifungal drugs in antibacterial agent of antibacterial product by using gas chromatography mass spectrometry Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 229940121375 antifungal agent Drugs 0.000 title claims abstract description 17
- 230000000843 anti-fungal effect Effects 0.000 title claims abstract description 15
- 239000003814 drug Substances 0.000 title claims abstract description 14
- 229940079593 drug Drugs 0.000 title claims abstract description 12
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 11
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 8
- 230000000844 anti-bacterial effect Effects 0.000 title claims description 11
- LEZWWPYKPKIXLL-UHFFFAOYSA-N 1-{2-(4-chlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl}imidazole Chemical compound C1=CC(Cl)=CC=C1COC(C=1C(=CC(Cl)=CC=1)Cl)CN1C=NC=C1 LEZWWPYKPKIXLL-UHFFFAOYSA-N 0.000 claims abstract description 19
- IIUZTXTZRGLYTI-UHFFFAOYSA-N Dihydrogriseofulvin Natural products COC1CC(=O)CC(C)C11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 IIUZTXTZRGLYTI-UHFFFAOYSA-N 0.000 claims abstract description 19
- UXWOXTQWVMFRSE-UHFFFAOYSA-N Griseoviridin Natural products O=C1OC(C)CC=C(C(NCC=CC=CC(O)CC(O)C2)=O)SCC1NC(=O)C1=COC2=N1 UXWOXTQWVMFRSE-UHFFFAOYSA-N 0.000 claims abstract description 19
- DDUHZTYCFQRHIY-UHFFFAOYSA-N Negwer: 6874 Natural products COC1=CC(=O)CC(C)C11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 DDUHZTYCFQRHIY-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960004022 clotrimazole Drugs 0.000 claims abstract description 19
- 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 claims abstract description 19
- 229960003913 econazole Drugs 0.000 claims abstract description 19
- 229960004884 fluconazole Drugs 0.000 claims abstract description 19
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229960002867 griseofulvin Drugs 0.000 claims abstract description 19
- DDUHZTYCFQRHIY-RBHXEPJQSA-N griseofulvin Chemical compound COC1=CC(=O)C[C@@H](C)[C@@]11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 DDUHZTYCFQRHIY-RBHXEPJQSA-N 0.000 claims abstract description 19
- 229960002722 terbinafine Drugs 0.000 claims abstract description 19
- DOMXUEMWDBAQBQ-WEVVVXLNSA-N terbinafine Chemical compound C1=CC=C2C(CN(C\C=C\C#CC(C)(C)C)C)=CC=CC2=C1 DOMXUEMWDBAQBQ-WEVVVXLNSA-N 0.000 claims abstract description 19
- OCAPBUJLXMYKEJ-UHFFFAOYSA-N 1-[biphenyl-4-yl(phenyl)methyl]imidazole Chemical compound C1=NC=CN1C(C=1C=CC(=CC=1)C=1C=CC=CC=1)C1=CC=CC=C1 OCAPBUJLXMYKEJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- BYBLEWFAAKGYCD-UHFFFAOYSA-N Miconazole Chemical compound ClC1=CC(Cl)=CC=C1COC(C=1C(=CC(Cl)=CC=1)Cl)CN1C=NC=C1 BYBLEWFAAKGYCD-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229960002206 bifonazole Drugs 0.000 claims abstract description 18
- 229960002509 miconazole Drugs 0.000 claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 59
- 150000002500 ions Chemical class 0.000 claims description 38
- 239000000523 sample Substances 0.000 claims description 34
- 230000014759 maintenance of location Effects 0.000 claims description 23
- 239000012224 working solution Substances 0.000 claims description 16
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- 150000001875 compounds Chemical class 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000003429 antifungal agent Substances 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 6
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- 238000007865 diluting Methods 0.000 claims description 5
- 238000004451 qualitative analysis Methods 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- -1 acetonitrile saturated n-hexane Chemical class 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000004445 quantitative analysis Methods 0.000 claims description 3
- 239000010453 quartz Substances 0.000 claims description 3
- 238000002098 selective ion monitoring Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000013076 target substance Substances 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 2
- 239000012982 microporous membrane Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 2
- 239000007789 gas Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 9
- 239000000126 substance Substances 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 7
- 239000006071 cream Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 1
- 241001071917 Lithospermum Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/72—Mass spectrometers
- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
-
- 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
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for measuring antifungal drugs in antibacterial agents and antifungal products by utilizing a gas chromatography mass spectrometry method. The invention can simply, conveniently and rapidly detect the content of antifungal drugs such as fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole, oxconazole and the like in the disinfection product, and has the characteristics of simplicity, convenience, good accuracy and repeatability and high sensitivity.
