CN114878721B - Method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics - Google Patents
Method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics Download PDFInfo
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- CN114878721B CN114878721B CN202210634126.9A CN202210634126A CN114878721B CN 114878721 B CN114878721 B CN 114878721B CN 202210634126 A CN202210634126 A CN 202210634126A CN 114878721 B CN114878721 B CN 114878721B
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- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 title claims abstract description 41
- KASDHRXLYQOAKZ-ZPSXYTITSA-N pimecrolimus Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](Cl)[C@H](OC)C1 KASDHRXLYQOAKZ-ZPSXYTITSA-N 0.000 title claims abstract description 41
- 229960005330 pimecrolimus Drugs 0.000 title claims abstract description 41
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 41
- 239000002537 cosmetic Substances 0.000 title claims abstract description 40
- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000011159 matrix material Substances 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 150000002500 ions Chemical class 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 238000012544 monitoring process Methods 0.000 claims description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- 239000005695 Ammonium acetate Substances 0.000 claims description 7
- 229940043376 ammonium acetate Drugs 0.000 claims description 7
- 235000019257 ammonium acetate Nutrition 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000011550 stock solution Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000013558 reference substance Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000006071 cream Substances 0.000 claims description 4
- 239000000839 emulsion Substances 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 239000013582 standard series solution Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000003921 oil Substances 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 238000007689 inspection Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000005173 quadrupole mass spectroscopy Methods 0.000 abstract description 3
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 206010012438 Dermatitis atopic Diseases 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 201000008937 atopic dermatitis Diseases 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 230000001861 immunosuppressant effect Effects 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 208000002029 allergic contact dermatitis Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- ZDQSOHOQTUFQEM-PKUCKEGBSA-N ascomycin Chemical class C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C\C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-PKUCKEGBSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229930182851 human metabolite Natural products 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 239000008041 oiling agent Substances 0.000 description 1
- 238000013310 pig model Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 208000037851 severe atopic dermatitis Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
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- 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
- G01N2030/047—Standards external
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- 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
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Abstract
The invention discloses a method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics, which adopts a high performance liquid chromatography-tandem triple quadrupole mass spectrometry method to simultaneously determine the qualitative and quantitative methods of tacrolimus and pimecrolimus in cosmetics. The method has the advantages of simple pretreatment, good precision and reproducibility, small matrix effect and strong applicability, fills up the blank of the standards and the inspection methods of the two components in the field of cosmetics inspection, establishes sound regulations, can rapidly, accurately and efficiently qualitatively and quantitatively measure the illegal addition of the tacrolimus and the pimecrolimus in the cosmetics, provides powerful support for the supervision department to timely find illegal addition of the components in the cosmetics, and ensures the life and property safety of people.
Description
Technical Field
The invention belongs to the technical field of cosmetic quality safety detection, and particularly relates to a method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics.
Background
Tacrolimus (Tacrolimus) is an immunosuppressant, is a fermentation product separated from streptomyces, is a macrolide antibiotic, is a powerful novel immunosuppressant, and is mainly used for comprehensively inhibiting the effect of T lymphocytes by inhibiting the release of interleukin-2 (IL-2) and is 100 times stronger than cyclosporin (CsA). FK506 also plays a positive role in the treatment of autoimmune diseases such as Atopic Dermatitis (AD), systemic Lupus Erythematosus (SLE), autoimmune eye diseases, and the like. Pimecrolimus is a lipophilic anti-inflammatory derivative of ascomycin macrolactam that can cell selectively inhibit the production and release of pro-inflammatory cytokines. Pimecrolimus exhibits potent anti-inflammatory activity following topical or systemic administration in animal models of skin inflammation. In a pig model of allergic contact dermatitis, pimecrolimus for external use acts quite well as a potent corticosteroid hormone.
