A method for simultaneously determining 150 kinds of stimulant substances in cosmetics by UPLC-MS / MS

Abstract

The present application relates to a kind of UPLC-MS / MS detection method for simultaneously determining 150 kinds of stimulant substances in cosmetics, belong to the technical field of stimulant detection.The detection method of the present application uses the pretreatment technology of solid phase extraction and QuECHERS, first, using the method of QuECHERS acetonitrile ultrasonic extraction, second, using the form of solid phase extraction to purify matrix, extraction tube selects filter type solid phase extraction column: Agilent Captiva EMR-Lipid filter column, unlike traditional HLB solid phase extraction column, the filter column does not need to activate and balance, can be self-eluted in the process of sample purification by gravity, and only need to add 20% pure water in the extraction liquid to achieve the purpose of activating filter column;While water can also be used as dispersing agent and impurity remover, the sample is dispersed while the impurities dissolved in water are also adsorbed.The detection method of the present application can meet the conditions of the pretreatment method of 150 kinds of stimulant substances in cosmetics under the condition of simultaneously extracting 150 kinds of stimulant substances in cosmetics, and realize the LC-MS / MS determination of 150 kinds of stimulant substances in cosmetics.

Description

Technical Field

[0001] This invention relates to a UPLC-MS / MS method for simultaneously determining 150 stimulant substances in cosmetics, belonging to the field of stimulant detection technology. Background Technology

[0002] Cosmetics and skincare products play a vital role in athletes' competitions and daily lives, but the doping safety of athletes using these products has always been a major concern. This is because these products may contain residual doping substances due to the use of raw materials (e.g., products containing traditional Chinese medicine or natural plant ingredients may contain ingredients like norcodine), the illegal addition of functional ingredients (hormonal substances), or the lack of a separate production line. Therefore, athletes using these products may test positive for doping substances in their urine. To prevent athletes from coming into contact with these contaminated products and protect their safety during competitions, it is urgent to strengthen the monitoring of doping substances in cosmetics and skincare products. However, currently, there is a lack of detection methods for doping substances in cosmetics both domestically and internationally, and there is no ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS / MS) method that can simultaneously detect hundreds of doping substances in cosmetics.

[0003] Cosmetic matrices are more complex than those in food. In addition to solvents (water or ethanol), ordinary cosmetics often contain a significant amount of water-soluble alcohols (e.g., glycerin, butylene glycol) as moisturizers, and esters or cyclopentamethoxysiloxanes as emulsifiers or skin-moisturizers. Functional skincare products, on the other hand, further contain active ingredients such as plant extracts, vitamins, and niacinamide. With the increasing diversification of functions in commercially available products, the matrix of cosmetics cannot be simply distinguished by whether it is an aqueous or oil phase. Most products have a matrix in the form of "water-in-oil" or "oil-in-water," which poses a challenge to sample purification methods.

[0004] Currently, research on pretreatment techniques for cosmetic testing both domestically and internationally mainly focuses on solvent extraction, direct ultrasonic extraction, solid-phase extraction (SPE), and the QuEChERS method. Solvent extraction is only suitable for cases with a small number of target substances; the simple transfer between two phases cannot simultaneously extract hundreds of stimulant substances. Direct ultrasonic extraction is more suitable for cases with a larger number of target substances; selecting appropriate broad-spectrum extraction reagents (such as methanol, acetonitrile, DMSO, etc.) can meet the extraction requirements of hundreds of stimulant substances in this invention. However, this method cannot cover pretreatment of cosmetics with all matrix types, and it is prone to strong matrix effects when dealing with samples with complex matrices, resulting in a reduced recovery rate of the analytes. Solid-phase extraction is relatively cumbersome and time-consuming. It requires pre-activating the SPE column with solvents such as methanol and water, and then adding the extraction solvent into the SPE column. The packing material in the SPE column adsorbs impurities from the sample matrix, and then the extraction solvent passes through the column under gravity to obtain a relatively clean sample. In practice, it takes 1-2 hours for a single sample to complete the purification process, which is very time-consuming and labor-intensive. QuECHERS is an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safety, representing a purification method that is "fast, simple, inexpensive, effective, stable, and safe," capable of processing 30-40 samples per hour. It's a pretreatment method based on matrix solid-phase dispersion technology. Different adsorbents (NaCl, anhydrous MgSO4, PSA, GCB) are added to the extraction solvent. After centrifugation to separate impurities from the extraction solvent, the purified supernatant can be directly analyzed by the instrument. However, this method requires manual addition of different types of adsorbents depending on the matrix type, and the variety of commercially available adsorbents is currently limited. Furthermore, controlling the amount added is very difficult; too much will adsorb the analyte, while too little will increase the matrix effect or even prevent instrument analysis. Furthermore, relying solely on centrifugation to extract solvents and adsorbents can lead to residual impurities in the matrix, affecting purification efficiency. Additionally, the adsorbent may be introduced during organic reagent extraction, resulting in trace amounts of non-volatile salts in the supernatant. Large-volume injections can damage the chromatographic column and the ion source of the mass spectrometer. Therefore, the single QuEChERS method is insufficient for pretreatment of cosmetics with diverse matrix types. Consequently, there is an urgent need to develop a pretreatment method for stimulant detection that can cover most cosmetic matrices.

[0005] Meanwhile, since the World Anti-Doping Agency (WADA) Prohibited List contains more than 300 doping substances (in 10 categories), this invention uses a pretreatment method to extract and determine 150 doping substances, including S2 (growth factors), S3 (β2 agonists), S4 (hormones and metabolic regulators), S5 (diuretics and masking agents), S9 (glucocorticoids), and P1 (β-blockers), based on the chemical properties of the different categories of substances on the WADA Prohibited List. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a UPLC-MS / MS detection method for simultaneously determining 150 stimulant substances in cosmetics. The technical problem to be solved by this invention is to provide a detection method for LC-MS / MS determination of the 150 stimulant substances under the condition of establishing a pretreatment method that can simultaneously meet the requirements of the extraction of 150 stimulant substances.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A UPLC-MS / MS method for the simultaneous determination of 150 stimulant substances in cosmetics, the method comprising the following steps:

[0009] (1) Add internal standard solution to the test sample, negative control sample and positive control sample respectively;

[0010] (2) The test sample, negative control sample and positive control sample were pretreated respectively. The supernatant obtained from the pretreatment was concentrated by nitrogen blowing, and a complex solution was added. After passing through a 0.22 μm aqueous phase membrane, ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was performed.