Description
Technical Field
The invention relates to the technical field of drug detection and analysis, in particular to a method for measuring antifungal drugs in an antibacterial agent of an antibacterial product by utilizing a gas chromatography mass spectrometry.
Background
The antifungal medicine mainly comprises fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole, oxconazole and the like. The antifungal medicine is a forbidden raw material regulated in the sanitary regulations of disinfection product production enterprises in China, and at present, no gas chromatography mass spectrometry detection method for measuring substances in the antibacterial agent with the' anti (anti) character size is available.
Disclosure of Invention
The invention aims to provide a method for measuring the content of antifungal drugs illegally added into antibacterial agents and antifungal products by utilizing a gas chromatography mass spectrometry method, which can simply, conveniently and rapidly detect the content of the antifungal drugs such as fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole, oxconazole and the like in the antibacterial products, and has the characteristics of simplicity, convenience, good accuracy and repeatability and high sensitivity.
The technical scheme of the invention is as follows:
a method for determining antifungal drugs in antibacterial agents of antibacterial products by utilizing gas chromatography mass spectrometry, comprising the following steps:
1) Dispersing a sample to be detected by a saturated sodium chloride solution to form a sample solution;
2) Extracting the sample liquid with acetonitrile, purifying the extract liquid with n-hexane, centrifuging, and filtering to obtain a sample solution;
3) Preparing a mixed standard working solution containing fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole;
4) Carrying out gas chromatographic analysis on the mixed standard working solution obtained in the step 3) to obtain the retention time, quantitative ion and qualitative ion information of the chromatograms and characteristic peaks of each compound;
5) Respectively sucking 50 mu L, 100 mu L, 200 mu L, 400 mu L, 500 mu L, 600 mu L and 800 mu L of mixed standard working solution, and diluting to 1mL by acetonitrile to obtain a series of mixed standard working solutions; taking a series of mixed standard working solutions for sequential sample injection, taking the peak area of a target quantitative ion peak as an ordinate and the corresponding target concentration as an abscissa, and drawing a standard curve, wherein the linear correlation coefficient R is more than or equal to 0.99;
6) Carrying out gas chromatographic analysis on the sample solution obtained in the step 2) under the same condition to obtain retention time, quantitative ion and qualitative ion information of a chromatogram and a characteristic peak, and carrying out qualitative analysis on the sample solution to be tested;
7) Substituting the peak area of the quantitative ion peak measured in the step 6) into the standard curve obtained in the step 5) to obtain the concentration of each compound;
8) Calculating to obtain the content of each antifungal drug.
Preferably, in the step 2), acetonitrile 2mL is added into the sample solution, and the mixture is uniformly mixed and subjected to ultrasonic extraction for 15min; then adding 2mL of acetonitrile saturated n-hexane, vortex shaking for 1min, and centrifuging for 10min at 4000r/min; filtering the middle layer by a filter membrane to obtain a sample solution.
Preferably, the step 3) accurately weighs a proper amount of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole to 0.1mg, and uses acetonitrile to dissolve the fluconazole, terbinafine, griseofulvin, clotrimazole, econazole and oxconazole, and respectively prepares standard stock solutions with the concentration of 1.0 mg/mL; and respectively sucking a proper amount of standard stock solutions of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole, diluting with acetonitrile, and filtering to prepare a mixed standard working solution of the antifungal drug with the concentration of 80 mug/mL.