At present, some illegal vendors are to make the use of the illicit, and various forbidden components such as hormone, antibiotics and the like are illegally added into the cosmetics to make the cosmetics play a role of so-called 'treatment'. Tacrolimus and pimecrolimus are two commonly used dermatological medicines, and are mainly used for treating moderate to severe atopic dermatitis in children and adults with poor or intolerance, but have certain side effects, such as local pruritus, erythema, stinging and the like, and are forbidden for children under 2 years old, belong to forbidden components which cannot be added in cosmetics, and no detection method for the two components is formulated in the related standards of the existing cosmetics.
Although the prior art and the prior art report on the detection of tacrolimus and/or pimecrolimus, the method is a detection method when the tacrolimus and/or pimecrolimus are used as medicines, such as the concentration detection of tacrolimus and/or pimecrolimus in blood plasma and human metabolites. The cosmetic comprises various formulations such as cream, emulsion, oil, water agent and the like, and the cosmetic has complex matrix and various components, so that the cosmetic cannot be detected by using the existing medicine detection method.
In order to make up for the defect of the inspection methods of tacrolimus and pimecrolimus in cosmetics, it is highly desirable to establish a quick, efficient and simple inspection method for the two components, control the safety risk and provide technical support for daily supervision of cosmetics.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics. The method realizes the qualitative and quantitative detection of the illegally added tacrolimus and pimecrolimus in the cosmetics, fills the blank of the standards and the detection methods of the two components in the field of cosmetics detection, and provides technical support for establishing sound regulations and supervision of cosmetics in the future.
The invention provides a method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics, which comprises the following steps:
1) Sample preparation: weighing a uniformly mixed sample in a colorimetric tube with a plug, adding methanol, oscillating until the sample is uniformly mixed with an extraction solvent, performing ultrasonic extraction, standing to room temperature, then using the methanol to fix the volume, shaking uniformly, centrifuging, filtering the supernatant by a filter membrane, and taking the filtrate as a solution to be detected;
2) Preparing a standard solution: weighing a proper amount of tacrolimus and pimecrolimus reference substances, placing the tacrolimus and pimecrolimus reference substances into a volumetric flask, adding methanol for dissolution and fixing the volume, and preparing a standard stock solution; absorbing a proper amount of standard stock solution, gradually diluting with methanol to prepare a standard series of solutions;
3) Liquid chromatography conditions: chromatographic column: c18 column (2.1 mm. Times.100 mm,1.7 μm) or equivalent chromatography column; column temperature: 45 ℃; flow rate: 0.3mL/min; sample injection amount: 2. Mu.L; mobile phase: an aqueous solution (A) containing 0.1% acetic acid-5 mmol/L ammonium acetate; methanol solution (B) containing 0.1% acetic acid-5 mmol/L ammonium acetate;
mass spectrometry conditions:
ion source: electrospray ion source (ESI source); monitoring mode: MRM multi-reaction positive ion monitoring mode; spray pressure: 20psi; drying gas flow rate: 14L/min; drying gas temperature: 200 ℃; capillary voltage: 3000V;
4) Qualitative and quantitative: under the same test conditions, the retention time of the mass chromatographic peak of the tested component in the sample is consistent with that of the corresponding component in the standard solution; the deviation between the relative abundance ratio of the selected monitoring ion pairs in the sample chromatogram and the relative abundance ratio of the ions of the standard solution with the equivalent concentration is not more than the specified range of the table, and the existence of the corresponding components in the sample can be judged;
maximum allowable deviation of relative ion abundance in qualitative
Drawing a standard curve by taking the standard series concentration of each component as an abscissa and the peak area as an ordinate, wherein the linear correlation coefficient of the standard curve is larger than 0.99;
the mass fractions of tacrolimus and pimecrolimus are calculated by the following formula:
wherein: omega- -mass fraction of tacrolimus and pimecrolimus in cosmetic, μg/g;
d- -dilution of sample (1 without dilution);
ρ - -the mass concentration of the component to be measured, ng/mL, is obtained from the standard curve;
v- -sample constant volume, mL;
m- -sample amount, g.
Preferably, the liquid chromatography in step 3) is a gradient elution, and the gradient elution procedure is as follows:
preferably: the triple quadrupole ion pair and the related voltage reference parameters in the step 3) are set as follows:
note that: ". Times" are quantitative ions.