[0011] (3) By comparing the analysis results of the test sample, negative control sample and positive control sample, the type of stimulant in the test sample is determined.

[0012] This invention provides a UPLC-MS / MS detection method for simultaneously determining 150 stimulant substances in cosmetics. First, different matrix categories are classified according to the type of cosmetics. Second, pretreatment methods are established and optimized according to different mechanism categories. Finally, dynamic multiple reaction monitoring (dMRM) mode is used to simultaneously determine the 150 extracted stimulant substances.

[0013] In a preferred embodiment of the detection method of the present invention, in step (1), the amount of the test sample, negative control sample and positive control sample is 0.5 mL or 0.5 g, and the amount of the internal standard solution is 100 ng; the negative control sample is a blank matrix control sample, and the positive control sample is a mixed standard working solution of 150 kinds of stimulant substances with 100 ng added to the blank matrix control sample, the concentration of the mixed standard working solution is 10 ng / μL, and the solvent is methanol or acetonitrile.

[0014] As a preferred embodiment of the detection method of the present invention, in step (1), the preparation step of the internal standard solution is as follows: take solid internal standards higenamine-D4, clenbuterol-D9, salbutamol-D3, furosemide-D5, and hydrocortisone-D3, dissolve them in methanol and dilute them to a stock solution of 100 ng / μL, and then dilute them to 10 ng / μL to obtain the internal standard solution.

[0015] As a preferred embodiment of the detection method of the present invention, in step (2), when the sample to be tested is an aqueous matrix sample, the pretreatment operation is as follows: add 5 mL of acetonitrile to each of the sample to be tested, the negative control sample and the positive control sample with added internal standard solution, extract by ultrasonication for 30 min, centrifuge for 8 min at a speed of 4000 rpm / min to obtain supernatant.

[0016] Cosmetic bases are classified into aqueous phase (Liquid), lotion (oil-in-water), cream (water-in-oil), and oil phase (Oil). Because aqueous phase bases have simpler components than the other three types, using only water and water-soluble moisturizers (butanediol, glycerin, etc.), they are analyzed using a direct ultrasonic extraction method.

[0017] As a preferred embodiment of the detection method of the present invention, in step (2), when the sample to be tested is an emulsion, cream or oil phase matrix sample, the pretreatment operation is as follows: 1.2 mL of deionized pure water is added to each of the sample to be tested, negative control sample and positive control sample with added internal standard solution for dispersion, then 4.8 mL of acetonitrile is added and vortexed for 10 s, ultrasonically extracted for 30 min, and the supernatant is added to a filter-type solid phase extraction column Agilent Captiva EMR-Lipid for elution. The specifications of the extraction column are 6 mL and 600 mg. The eluent flows into a glass test tube with 0.3 g of anhydrous MgSO4 added. The eluent is centrifuged for 8 min at a speed of 4000 rpm / min to obtain the supernatant after centrifugation.

[0018] The matrix compositions of emulsions, creams, and oils are complex, containing not only solvents but also moisturizers, emollients, thickeners, and antioxidants. These three types of matrices are analyzed using a combination of solid-phase extraction and QuEChERS methods. After the aforementioned pretreatment, all four types of matrices meet the requirements for UPLC-MS / MS detection in terms of flowability and turbidity.

[0019] In a preferred embodiment of the detection method of the present invention, in step (2), the nitrogen blowing concentration temperature is 65°C, the volume of the reconstitution solution is 500 μL, and the reconstitution solution is 10 mmol·L⁻¹ of formic acid containing 0.05% formic acid. -1 A mixture of ammonium formate solution and acetonitrile, containing 0.05% formic acid at a concentration of 10 mmol·L⁻¹. -1 The volume ratio of ammonium formate solution to acetonitrile is 90:10, containing 0.05% formic acid in a 10 mmol·L solution. -1 In an ammonium formate solution, formic acid accounts for 0.05% of the water by volume.

[0020] As a preferred embodiment of the detection method of the present invention, in step (2), the liquid chromatography conditions in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry test are as follows:

[0021] The liquid chromatography column was an Agilent ZORBAX Eclipse Plus C18 column with dimensions of 2.1 × 100 mm, a packing particle size of 1.8 μm, and a column temperature of 40 °C.

[0022] The mobile phase of the liquid chromatography includes mobile phase A and mobile phase B; mobile phase A is ammonium formate-formic acid buffer; mobile phase B is acetonitrile solution;

[0023] The ammonium formate-formic acid buffer solution contains ammonium formate, formic acid, and water, with an ammonium formate concentration of 10 mmol·L⁻¹. -1 Formic acid accounts for 0.05% of the water by volume;

[0024] The gradient elution program for liquid chromatography was as follows: from 0.0 to 12.0 min, the volume of mobile phase B increased from 10% to 50% at a flow rate of 0.25 mL / min; from 12.0 to 12.01 min, the volume of mobile phase B remained constant at 50% at a flow rate increasing from 0.25 mL / min to 0.4 mL / min; from 12.01 to 16.0 min, the volume of mobile phase B remained constant at 50% at a flow rate of 0.4 mL / min. From 16.0 to 17.0 min, the volume of mobile phase B increased from 50% to 95% at a flow rate of 0.4 mL / min; from 17.0 to 18.0 min, mobile phase B remained at 95% at a flow rate of 0.4 mL / min; from 18.0 to 18.1 min, mobile phase B decreased from 95% to 10% at a flow rate of 0.25 mL / min; from 18.1 to 20.0 min, mobile phase B remained at 10%.

[0025] The flow rate was 0.25 mL / min;

[0026] The injection volume was 5 μL.

[0027] In a preferred embodiment of the detection method of the present invention, in step (2), the mass spectrometry conditions for ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry testing are as follows:

[0028] The ionization mode for mass spectrometry analysis was electrospray ionization, with an Agilent AJS ESI ion source and dynamic multiple reaction monitoring (MRM). The nebulizer voltage was 45 psi, the sheath gas flow rate was 11 L / min, the dryer flow rate was 14 L / min, the dryer temperature was 200 °C, the sheath gas temperature was 250 °C, the nozzle voltage was 500 V(+) / 500 V(-), the capillary voltage was 4000 °C(+) / 3500 °C(-), the high-pressure iFunnel RF was 150 V(+) / 90 V(-), and the low-pressure iFunnel RF was 60 V(+) / 60 V(-).