Preferably, the filtration membrane is a 0.22 μm organic microporous filtration membrane.
Preferably, the gas chromatography parameters in step 3) include: chromatographic column: DB-5MS quartz capillary column or other chromatographic column with equivalent column effect; the column size was 30 m.times.250. Mu.m.times.0.25. Mu.m; chromatographic column temperature program: keeping at 170 ℃ for 2min, and then heating to 210 ℃ at 10 ℃/min; heating to 235 ℃ at 25 ℃ per minute, and keeping for 2 minutes; then heating to 280 ℃ at 45 ℃/min; heating to 300 ℃ at 10 ℃/min, and keeping for 4min; carrier gas: helium with purity more than or equal to 99.999%, constant flow mode and flow rate of 1mL/min; sample inlet temperature: 280 ℃; sample injection amount: 1 μl; sample injection mode: the split ratio is 20:1, and the split flow is 20mL/min; ion source: EI source, 70eV; mass acquisition range: 40amu to 500amu; ion source temperature: 230 ℃; interface temperature: 260 ℃.
Preferably, the retention time of fluconazole in the step 4) is 9.254min, the retention time of terbinafine is 10.105min, the retention time of griseofulvin is 12.739min, the retention time of clotrimazole is 13.106min, the retention time of econazole is 13.602min, the retention time of bifonazole is 14.068min, the retention time of miconazole is 14.552min, and the retention time of oxconazole is 14.698min.
Preferably, in step 7), the concentration of each compound is calculated according to the following formula:
wherein:
omega-the content of target in the sample in micrograms per gram (μg/g);
c-obtaining the concentration of the target substance from the standard curve, wherein the unit is micrograms per milliliter (mug/mL);
v-volume extracted by adding acetonitrile in milliliters (mL);
d, dilution ratio, if not diluted, is 1;
m-sample amount in grams (g).
Preferably, step 9) is further included: the same sample was subjected to 2 parallel test determinations, the content of each compound was calculated, and the relative phase difference was calculated according to the following formula:
wherein:
RD-relative phase difference;
ω 1 、ω 2 -content values of two samples quantitatively determined in parallel;
-average value of content of two samples measured quantitatively in parallel;
when RD is less than or equal to 10%, the data is valid; when RD > 10%, re-detection is required.
Compared with the prior art, the invention has the following beneficial effects:
the invention is used for measuring antifungal drugs fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole in water aqua and cream anti (antibacterial) preparations with 'eliminating' character size, and has the advantages of simple operation, low detection limit and reliable result.
Drawings
FIG. 1 is a total ion flow chromatogram of 8 antifungal agents;
FIG. 2 is a chromatogram of the test of example 1;
FIG. 3 is a chromatogram of the test of example 2;
FIG. 4 is a chromatogram of the test of example 3;
FIG. 5 is a chromatogram of the test of example 4;
FIG. 6 is a chromatogram of the test of example 5;
FIG. 7 is a chromatogram of the test of example 6.
Detailed Description
The present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are within the scope of the invention.
The reagents used in the assays of the following examples of the invention were all analytically pure, and included:
acetonitrile: chromatographic purity;
sodium chloride;
n-hexane;
water: meets the first-grade water specified in GB/T6682.
Saturated sodium chloride solution: taking 40.0g of sodium chloride, adding 100mL of water to dissolve, standing, and taking supernatant for later use;
acetonitrile saturated n-hexane: respectively taking 20mL of acetonitrile and 50mL of n-hexane in the same conical flask, uniformly mixing, standing, and obtaining the acetonitrile saturated n-hexane as the upper layer;
standard substance: the Chinese names, english names, CAS numbers, molecular formulas and relative molecular weights of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole are shown in Table 1. The purity of each standard substance is more than or equal to 98 percent, and the standard substance is a proven standard substance or a traceable secondary standard substance.