Preferably: the centrifugation in the step 1) is 4000r/min.
Preferably: the supernatant in step 1) is filtered through a 0.22 μm filter membrane.
Preferably: in the step 2), standard series solutions with the concentration of 0.5,1.0,2.0,5.0, 20 and 50ng/mL are prepared.
The second purpose of the invention is to provide the application of the method in detecting tacrolimus and pimecrolimus in other cream, emulsion, water agent and oil agent products.
The invention has the beneficial effects that:
the method for simultaneously determining the tacrolimus and pimecrolimus in the cosmetics by using the high performance liquid chromatography tandem triple quadrupole mass spectrometry for the first time has the advantages of simple pretreatment, good precision and reproducibility, small matrix effect and strong applicability, fills up the blank of the standards and methods of the two components in the field of cosmetics inspection, establishes a sound rule, can rapidly, accurately and efficiently determine the tacrolimus and pimecrolimus in the cosmetics qualitatively and quantitatively, provides powerful support for the supervision department to discover illegal addition of forbidden components in time by a powerful technical means, and ensures the life and property safety of people.
The method provided by the invention is verified by methodology, and the obtained results meet the requirements of the technical specification of the verification of forbidden substances and limited substances in cosmetics and the working specification of the management of the cosmetic supplement inspection method.
The method effectively avoids the influence of various product types such as cream, emulsion, oiling agent, water agent and the like on the cosmetic matrix, and accurately carries out qualitative and quantitative detection on the tacrolimus and the pimecrolimus.
Drawings
Fig. 1 is a tacrolimus and pimecrolimus Mo Sizong ion flow diagram (TIC);
FIG. 2 is a Tacrolimus extraction ion flow diagram (MRM);
fig. 3 is a pimecrolimus extraction ion flow diagram (MRM).
Detailed Description
In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. It will be apparent that the following embodiments are part, but not all, of the embodiments of the present application and, therefore, the following detailed description of the embodiments does not limit the scope of the application claimed, and equivalents and modifications thereof by one of ordinary skill in the art without making any inventive effort are intended to be included in the scope of the present invention.
1 reagents and materials
Unless otherwise indicated, the reagents used in the method are all analytically pure, and the water is primary water specified in GB/T6682;
1.1 methanol (CH) 3 OH): a chromatographic grade;
1.2 ammonium acetate (CH) 3 COONH 4 ): a mass spectrum stage;
1.3 acetic acid (CH) 3 COOH): mass spectrum level.
2 instruments and apparatus
2.1 high performance liquid chromatography-triple quadrupole mass spectrometer, electric spray ion source (ESI);
2.2 analytical balance: the sensing amount is 0.0001g and 0.00001g;
2.3 high speed centrifuge;
2.4 an ultrasonic cleaner;
2.5 vortex mixer.
3 analysis step
3.1 preparation of standard series solutions
About 10mg (accurate to 0.01 mg) of tacrolimus and pimecrolimus reference substances are precisely weighed, placed in a 10mL volumetric flask, dissolved by adding methanol, and fixed to a scale, so as to prepare a standard stock solution.
The standard stock solution was taken up in appropriate amounts and gradually diluted with methanol to give standard series of solutions at concentrations of 0.5,1.0,2.0,5.0, 20, 50ng/mL.
3.2 sample solution preparation
Weighing about 0.2g (accurate to 0.001 g) of a uniformly mixed sample into a 10mL colorimetric tube with a plug, adding 8mL of methanol, oscillating on a vortex mixer for 30s until the sample and an extraction solvent are uniformly mixed, ultrasonically extracting for 15min, standing to room temperature, then using methanol to fix the volume to scale, shaking uniformly, centrifuging at 4000r/min for 5min, filtering the supernatant by a 0.22 mu m filter membrane, and taking the filtrate as a solution to be detected.