[0029] As a preferred embodiment of the detection method described in this invention, the 150 stimulant substances include medocarbamate, acalcidin, orsinaline, salbutamol, terbutaline, atenolol, cimaterol, amiloride, sotalol, procaterol, acetazolamide, dazolide, norcodone, 4-amino-6-chloro-1,3-benzenesulfonamide, fenoterol, chlorothiazide, ritodrine, hydrochlorothiazide, trimetazidine, carteolol, nadolol, indololol, triamterene, flumethiazide, tratoquinol, ractopamine, mefenolol, hydrofluorothiazide, brinzolamide, timolol, acebutolol, metoprolol, tobacterium, and clenbuterol. Tarceva, Formoterol, Afortrol, Phenylephrine, Aminoglutate, Triamcinolone, Levobuprofen, Penbuprofen, Odaterol, Esmolol, Chlorthalidone, Celivolol, Bambutrol, Isosorbide, Oxyphenidyl, Mabutrol, Caralolol, Labetalol, Bisoprolol, Torasemi, Metenolol, Clopamide, Propranolol, Trichlorothiazide, Alpralonol, Clorazani, Blavalolol, Mozafatan, Methoxythiazide, Prednisolone, Betalol, Fluprednisolone, Isoflurane, Prednisone, Hydrocortisone, Furosemide, Zipatero, Butifrolol, Carmorelin, Raloxifene, Indaterol, Metoprazone, Althiazide Azosemide, Methylprednisolone, Buthiazine, Dexamethasone / Betamethasone, Carvedilol, Eplerenone, MK677, Badoxifene, Flumethasone, Epilathiazide, Letrozole, Anastrozole, Benzylchlorothiazide, Conivevatan, Indapamide, Beclomethasone, Vilanterol, Nebivolol, Benzylchlorothiazide, Dexamethasone Acetate, Flumethasone, Triamcinolone, Desodium, Flunisolone, Ethacrylic Acid, Cyclothiazide, Pyrotartanone, Prednisolone Acetate, Flumethasone, Salmeterol, Methylprednisolone Succinate, Flucorone, Hydrocortisone Acetate, Flucortisone Acetate, Porrithiazine, Prednisolone Acetate, Androstriene Dione, Benzylfluorothiazide, Defcote, Cyclopentazoline Zine, Zelenol, Sipamide, Probenecid, Cortisone Acetate, Bumetanide, Andarine, Mebthiazide, Deoxycorticosterone, Budesonide, Hydrocortisone Butyrate, Exemestane, Spironolactone, Canrone, Tolvaptan, Triamcinolone Acetate, Mometasone, Fluticasone, Ostarine, Fluocinolone Acetate, Clomiphene Citrate, Toremiphene, Arimistane, Tamoxifen Citrate, Halcinonide, Clobetasol Propionate, Fluticasone Propionate, Beclomethasone Dipropionate, GW-1516, Flucosolone Trimethylacetate, GW-0742, Fluvestrantraniliprole, SR-9009, Cyclofenib, Cyclosonesone.

[0030] As a preferred embodiment of the detection method of the present invention, in step (2), the instrument for ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry is an Agilent 6495C.

[0031] Compared with existing technologies, the UPLC-MS / MS detection method for simultaneously determining 150 stimulant substances in cosmetics provided by this invention can achieve at least one of the following beneficial effects:

[0032] (1) The detection method of the present invention adopts a pretreatment method combining solid phase extraction and QuECHERS. First, acetonitrile ultrasonic extraction is used in the QuECHERS method. Then, solid phase extraction is used to purify the matrix. The extraction tube uses a filterable solid phase extraction column: Agilent Captiva EMR-Lipid filter column. Unlike the traditional HLB solid phase extraction column, this filter column does not require activation and equilibration. It can be gravity-driven to self-elute during the sample purification process. Only 20% pure water needs to be added to the extract to activate the filter column. Water can also be used as a dispersant and impurity remover at the same time. It can disperse the sample and adsorb water-soluble impurities.

[0033] (2) The detection method of the present invention adopts a novel matrix purification method, which combines the advantages of solid phase extraction column method and QuEChERS method. This method can ensure that the pretreatment method is simple to operate, and can ensure that all 150 stimulant substances have high recovery rates and accurate subsequent UPLC-MS / MS detection results, effectively improving the detection sensitivity and significantly improving the detection efficiency.

[0034] (3) The detection method of the present invention does not require the addition of complex adsorbents during sample pretreatment, thus avoiding column contamination and improving column life.

[0035] (4) In the detection method of the present invention, during the UPLC-MS / MS detection process, the mass spectrometry detection adopts dynamic multiple reaction monitoring (dMRM) scanning, which can simultaneously detect 150 kinds of stimulant substances. The detection time is shortened from 50 minutes using multiple reaction monitoring (MRM) mode to 23 minutes, saving detection time and improving detection efficiency. Attached Figure Description

[0036] Figure 1 This is a chromatogram of representative substances in the aqueous matrix (Liquid).

[0037] Figure 2 This is a chromatogram of representative substances in the emulsion matrix (Lotion).

[0038] Figure 3 This is a chromatogram of representative substances in the cream matrix.

[0039] Figure 4 This is a chromatogram of representative substances in the oil phase matrix.

[0040] Figure 5This is a bar chart comparing the response values ​​of 20 representative stimulant substances in the emulsion matrix of Example 6 under different masses of anhydrous magnesium sulfate.

[0041] Figure 6 This is a bar chart comparing the response values ​​of 20 representative stimulant substances in the cream matrix of Example 6 under different masses of anhydrous magnesium sulfate.

[0042] Figure 7 This is a bar chart comparing the response values ​​of 20 representative stimulant substances in the oil phase matrix of Example 6 under different masses of anhydrous magnesium sulfate.

[0043] Figure 8 A bar chart comparing the response values ​​of 18 representative stimulant substances after using the purification method of the present invention and the QuECHERS method on the emulsion matrix in Comparative Example 1. Detailed Implementation

[0044] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0045] The specific instrument models and reagent sources used in the embodiments of this invention are as follows:

[0046] (1) Instruments:

[0047] Agilent 1290 Infinity III ultra-high performance liquid chromatograph (Agilent Technologies, USA); Agilent 6495C triple quadrupole mass spectrometer (Agilent Technologies, USA); Milli-Q ultrapure water preparation system (Merck, Germany); Multifuge X3R centrifuge (Thermo Fisher Scientific, USA); Dri-Block nitrogen evaporator (Techne, UK); Genie Vortex-2 vortex mixer (Scientific Industries).