TABLE 1 antifungal drug Standard substance information
The invention adopts the instrument and the equipment
Gas chromatograph-mass spectrometer: an electron bombardment source (EI);
analytical balance (minimum graduation values 0.1mg and 0.01 mg);
centrifuge: the highest rotating speed is not lower than 4000r/min;
a vortex oscillator;
a pipette;
an ultrasonic cleaner;
nitrogen blowing instrument.
The method for measuring antifungal drugs used in the following examples of the present invention comprises the steps of:
1 preparation of sample solution
Sample 0.2g (accurate to 0.001 g) is taken in a centrifuge tube with a cover, 3mL of saturated sodium chloride solution is added, the sample is dispersed by vortex, then 2mL of acetonitrile is added, the mixture is uniformly mixed, and ultrasonic extraction is carried out for 15min. Then, 2mL of acetonitrile-saturated n-hexane was added, and the mixture was vortexed and shaken for 1min, followed by centrifugation at 4000r/min for 10min. Filtering the intermediate layer with 0.22 μm organic microporous membrane, collecting the subsequent filtrate, and analyzing with gas chromatograph-mass spectrometer.
2 preparation of mixed standard working solution
Accurately weighing proper amounts of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole to 0.1mg, and dissolving the materials with acetonitrile to prepare standard stock solutions with the concentration of 1.0 mg/mL; and respectively sucking a proper amount of standard stock solutions of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole, diluting with acetonitrile, and filtering with a 0.22 mu m organic microporous filter membrane to prepare a mixed standard working solution of the antifungal drug with the concentration of 80 mu g/mL. And (3) operating in parallel with the sample solution to obtain a quality control sample extracting solution for instrument detection.
3 gas chromatograph-mass spectrometer reference conditions
Chromatographic column: DB-5MS (30 m 250 m 0.25 m) quartz capillary column or other column effect equivalent chromatographic column;
chromatographic column temperature program: keeping at 170 ℃ for 2min, and then heating to 210 ℃ at 10 ℃/min; heating to 235 ℃ at 25 ℃ per minute, and keeping for 2 minutes; then heating to 280 ℃ at 45 ℃/min; heating to 300 ℃ at 10 ℃/min, and keeping for 4min;
carrier gas: helium (purity is more than or equal to 99.999%), constant flow mode, and flow rate of 1mL/min;
sample inlet temperature: 280 ℃;
sample injection amount: 1 μl;
sample injection mode: the split ratio is 20:1, and the split flow is 20mL/min;
ion source: EI source, 70eV;
mass acquisition range: 40amu to 500amu;
ion source temperature: 230 ℃;
interface temperature: 260 ℃;
scanning mode: the qualitative analysis adopts a full Scanning (SCAN) mode; the quantitative analysis uses a Selective Ion Monitoring (SIM) mode, and the retention time, quantitative ion, and qualitative ion of each compound are shown in table 2. The total ion flow chromatogram of 8 antifungal agents is shown in figure 1.
TABLE 2 Standard substance retention time and Mass Spectrometry characteristic ion
Note that: * And quantifying ions.
4 qualitative analysis
The measurement is carried out under the same condition, compared with the target in the standard working solution with the concentration close to that of the target, the retention time of chromatographic peaks is consistent (the relative error is within +/-2 percent), the mass spectrum characteristic ions (not less than 3) are consistent, the relative deviation of the abundance ratio of each ion is not more than the range specified in the table 3, and the detection of the target in the sample can be judged without interference.
TABLE 3 maximum allowable deviation of relative ion abundance in qualitative determinations
5 quantitative analysis
Taking serial mixed standard working solutions, sequentially sampling, drawing a standard curve by taking the peak area of a target quantitative ion peak as an ordinate and the corresponding target concentration as an abscissa, wherein the linear correlation coefficient R is more than or equal to 0.99. Taking a sample solution for sample injection, measuring the peak area of a quantitative ion peak, and substituting the peak area into a standard curve to obtain the concentration of each compound. The concentration of each compound was calculated according to the following formula:
wherein:
omega-the content of target in the sample in micrograms per gram (μg/g);
c-obtaining the concentration of the target substance from the standard curve, wherein the unit is micrograms per milliliter (mug/mL);
v-volume extracted by adding acetonitrile in milliliters (mL);
d, dilution ratio, if not diluted, is 1;
m-sample amount in grams (g).