3.3 instrument reference conditions
3.3.1 liquid chromatography conditions:
chromatographic column: c18 column (2.1 mm. Times.100 mm,1.7 μm) or equivalent chromatography column; column temperature: 45 ℃; flow rate: 0.3mL/min; sample injection amount: 2. Mu.L; mobile phase: an aqueous solution (A) containing 0.1% acetic acid-5 mmol/L ammonium acetate; methanol solution (B) containing 0.1% acetic acid-5 mmol/L ammonium acetate.
TABLE 1 gradient elution procedure
3.3.2 Mass Spectrometry conditions:
ion source: electrospray ion source (ESI source); monitoring mode: MRM multi-reaction positive ion monitoring mode; spray pressure: 20psi; drying gas flow rate: 14L/min; drying gas temperature: 200 ℃; capillary voltage: 3000V.
The triple quadrupole ion pairs and associated voltage reference parameters are set forth in the following table.
Table 2 ion pairs and related mass spectra
Note that: * To quantify ions
2. When using different mass spectrometry instruments, there may be differences in instrument parameters, and mass spectrometry parameters should be optimized to be optimal prior to measurement.
3.4 assays, as shown in figures 1 to 3.
3.4.1 characterization
The qualitative judgment is carried out on the sample by using a liquid chromatography-tandem mass spectrometry method, and under the same test condition, the retention time of the mass chromatographic peak of the tested component in the sample is consistent with the retention time of the mass chromatographic peak of the corresponding component in the standard solution; the relative abundance ratio of the selected monitoring ion pairs in the sample chromatogram does not deviate from the relative abundance ratio of the ions of the standard solution with the equivalent concentration by more than the range specified in the following table, and the presence of the corresponding components in the sample can be judged.
TABLE 3 maximum allowable deviation of relative ion abundance for characterization
3.4.2 quantitative determination
Under the condition of '3.3' liquid chromatography-triple quadrupole mass spectrometry, taking mixed standard series solution (3.1) for sample injection respectively, drawing a standard curve by taking the standard series concentration of each component as an abscissa and the peak area as an ordinate, wherein the linear correlation coefficient is larger than 0.99.
Taking the solution to be detected under the item of '3.2', substituting the peak area of each component into a standard curve to calculate the concentration, and calculating the content of each component in the sample according to the item of '4'.
4 representation of the analysis results
4.1 calculation
Wherein: omega- -mass fraction of tacrolimus and pimecrolimus in cosmetic, μg/g;
d- -dilution of sample (1 without dilution);
ρ -obtaining the mass concentration of the component to be measured, ng/mL from the standard curve;
v- -sample constant volume, mL;
m- -sample amount, g.
The absolute difference between the two independent measurements obtained under repetitive conditions must not exceed 10% of the arithmetic mean.
4.2 recovery and precision
The recovery rate of the method verified by a plurality of laboratories is 80% -120%, and the relative standard deviation is less than 10%.
The foregoing is merely illustrative and explanatory of the invention, as it is well within the scope of the invention as claimed, as it relates to various modifications, additions and substitutions for those skilled in the art, without departing from the inventive concept and without departing from the scope of the invention as defined in the accompanying claims.