[0048] (2) Standards and reagents

[0049] All standards were purchased from Tianjin Alta Technology Co., Ltd. Methanol, formic acid, and ammonium formate were chromatographically pure reagents; methanol and formic acid were purchased from Dikma (USA), and ammonium formate from Sigma-Aldrich (Switzerland). 15 mL polypropylene centrifuge tubes and 10 mL Plyrex glass tubes were purchased from Corning (USA). Captivia EMR-Lipid filter-type solid-phase extraction columns (6 mL, 600 mg) were purchased from Agilent Technologies (USA). Anhydrous magnesium sulfate (salt package) was purchased from Agilent Technologies (USA), and PSA and C18 were purchased from CNW Technologies (Germany). 2 mL brown sample vials and 200 μL bayonet micro-sample vials were purchased from Thermo Fisher Scientific (USA).

[0050] (3) Mixed standard substance working solution

[0051] Spironolone, Acebutolol, Methylprednisolone Hemisuccinate, Triamcinolone Acetonide, Fluranandrenolide, Flunisolide, Prednisolone Acetate, Hydrocortisone Acetate, Fludrocortisone Acetate, Prednisone Acetate, Budesonide, Ciclesonide, Desonide, Fluocinonide Acetate, Clobetasol Propionate, Fluticasone Propionate, Beclomethasone Dipropionate Dipropionate, Fluocortolone Pivalate, Cortisone Acetate, Dexamethasone Acetate, Hydrocortisone Butyrate, Triamcinolone Acacetonide Acetate, Halcinonide, and AICAR: These 25 substances were prepared by dissolving solid standards in acetonitrile to prepare stock solutions with a concentration of 1 mg / mL. The stock solutions were then diluted with the same solvent to a mass concentration of 100 ng / μL (0.1 mg / mL). 100 μL of each of the 100 ng / μL solutions was added to a 2 mL brown injection bottle and diluted with solvent to a total volume of 1 mL for mixed standard working solution (10 ng / μL).

[0052] In addition to the 25 substances mentioned above, the remaining substances were prepared by dissolving solid standards in methanol to prepare stock solutions with a concentration of 1 mg / mL. The stock solutions were then diluted with the same solvent to a mass concentration of 100 ng / μL. 100 μL of the 100 ng / μL solution was added to 2 mL brown injection bottles and diluted with solvent to a total volume of 1 mL for mixed standard working solution (10 ng / μL).

[0053] (4) Preparation of internal standard solution

[0054] Dissolve and dilute solid higenamine-D4, clenbuterol-D9, salbutamol-D3, furosemide-D5, and hydrocortisone-D3 in methanol to prepare a stock solution of 100 ng / μL, and then dilute to 10 ng / μL to obtain an internal standard solution.

[0055] Example 1

[0056] This embodiment uses the detection method of the present invention, including the following steps:

[0057] (1) Sample taking: 0.5g (±0.02g) of each matrix sample (Liquid, Lotion, Cream, Oil) was taken.

[0058] (2) Sample pretreatment with the aqueous (Liquid) matrix: After artificially adding 100 ng of internal standard solution and 100 ng of a mixed standard working solution of 150 stimulant substances, 5 mL of acetonitrile was added to the sample to disperse the matrix, followed by ultrasonic extraction for 30 min. After ultrasonic extraction, the sample was centrifuged at 4000 rpm / min for 8 min. The supernatant was then transferred to a 10 mL glass tube and dried under nitrogen at 65 °C. 500 μL of the initial mobile phase was added to reconstitute the solution, which was a 10 mmol·L⁻¹ solution containing 0.05% formic acid. -1 A mixture of ammonium formate solution and acetonitrile, containing 0.05% formic acid at a concentration of 10 mmol·L⁻¹. -1 The volume ratio of ammonium formate solution to acetonitrile is 90:10, containing 0.05% formic acid in a 10 mmol·L solution. -1 In the ammonium formate solution, the formic acid accounted for 0.05% of the water by volume. The reconstituted solution was passed through a 0.22 μm aqueous membrane and then subjected to UPLC-MS / MS analysis.

[0059] (3) Sample pretreatment of emulsion / cream / oil matrix: After artificially adding 100 ng of internal standard solution and 100 ng of mixed standard working solution of 150 kinds of stimulant substances, 1.2 mL of deionized water was added to the sample to disperse the matrix, and then 4.8 mL of acetonitrile was added. After mixing, ultrasonic extraction was performed for 30 min. After ultrasonic extraction, the sample was centrifuged at 4000 rpm for 8 min. The supernatant was then transferred to a filter-type solid-phase extraction column (Agilent Captivia EMR-Lipid, 6 mL, 600 mg). After passing through the column, the supernatant was transferred to a 10 mL glass tube (containing 0.3 g of anhydrous magnesium sulfate) placed below the column. The glass tube was then centrifuged at 4000 rpm for 8 min. The supernatant was then transferred to a clean 10 mL glass tube and dried under nitrogen at 65 °C. 500 μL of the initial mobile phase was added to redissolve the supernatant. The redissolved solution was then filtered through a 0.22 μm aqueous membrane and analyzed by UPLC-MS / MS.

[0060] Regarding the amount of acetonitrile used: Considering that the maximum capacity of the Agilent Captivia EMR-Lipid filtration-type solid-phase extraction column is 6 mL of acetonitrile-water, and based on the optimal acetonitrile-water ratio in this column (acetonitrile:water = 1:4), 4.8 mL of acetonitrile and 1.2 mL of deionized water were selected. Furthermore, experiments showed that 10 mL of acetonitrile and 6 mL of acetonitrile-water had comparable extraction effects on the target substances (19 representative stimulant substances were selected as targets), with no significant difference in the response values ​​of the target substances.

[0061] (4) Perform ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS) tests:

[0062] The liquid chromatography column was an Agilent ZORBAX Eclipse Plus C18 column with dimensions of 2.1 × 100 mm, a packing particle size of 1.8 μm, and a column temperature of 40 °C.