The same sample was subjected to 2 parallel test determinations, the content of each compound was calculated, and the relative phase difference was calculated according to the following formula:
wherein:
RD-relative phase difference;
ω 1 、ω 2 -content values of two samples quantitatively determined in parallel;
-average value of content of two samples measured quantitatively in parallel;
when RD is less than or equal to 10%, the data is valid; when RD > 10%, re-detection is required.
Example 1
A commercial skin antibacterial cream product is extracted according to the preparation method of the sample, detected by a gas chromatograph mass spectrometer, and detected to obtain terbinafine and griseofulvin components, wherein the quantitative ion peak areas are 7907107.58 and 5639490.47 respectively, and are brought into a standard curve according to the formula as shown in figure 2Calculated terbinafine content is 0.38g/100g, griseofulvin content is 0.52g/100g.
Example 2
Some commercial seedling Fang Baobao skin cream products are extracted according to the preparation method of the test sample, and detected by a gas chromatograph mass spectrometer to detect the components of clotrimazole and econazole, as shown in figure 3. The quantitative ion peak areas are 2161642.98 and 4046714.71 respectively, and are brought into a standard curve according to the formulaCalculated to obtain the clotrimazole content of 0.26g/100g and the econazole content of 0.46g/100g.
Example 3
The commercial platinum antibacterial cream product is extracted according to the preparation method of the sample, and detected by a gas chromatograph mass spectrometer to detect the miconazole component, as shown in figure 4. The quantitative ion peak area is 7518325.61, which is brought into a standard curve, according to the formulaThe calculated miconazole content is 0.86g/100g.
Example 4
Some commercial infant antibacterial cream product is extracted according to the preparation method of the sample, and is subjected to gas chromatography mass spectrometryThe instrument detects the fluconazole composition as shown in fig. 5. The quantitative ion peak area is 6414900.82, which is brought into a standard curve, according to the formulaThe calculated fluconazole content is 0.78g/100g.
Example 5
Some commercial baby eczema ointment is extracted according to the preparation method of the test sample, and the components of the oxconazole are detected by a gas chromatograph mass spectrometer, as shown in figure 6. The quantitative ion peak area is 3214221.29 and is brought into a standard curve, and the method is based on the formulaThe calculated oxconazole content is 0.71g/100g.
Example 6
Some commercial lithospermum antibacterial cream is extracted according to the preparation method of the sample, and the bifonazole component is detected by a gas chromatograph mass spectrometer, as shown in figure 7. The quantitative ion peak area is 471436.34 and is brought into a standard curve, and the method is based on the formulaThe calculated content of the bifonazole is 0.88mg/100g.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (6)
1. A method for determining antifungal drugs in antibacterial agents of an antibacterial product by utilizing a gas chromatography mass spectrometry method, which is characterized by comprising the following steps:
1) Dispersing a sample to be detected by a saturated sodium chloride solution to form a sample solution;
2) Adding acetonitrile 2mL into the sample liquid, uniformly mixing, and carrying out ultrasonic extraction for 15min; adding acetonitrile saturated n-hexane 2mL, vortex oscillating for 1min, and centrifuging for 10min at 4000r/min; filtering the middle layer by a filter membrane to obtain a sample solution;
3) Preparing a mixed standard working solution containing fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole;
4) Carrying out gas chromatography mass spectrometry on the mixed standard working solution obtained in the step 3) to obtain the retention time, quantitative ion and qualitative ion information of the chromatograms and characteristic ion peaks of each compound;
the gas chromatography mass spectrometry parameters included: chromatographic column: DB-5MS quartz capillary column; the column size was 30 m.times.250. Mu.m.times.0.25. Mu.m;