Claims (8)
1. A method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics is characterized by comprising the following steps: comprises the following steps:
1) Sample preparation: weighing a uniformly mixed sample in a colorimetric tube with a plug, adding methanol, oscillating until the sample is uniformly mixed with an extraction solvent, performing ultrasonic extraction, standing to room temperature, then using the methanol to fix the volume, shaking uniformly, centrifuging, filtering the supernatant by a filter membrane, and taking the filtrate as a solution to be detected;
2) Preparing a standard solution: weighing a proper amount of tacrolimus and pimecrolimus reference substances, placing the tacrolimus and pimecrolimus reference substances into a volumetric flask, adding methanol for dissolution and fixing the volume, and preparing a standard stock solution; absorbing a proper amount of standard stock solution, gradually diluting with methanol to prepare a standard series of solutions;
3) Liquid chromatography conditions: chromatographic column: c18 column (2.1 mm. Times.100 mm,1.7 μm) or equivalent chromatography column; column temperature: 45 ℃; flow rate: 0.3mL/min; sample injection amount: 2. Mu.L; mobile phase: an aqueous solution (A) containing 0.1% acetic acid-5 mmol/L ammonium acetate; methanol solution (B) containing 0.1% acetic acid-5 mmol/L ammonium acetate;
mass spectrometry conditions:
ion source: electrospray ion source (ESI source); monitoring mode: MRM multi-reaction positive ion monitoring mode; spray pressure: 20psi; drying gas flow rate: 14L/min; drying gas temperature: 200 ℃; capillary voltage: 3000V;
4) Qualitative: under the same test conditions, the retention time of the mass chromatographic peak of the tested component in the sample is consistent with that of the corresponding component in the standard solution; the deviation between the relative abundance ratio of the selected monitoring ion pairs in the sample chromatogram and the relative abundance ratio of the ions of the standard solution with the equivalent concentration is not more than the specified range of the table, and the existence of the corresponding components in the sample can be judged;
maximum allowable deviation of relative ion abundance in qualitative
。
2. The method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: the method also comprises the step 5) of quantitatively detecting, wherein the standard serial concentration of each component is taken as an abscissa, the peak area is taken as an ordinate, and a standard curve is drawn, and the linear correlation coefficient is larger than 0.99;
the mass fractions of tacrolimus and pimecrolimus are calculated by the following formula:
wherein: omega- -mass fraction of tacrolimus and pimecrolimus in cosmetic, μg/g;
d- -dilution of sample (1 without dilution);
ρ - -the mass concentration of the component to be measured, ng/mL, is obtained from the standard curve;
v- -sample constant volume, mL;
m- -sample amount, g.
3. The method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: the centrifugation in the step 1) is 4000r/min.
4. The method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: the supernatant in step 1) is filtered through a 0.22 μm organic filter membrane.
5. The method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: in the step 2), standard series solutions with the concentration of 0.5,1.0,2.0,5.0, 20 and 50ng/mL are prepared.
6. The method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: the liquid chromatography in the step 3) is gradient elution, and the gradient elution procedure is as follows:
。
7. the method for simultaneously detecting tacrolimus and pimecrolimus in cosmetics according to claim 1, characterized in that: the triple quadrupole ion pair and the related voltage reference parameters in the step 3) are set as follows:
note that: * To quantify ions.
8. Use of the method according to any one of claims 1-7 for simultaneous detection of tacrolimus and pimecrolimus in other cosmetic products of matrix type such as creams, emulsions, aqueous solutions, oils, etc.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101419225A (en) * | 2008-11-06 | 2009-04-29 | 复旦大学附属华山医院 | Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human urine |
| CN112697918A (en) * | 2020-12-21 | 2021-04-23 | 北京和合医学诊断技术股份有限公司 | Tacrolimus detection method |
| CN113533555A (en) * | 2021-06-17 | 2021-10-22 | 杭州凯莱谱精准医疗检测技术有限公司 | Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof |
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| US10126316B2 (en) * | 2016-03-22 | 2018-11-13 | Rhode Island Council on Postsecondary Education, Statutory Sucessor to the Rhode Island Board of Education, and Rhode Island Board of Governors for Higher Education | Systems and methods for the measurement of tacrolimus in oral fluids by liquid chromatography tandem mass spectrometry |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419225A (en) * | 2008-11-06 | 2009-04-29 | 复旦大学附属华山医院 | Method for simultaneously determining mycophenolic acid ester, mycophenolic acid and metabolite thereof in human urine |
| CN112697918A (en) * | 2020-12-21 | 2021-04-23 | 北京和合医学诊断技术股份有限公司 | Tacrolimus detection method |
| CN113533555A (en) * | 2021-06-17 | 2021-10-22 | 杭州凯莱谱精准医疗检测技术有限公司 | Detection kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 高效液相色谱法检测化妆品中非法添加的6种抗角化药物;刘莹;李启艳;王小兵;李俊婕;林钰镓;于海英;;卫生研究;20170930(第05期);全文 * |
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