[0063] The mobile phase of liquid chromatography includes mobile phase A and mobile phase B; mobile phase A has a concentration of 10 mmol·L⁻¹. -1 Ammonium formate aqueous solution (formic acid accounts for 0.05% of water by volume); mobile phase B is acetonitrile solution.

[0064] The gradient elution program for liquid chromatography was as follows: from 0.0 to 12.0 min, the volume of mobile phase B increased from 10% to 50% at a flow rate of 0.25 mL / min; from 12.0 to 12.01 min, the volume of mobile phase B remained constant at 50% at a flow rate increasing from 0.25 mL / min to 0.4 mL / min; from 12.01 to 16.0 min, the volume of mobile phase B remained constant at 50% at a flow rate of 0.4 mL / min; from 16.0 to... At 17.0 min, the volume of mobile phase B increased from 50% to 95% at a flow rate of 0.4 mL / min; from 17.0 to 18.0 min, mobile phase B remained at 95% at a flow rate of 0.4 mL / min; from 18.0 to 18.1 min, mobile phase B decreased from 95% to 10% at a flow rate of 0.25 mL / min; from 18.1 to 20.0 min, mobile phase B remained at 10% at a flow rate of 0.25 mL / min. The injection volume was 5 μL.

[0065] The conditions for mass spectrometry analysis are as follows: the ionization mode of the mass spectrometry analysis is electrospray ionization, the ion source is Agilent AJS ESI, dynamic multiple reaction monitoring (dMRM) scanning is used, the nebulizer voltage is 45 psi, the sheath gas flow rate is 11 L / min, the dryer flow rate is 14 L / min, the dryer temperature is 200℃, the sheath gas temperature is 250℃, the nozzle voltage is 500V(+) / 500V(-), the capillary voltage is 4000℃(+) / 3500℃(-), the high-pressure iFunnel RF is 150V(+) / 90V(-), and the low-pressure iFunnel RF is 60V(+) / 60V(-).

[0066] The dMRM model parameters of 150 doping substances are shown in Table 1.

[0067] Table 1. dMRM model parameters for 150 doping substances

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Example 2

[0075] This embodiment confirms the matrix effect of each sample matrix in the specific method of Example 1.

[0076] In this embodiment, the four matrices were pretreated according to the specific method of Example 1 to obtain four blank matrix control samples. The standard working solution was then diluted and mixed using these four blank matrix control samples to obtain the test solution. This test solution was then analyzed by UPLC-MS / MS according to the method of Example 1, yielding chromatograms of 150 stimulant substances. The chromatograms of representative substances in the aqueous matrix (Liquid) are shown below. Figure 1 As shown, the chromatograms of representative substances in the emulsion matrix (Lotion) are as follows: Figure 2 As shown, the chromatograms of representative substances in the cream matrix are as follows: Figure 3 As shown, the chromatograms of representative substances in the oil phase matrix are as follows: Figure 4 As shown, ND indicates that the substance is undetectable. Qualitative analysis can be performed on each doping substance based on the chromatogram. Twenty-three doping substances were selected as representative substances, and the peak area was calculated using internal standard normalization according to Formulas 1 and 2 below. The matrix effect (ME) was then evaluated.

[0077] Formula 1: Normalized peak area = Internal standard peak area / Target peak area;

[0078] Formula 2: ME (%) = (Normalized peak area of ​​standard substance in blank matrix / Normalized peak area of ​​standard substance in solvent) × 100%;

[0079] The 23 substances are salbutamol, sotalol, higenamine, hydrochlorothiazide, trimetazidine, carteolol, brinzolamide, tulobuterol, prednisolone, hydrocortisone, and furosemide. Carvedilol, Bazedoxifen, Flumethasone, Indapamide, Androstatrienedione, Zeranol, Budesonide, Mometasone, Tolvaptan, Ostarine, Toremifene, and Clobetasol Propionate.

[0080] The calculation results are shown in Table 2. The results show that in each of the 23 stimulant aqueous and oil matrices, two substances had a median efficiency (ME) < 80%, while the MEs of the remaining substances were between 80% and 120%. In the lotion matrix, four substances had an ME < 80%, and five substances had an ME > 120%. In the cream matrix, four substances had an ME < 80%, and one substance had an ME > 120%. These results indicate that the four matrices still exhibit certain matrix inhibition and enhancement effects on the detection of stimulant substances. To eliminate the influence of the matrix and ensure the accuracy of the analytical results, the method of this invention uses a blank matrix control sample to dilute the standard solution to prepare a series of matrix standard working solutions, and uses a matrix-matched standard curve and internal standard method for quantitative calculation.

[0081] Table 2. Matrix effects of 23 representative doping substances in 4 matrices

[0082]

[0083] Example 3

[0084] This embodiment confirms the linear range and limit of detection (LOD) of the specific method in Embodiment 1.

[0085] Linearity refers to the ability of experimental results to exhibit a linear relationship with the concentration of the analyte in a sample within a certain concentration range. Within this range, signal-to-concentration conversion can be performed based on this linear relationship. This implementation case uses a method of diluting the mixed standard working solution with a blank matrix control sample to examine the linear relationship of the target substance. The mixed standard working solution with a concentration of 10 ng / μl was diluted to 400 ng / ml, 200 ng / ml, 100 ng / ml, 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 2 ng / ml before being tested. The results showed that most stimulant substances exhibited good linearity in the range of 2-400 ng / ml in aqueous, emulsion, cream, and oil-based matrices (results are shown in Tables 3-6).

[0086] The limit of detection (LOD) refers to the lowest concentration or content of a target substance that can be reliably detected after pretreatment. It is the threshold obtained when the signal-to-noise ratio (S / N) of the target substance is ≥3 (S / N ≥ 3). The results are shown in Table 3-6. In aqueous matrices, except for thiazides and azosemide, the LODs of strongly polar and moderately polar analytes were 0.02-2 μg / kg, but weakly polar analytes (such as beclomethasone dipropionate and ciclosonide) were undetectable. In emulsion and cream matrices, the LODs of moderately polar analytes could also reach 0.02-2 μg / kg, but the LODs of strongly polar analytes (such as Meldonium and Orciprenaline) and most weakly polar analytes (such as esters and cyclic compounds) were higher, reaching 100-200 μg / kg. Due to the low mass number of fragment ions in Toremifene, it is easily affected by background interference from complex matrices, resulting in a LOD of 400 μg / kg. In oil-based matrices, the LOD of most moderately polar analytes can reach 0.2-50 μg / kg. However, the LOD of strongly polar, weakly polar, and Vilanterol and Salmeterol is as high as 400 μg / kg, or even undetectable. The possible reasons for this are as follows: 1. The high oil content in oil-based samples may result in incomplete adsorption of oil by the EMR column. Residual oil can easily inhibit the ion production of weakly polar substances, thus significantly affecting the matrix. 2. Vilanterol and Salmeterol contain long-chain alkyl groups in their chemical structures, with LogP values ​​of approximately 4.2 and 3.8, respectively, exhibiting extremely high lipophilicity, leading to very low extraction and recovery rates during matrix purification and oil removal. 3. Oil-based samples may form microemulsion particles in the extraction solvent (acetonitrile water), which can easily encapsulate highly polar substances, preventing solvent contact and resulting in extremely low extraction recovery rates.