chromatographic column temperature program: 170. maintaining at the temperature for 2min, and then heating to 210 ℃ at 10 ℃/min; heating to 235 ℃ at 25 ℃ per minute, and keeping for 2 minutes; then heating to 280 ℃ at 45 ℃/min; heating to 300 ℃ at 10 ℃/min, and keeping for 4min; carrier gas: helium with purity more than or equal to 99.999%, constant flow mode and flow rate of 1mL/min; sample inlet temperature: 280. the temperature is lower than the temperature; sample injection amount: 1. mu L; sample injection mode: the split ratio is 20:1, and the split flow is 20mL/min; ion source: EI source, 70eV; mass acquisition range: 40 amu-500 amu; ion source temperature: 230. the temperature is lower than the temperature; interface temperature: 260. the temperature is lower than the temperature;
the qualitative analysis adopts a full scanning mode; the quantitative analysis adopts a selective ion monitoring mode; the quantitative ions of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole are 224 m/z, 141 m/z, 352 m/z, 277 m/z, 125 m/z, 243 m/z, 159 m/z and 159 m/z respectively;
5) Respectively sucking 50 mu L, 100 mu L, 200 mu L, 400 mu L, 500 mu L, 600 mu L and 800 mu L of mixed standard working solution, and diluting to 1mL by acetonitrile to obtain a series of mixed standard working solutions; taking a series of mixed standard working solutions for sequential sample injection, taking the peak area of a target quantitative ion peak as an ordinate and the corresponding target concentration as an abscissa, and drawing a standard curve, wherein the linear correlation coefficient R is more than or equal to 0.99;
6) Carrying out gas chromatography mass spectrometry on the sample solution obtained in the step 2) under the same condition to obtain retention time, quantitative ion and qualitative ion information of a chromatogram and characteristic ion peaks, and carrying out qualitative analysis on the sample solution to be tested;
7) Substituting the peak area of the quantitative ion peak measured in the step 6) into the standard curve obtained in the step 5) to obtain the concentration of each compound;
8) Calculating to obtain the content of each antifungal drug.
2. The method according to claim 1, wherein said step 3) accurately weighs a proper amount of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole, oxconazole to 0.1mg, and uses acetonitrile to dissolve the materials to prepare standard stock solutions with a concentration of 1.0 mg/mL; and respectively sucking a proper amount of standard stock solutions of fluconazole, terbinafine, griseofulvin, clotrimazole, econazole, bifonazole, miconazole and oxconazole, diluting with acetonitrile, and filtering to prepare a mixed standard working solution of the antifungal drug with the concentration of 80 mug/mL.
3. The method according to claim 1, wherein the filtration membrane is a 0.22 μm organic microporous membrane.
4. The method according to claim 1, wherein the retention time of fluconazole in step 4) is 9.254min, the retention time of terbinafine is 10.105min, the retention time of griseofulvin is 12.739min, the retention time of clotrimazole is 13.106min, the retention time of econazole is 13.602min, the retention time of bifonazole is 14.068min, the retention time of miconazole is 14.552min, and the retention time of oxconazole is 14.698min.
5. The method according to claim 1, wherein in step 7), the concentration of each compound is calculated according to the following formula:
wherein:
omega-the content of target in the sample in micrograms per gram (μg/g);
c-obtaining the concentration of the target substance from the standard curve, wherein the unit is micrograms per milliliter (mug/mL);
v-volume extracted by adding acetonitrile in milliliters (mL);
d, dilution ratio, if not diluted, is 1;
m-sample amount in grams (g).
6. The method according to claim 1, further comprising step 9): the same sample was subjected to 2 parallel test determinations, the content of each compound was calculated, and the relative phase difference was calculated according to the following formula:
wherein:
RD -relative phase difference;
ω 1 、ω 2 -content values of two samples quantitatively determined in parallel;
-average value of content of two samples measured quantitatively in parallel;
when (when)RDWhen the data is less than or equal to 10 percent, the data is valid; when (when)RDAt > 10%, re-detection is required.
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| WO2016086022A2 (en) * | 2014-11-26 | 2016-06-02 | Channel Therapeutics, Inc. | Heteroaromatic imidazolyl compounds and methods for treating cancer |
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