[0087] Table 3. Linearity and Limit of Detection (LOD) of 150 Doping Substances in Aqueous Liquid Matrix

[0088]

[0089]

[0090]

[0091]

[0092] ND indicates that the substance cannot be detected.

[0093] Table 4. Linearity and Limit of Detection (LOD) of 150 Doping Substances in Emulsion Matrix

[0094]

[0095]

[0096]

[0097]

[0098] ND indicates that the substance cannot be detected.

[0099] Table 5. Linearity and Limit of Detection (LOD) of 150 Doping Substances in Cream Matrix

[0100]

[0101]

[0102]

[0103]

[0104] ND indicates that the substance cannot be detected.

[0105] Table 6. Linearity and Limit of Detection (LOD) of 150 Doping Substances in Oil Phase

[0106]

[0107]

[0108]

[0109]

[0110] ND indicates that the substance cannot be detected.

[0111] Example 4

[0112] This embodiment uses the method of Example 1. Based on the linear range and detection limit obtained in Example 2, three concentrations (100 μg / kg, 200 μg / kg, and 400 μg / kg) of mixed standard working solution were added to four blank matrix control samples. Each concentration was measured in parallel six times. The "three-point calibration" method was used to verify the recovery rate and relative standard deviation (RSD) of 150 doping substances in each matrix under this detection method.

[0113] Experimental results showed that, excluding undetectable and substances below the integration limit, the recoveries of 144 compounds in the aqueous matrix ranged from 62.74% to 119.51%, with precision (expressed as relative standard deviation, RSD) ranging from 1.83% to 18.78%; the recoveries of 148 compounds in the lotion matrix ranged from 60.36% to 119.61%, with precision ranging from 1.29% to 19.87%; the recoveries of 148 compounds in the cream matrix ranged from 71.71% to 119.84%, with precision ranging from 0.94% to 19.88%; and the recoveries of 136 compounds in the oil matrix ranged from 65.57% to 119.74%, with precision ranging from 1.74% to 19.00%. Specific data are shown in Tables 7 to 10.

[0114] Experimental results show that the method of the present invention achieves recoveries of stimulant substances reaching the integration limit (≥3*LOD) within the range of 60%-120% as specified in the current national standard GB / T 27404-2008 for all four matrices, with precision within 30%. This indicates that the method has good accuracy and precision and can be used for the rapid detection of 150 stimulant substances in cosmetics containing the matrices. The recovery rate and precision are calculated according to the following formulas 2 and 3:

[0115] Formula 2: Recovery rate = (Measured value / Spike amount) × 100%;

[0116] Formula 3: Precision (RSD) = (Standard deviation / Arithmetic mean of the calculated results) × 100%.

[0117] Table 7. Recovery rates and precision (RSD) of 150 stimulant substances in an aqueous liquid matrix.

[0118]

[0119]

[0120]

[0121] NA indicates that the concentration is below the integration limit.

[0122] Table 8. Recovery rates and (RSD) of 150 stimulant substances in the emulsion matrix (Lotion)

[0123]

[0124]

[0125]

[0126]

[0127] NA indicates that the concentration is below the integration limit.

[0128] Table 9. Recovery rates and RSDs of 150 stimulant substances in cream matrices.

[0129]

[0130]

[0131]

[0132]

[0133] NA indicates that the concentration is below the integration limit.

[0134] Table 10. Recovery rates and RSDs of 150 stimulant substances in the oil phase matrix.

[0135]

[0136]

[0137]

[0138]

[0139] NA indicates that the concentration is below the integration limit.

[0140] Example 5

[0141] This embodiment analyzes and tests commercially available toners and lotions, including the following steps:

[0142] (1) Weigh 0.5g (±0.02g) each of the test samples (toner and lotion), positive control samples (blank toner and lotion base spiked) and negative control samples (blank toner and lotion base).

[0143] (2) Add 100 ng of internal standard solution to each sample. Add 100 ng of a mixed standard solution of 150 stimulant substances to the blank toner and lotion base to prepare positive control samples. The blank toner and lotion base are negative control samples.

[0144] (3) Detection of toner samples: Add 5 mL of acetonitrile to the above-mentioned test sample, positive control sample and negative control sample, mix well and then perform ultrasonic extraction for 30 min. After ultrasonic extraction, centrifuge at 4000 rpm / min for 8 min. After centrifugation, take the supernatant into a 10 mL glass test tube, dry the supernatant under nitrogen at 65℃, add 500 μL of the initial mobile phase to redissolve, filter the redissolved solution through a 0.22 aqueous membrane and perform UPLC-MS / MS test.

[0145] (4) Detection of emulsion samples: 1.2 mL of deionized water was added to the above-mentioned test sample, positive control sample and negative control sample respectively. After dispersing the matrix, 4.8 mL of acetonitrile was added, mixed well and then ultrasonically extracted for 30 min. After ultrasonic extraction, the sample was centrifuged at 4000 rpm / min for 8 min. After centrifugation, the supernatant was transferred to a filter-type solid phase extraction column (Agilent Captivia EMR-Lipid, specification 6 mL, 600 mg). After passing through the column, the supernatant was transferred to a 10 mL glass tube (0.3 g of anhydrous magnesium sulfate was added to the glass tube) placed below the column. The glass tube was then centrifuged at 4000 rpm / min for 8 min. After centrifugation, the supernatant was transferred to a clean 10 mL glass tube and dried under nitrogen at 65 °C. 500 μL of the initial mobile phase was added to redissolve the supernatant. The redissolved solution was filtered through a 0.22 μm aqueous membrane and then tested by UPLC-MS / MS.

[0146] (5) The analysis results of the test drug, negative control drug and positive control drug were compared. The results showed that trace amounts of norcodonine (concentration of 195 ng / g) were detected in the cosmetic water and trace amounts of norcodonine (concentration of 30.13 ng / g) were detected in the emulsion sample.

[0147] Example 6

[0148] This optimization example compares and optimizes the mass fraction of anhydrous magnesium sulfate used in the pretreatment of emulsions, creams, and oil phase (Lotion, Cream, Oil) matrices.

[0149] Anhydrous magnesium sulfate is used as a dehydrating agent in the pretreatment process to adsorb excess water, which is beneficial for the nitrogen blowing process. However, too little anhydrous magnesium sulfate will lead to incomplete water removal, while too much will lead to the adsorption of the target substance. Therefore, this optimization example selected 0.1g, 0.3g, 0.5g, 0.7g, and 1g of anhydrous magnesium sulfate as dehydrating agents for comparative experiments to ensure complete water removal while reducing the adsorption of the target substance. The experimental results are as follows. Figures 5 to 7 As shown.

[0150] Depend on Figures 5 to 7 It can be known that:

[0151] (1) In the emulsion matrix, the response of representative stimulant substances decreases with increasing mass of anhydrous magnesium sulfate, suggesting that anhydrous magnesium sulfate may have some adsorption effect on the target substances. However, when the mass of anhydrous magnesium sulfate is 0.1g, a small amount of moisture is still found during the nitrogen blowing process, leading to a prolonged nitrogen blowing time and an increase in nitrogen blowing temperature. When the mass of anhydrous magnesium sulfate is 0.3g, no moisture is found during the nitrogen blowing process, and the response of the target substances is comparable to that at 0.1g. Therefore, 0.3g of anhydrous magnesium sulfate is selected as the optimal mass.

[0152] (2) In both the cream and oil matrices, the responses of representative stimulant substances did not show a significant decreasing trend with increasing mass of anhydrous magnesium sulfate, and the differences in response values ​​were negligible, indicating that the adsorption effect of anhydrous magnesium sulfate on the target substances was small. Therefore, theoretically, the mass of anhydrous magnesium sulfate used should be as small as possible to ensure complete water adsorption, thereby reducing the adsorption of target substances. Therefore, 0.3 g of anhydrous magnesium sulfate was selected as the optimal mass for the pretreatment of these two matrices.

[0153] Comparative Example 1

[0154] This comparative example uses the traditional QuECHERS method to purify emulsions, creams, and oil phase (Lotion, Cream, Oil) matrices.

[0155] 0.5 g (±0.02 g) of each matrix sample was taken. After manually adding 100 ng of internal standard solution, 5 mL of acetonitrile was added to the sample, vortexed, and then ultrasonically extracted for 30 min. After ultrasonic extraction, 0.3 g of anhydrous magnesium sulfate, 0.05 g of PSA (N-propylethylenediamine solid-phase adsorbent), and 0.05 g of C18 adsorbent were added to a centrifuge tube. After vortexing, the sample was centrifuged at 4000 rpm / min for 8 min. The supernatant was then transferred to a 10 mL glass tube and dried under nitrogen at 65 °C. 500 μL of the initial mobile phase was added to redissolve the supernatant. The redissolved solution was filtered through a 0.22 μm aqueous membrane and then analyzed by UPLC-MS / MS.

[0156] Experimental results show that after using this purification method, 1-2 mL of oil remains undried in the oil phase matrix, preventing reconstitution and instrumentation testing. For cream-based matrices, after reconstitution with the initial mobile phase, flocculent precipitate forms, also preventing instrumentation testing. For lotion-based matrices, after using this purification method and reconstitution with the initial mobile phase, instrumentation testing is possible, but the response values ​​of the target substances (selected from 18 representative stimulant substances) are all lower than those of the pretreatment purification method of this invention. (See attached figures). Figure 8 .

[0157] Therefore, the pretreatment purification method in this invention is superior to the conventional QuECHERS method in terms of ease of operation, practicality, and target substance extraction rate.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A UPLC-MS / MS detection method for simultaneously measuring 150 stimulant substances in cosmetics, characterized by, The method comprises the following steps: (1) adding an internal standard solution to the to-be-tested sample, the negative control sample, and the positive control sample, respectively; (2) performing pretreatment operations on the to-be-tested sample, the negative control sample, and the positive control sample, respectively, performing nitrogen blowing concentration on supernatant obtained through the pretreatment operations, adding a redissolving solution, and performing ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry after passing through a 0.22 μm water phase membrane; In the step (2), when the to-be-tested sample is an emulsion, cream, or oil phase matrix sample, the pretreatment operation is specifically as follows: 1.2 mL of deionized pure water is added to each of the to-be-tested sample, the negative control sample, and the positive control sample to which the internal standard solution is added for dispersion, 4.8 mL of acetonitrile is further added for mixing and vortexing for 10 s, ultrasonic extraction is performed for 30 min, the supernatant is added to a filter type solid phase extraction column Agilent Captiva EMR-Lipid for elution, the specification of the extraction column is 6 mL and 600 mg, the eluent flows into a glass test tube in which 0.3 g of anhydrous MgSO4 has been added, the eluent is centrifuged for 8 min at a speed of 4000 rpm / min, and the supernatant after centrifugation is obtained; The amount of the redissolving solution is 500 μL; (3) comparing the analysis results of the to-be-tested sample, the negative control sample, and the positive control sample to determine the type of the stimulant in the to-be-tested sample.

2. The detection method of claim 1, wherein, In the step (1), the amount of the to-be-tested sample, the negative control sample, and the positive control sample is 0.5 mL or 0.5 g, and the amount of the internal standard solution is 100 ng; the negative control sample is a blank matrix control sample, and the positive control sample is a mixed standard substance working solution in which 100 ng of 150 kinds of stimulant substances is added to the blank matrix control sample; the concentration of the mixed standard substance working solution is 10 ng / μL, and the solvent is methanol or acetonitrile.

3. The method of claim 1, wherein the detecting is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. In the step (1), the preparation procedure of the internal standard solution is as follows: solid norcoclaurine-D4, clenbuterol-D9, salbutamol-D3, furosemide-D5, and hydrocortisone-D3 are dissolved and diluted with methanol to obtain a stock solution with a concentration of 100 ng / μL, and then diluted to 10 ng / μL to obtain the internal standard solution.

4. The method of claim 1, wherein the detecting is performed by a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, and chromatography. In the step (2), when the to-be-tested sample is a water phase matrix sample, the pretreatment operation is specifically as follows: 5 mL of acetonitrile is added to each of the to-be-tested sample, the negative control sample, and the positive control sample to which the internal standard solution is added, ultrasonic extraction is performed for 30 min, and centrifugation is performed for 8 min at a speed of 4000 rpm / min to obtain the supernatant.

5. The detection method as described in claim 1, characterized in that, The temperature of nitrogen blowing concentration in the step (2) is 65℃, and the re-dissolution solution is 10mmolˑL containing 0.05% formic acid -1 The mixture of ammonium formate solution and acetonitrile contains 10mmolˑL of 0.05% formic acid -1 The volume ratio of ammonium formate solution to acetonitrile is 90:10, and the concentration of 10mmolˑL contains 0.05% formic acid -1 The volume percentage of formic acid in ammonium formate solution is 0.05%.

6. The method of claim 1, wherein, In the step (2), in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, the liquid chromatography conditions are as follows: The liquid chromatography column is an Agilent ZORBAX Eclipse Plus C18 chromatography column with a specification of 2.1×100 mm and a filler particle size of 1.8 μm, and the column temperature is 40℃; the mobile phase of the liquid chromatography comprises a mobile phase A and a mobile phase B; the mobile phase A is a formic acid ammonium-formic acid buffer solution; and the mobile phase B is an acetonitrile solution; The ammonium formate-formic acid buffer contains ammonium formate, formic acid and water, the concentration of ammonium formate is 10 mmol·L -1 , and the volume percentage of formic acid in water is 0.05%. The gradient elution procedure of liquid chromatography is as follows: from 0.0 to 12.0 min, the volume of the mobile phase B is increased from 10% to 50%, the flow rate is 0.25 mL / min; from 12.0 to 12.01 min, the volume of the mobile phase B is kept at 50%, the flow rate is increased from 0.25 mL / min to 0.4 mL / min; from 12.01 to 16.0 min, the volume of the mobile phase B is kept at 50%, the flow rate is 0.4 mL / min; from 16.0 to 17.0 min, the volume of the mobile phase B is increased from 50% to 95%, the flow rate is 0.4 mL / min; from 17.0 to 18.0 min, the volume of the mobile phase B is kept at 95%, the flow rate is 0.4 mL / min; from 18.0 to 18.1 min, the volume of the mobile phase B is decreased from 95% to 10%, the flow rate is decreased to 0.25 mL / min; from 18.1 to 20.0 min, the volume of the mobile phase B is kept at 10%, the flow rate is kept at 0.25 mL / min; The flow rate is 0.25 mL / min. The injection volume is 5 μL.

7. The detection method as described in claim 1, characterized in that, In the step (2), in the test of ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, the conditions of mass spectrometry are as follows: The mode of mass spectrometry analysis is electrospray ionization, the ion source is Agilent AJS ESI, dynamic multiple reaction monitoring scanning is adopted, the voltage of sprayer is 45 psi, the flow rate of sheath gas is 11 L / min, the flow rate of dryer is 14 L / min, the temperature of dryer is 200℃, the temperature of sheath gas is 250℃, the voltage of nozzle is 500 V (+) / 500 V (-), the voltage of capillary is 4000℃ (+) / 3500℃ (-), the high-pressure iFunnel RF is 150 V (+) / 90 V (-), and the low-pressure iFunnel RF is 60 V (+) / 60 V (-).

8. The method of claim 1, wherein, The 150 stimulants include medoxomil, akathesin, orciprenaline, salbutamol, terbutaline, atenolol, cimaterol, amiloride, sotalol, procaterol, acetazolamide, dorzolamide, norepinephrine, 4-amino-6-chloro-1,3-benzenedisulfonamide, fenoterol, chlorothiazide, ritodrine, hydrochlorothiazide, trimethazidine, carteolol, nadolol, pindolol, aminophylline, flumethiazide, tritoqual, ractopamine, mepindolol, hydroflumethiazide, brinzolamide, timolol, acebutolol, metoprolol, tulobuterol, clenbuterol, formoterol, arformoterol, benofurone, aminoglutethimide, triamcinolone, levobunolol, penbutolol, olodaterol, esmolol, chlorthalidone, celiprolol, bambuterol, isoxsuprine, oxprenolol, mabuterol, carazolol, labetalol, bisoprolol, torsemide, metolazone, propranolol, trichlormethiazide, alprenolol, clorazepate, bupranolol, moxonidine, methylchlorothiazide, prednisolone, betaxolol, fluprednisolone, isoflupredone, prednisone, hydrocortisone, furosemide, zilpaterol, bufurol, carmoterol, raloxifene, indacaterol, metolazone, althiazide, azosemide, methylprednisolone, buthiazide, dexamethasone / betamethasone, carvedilol, eplerenone, MK677, bazedoxifene, flumethasone, epitiostanol, letrozole, anastrozole, bendroflumethiazide, conivaptan, indapamide, beclometasone, vilanterol, nebivolol, benzthiazide, dexamethasone acetate, flurandrenolide, triamcinolone acetonide, desonide, flunisolide, ethacrynic acid, cyclothiazide, pyrazolamine, prednisolone acetate, fluorometholone, salmeterol, methylprednisolone succinate, fluocortolone, hydrocortisone acetate, fluoro hydrocortisone acetate, polythiazide, prednisone acetate, androstenedione, bendroflumethiazide, deflazacort, cyclothiazide, zeranol, hippuran, probenecid, cortisone acetate, bumetanide, Andarine, mebutizide, deoxycortone, budesonide, hydrocortisone buteprate, exemestane, spironolactone, canrenone, tolvaptan, triamcinolone acetonide acetate, mometasone, fluticasone, Ostarine, fluocinolone acetonide, chloromethine citrate, toremifene, Arimistane, tamoxifen citrate, halcinonide, clobetasol propionate, fluticasone propionate, beclometasone dipropionate, GW-1516, flurandrenolide, GW-0742, fulvestrant, SR-9009, cyclofenine, ciclesonide.

9. The method of claim 1, wherein, In the step (2), the instrument of ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry is Agilent 6495C.

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