UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) detection method for simultaneously determining 150 stimulant substances in cosmetics

Abstract

The invention relates to a UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) detection method for simultaneously determining 150 stimulant substances in cosmetics, and belongs to the technical field of stimulant detection. According to the detection method, a solid-phase extraction and QuECHERS combined pretreatment technology is adopted, firstly, an acetonitrile ultrasonic extraction mode in a QuECHERS method is adopted, secondly, a matrix is purified in a solid-phase extraction mode, an extraction tube adopts a filter type solid-phase extraction column, namely an Agilent Captiva EMR-Lipid filter column, different from a traditional HLB solid-phase extraction column, the filter column does not need to be activated and balanced, and the detection method is simple and convenient to operate. Gravity self-elution can be achieved in the sample purification process, and only 20% of pure water needs to be added into an extracting solution to achieve the purpose of activating a filtering column; and water can also be used as a dispersing agent and an impurity removing agent at the same time, so that impurities dissolved in water can be adsorbed while the sample is dispersed. The detection method provided by the invention can realize LC-MS / MS determination of 150 stimulant substances in cosmetics under the condition of simultaneously satisfying a pretreatment method for extracting 150 stimulant substances in cosmetics.

Description

Technical Field

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

[0002] Cosmetics and skincare products play a vital role in athletes' competitions and daily lives, but the safety of doping in cosmetics used by athletes has long been a source of concern. This is because these products can contain residual doping substances during the use of raw materials (for example, products containing Chinese herbal medicines or natural plant ingredients may contain ingredients such as higenamine), the illegal addition of functional ingredients (hormones), or the lack of dedicated production lines during the manufacturing process (where separate production lines are not available). Consequently, athletes who use these products may test positive for doping in their urine. To prevent athletes from coming into contact with these contaminated products and to protect their safety, there is an urgent need to strengthen monitoring of doping substances in cosmetics and skincare products. However, there is a lack of methods for detecting doping substances in cosmetics both domestically and internationally, and there is no ultra-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 food matrices. In addition to solvents (water or ethanol), ordinary cosmetics contain more alcohols that are easily soluble in water (for example, glycerin, butylene glycol, etc.) as moisturizers, as well as esters or cyclopentasiloxane as emulsifiers or moisturizers. Functional skin care products will continue to add active ingredients such as plant extracts, vitamins, niacinamide, etc. With the diversification of the functions of commercially available products, the matrix of cosmetics cannot be distinguished only by the water phase or the oil phase. The matrix of most products exists in the form of "water-in-oil" or "oil-in-water", which poses a challenge to the sample purification method.

[0004] At present, the pre-treatment technology research of cosmetics detection at home and abroad mainly concentrates on solvent extraction, ultrasonic direct extraction, solid phase extraction and QuEChERS method.Solvent extraction method is only suitable for the situation that target substance is less, and the transfer between simple two phases can not satisfy the extraction of hundreds of doping substances simultaneously.Direct ultrasonic extraction rule is more suitable for the situation that more target substances extract, selects suitable broad spectrum extraction reagent (as methanol, acetonitrile, DMSO etc.) to satisfy the extraction of hundreds of doping substances in the present invention, but this method can't cover the cosmetics pre-treatment of all matrix types, easily produces strong matrix effect when facing the sample of matrix complexity, thereby causes the analyte extraction recovery rate to reduce.Solid phase extraction method operation is comparatively loaded down with trivial details and time-consuming, need to use methanol, water equal solvent activation SPE post in advance, then extraction solvent is added in SPE post, the filler in SPE post can adsorb the impurity component in sample matrix, then wait for extraction solvent to be subjected to the effect of gravity by post to obtain comparatively clean sample, actual situation one sample need just can complete purification process through 1-2 hour time, very consuming time and manpower. QuECHERS is an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safety, which stands for a "fast, easy, cheap, effective, stable, and safe" purification method that can process 30-40 samples in 1 hour. It is 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 the impurities in the matrix and the extraction solvent, the purified supernatant can be directly analyzed by the instrument. However, this method requires manual addition of different types of adsorbents according to the matrix type, and the types of adsorbents currently available on the market are limited. At the same time, the amount of addition is also very difficult to control. Too much will adsorb the analyte, and too little will increase the matrix effect or even make it impossible to use the instrument. Furthermore, relying solely on centrifugal separation to separate the extraction solvent and adsorbent can also result in residual impurities in the matrix, affecting the purification effect. Alternatively, the adsorbent may be carried over during the extraction of organic reagents, resulting in trace amounts of non-volatile salts in the supernatant. Large-scale sample injections can damage the chromatographic column and the mass spectrometer's ion source. Therefore, a single QuEChERS method is difficult to meet the needs of cosmetic pretreatment for a wide range of matrices. Therefore, there is an urgent need to develop a pretreatment method for doping detection that can cover a wide range of cosmetic matrices.

[0005] At the same time, since there are more than 300 stimulant substances (10 categories in total) listed in the World Anti-Doping Agency (WADA) banned list, the present invention uses a pretreatment method to extract and UPLC-MS / MS a total of 150 stimulant substances, including S2: growth factors, S3: β2 agonists, S4: hormones and metabolic regulators, S5: diuretics and masking agents, S9: glucocorticoids, and P1: β-blockers, according to the chemical properties of different categories of substances in the WADA banned list. Summary of the Invention

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

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

[0008] A UPLC-MS / MS method for simultaneously determining 150 stimulant substances in cosmetics comprises the following steps:

[0009] (1) adding 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 separately, and the supernatant obtained by the pretreatment operation was concentrated by nitrogen blowing, and the reconstitution 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) Comparing the analysis results of the test sample, the negative control sample, and the positive control sample to determine the type of stimulant in the test sample.

[0012] The present invention provides a UPLC-MS / MS detection method for the simultaneous determination of 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, the dynamic multiple reaction monitoring (dMRM) mode is used to simultaneously determine the 150 extracted stimulant substances.

[0013] As 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 substance working solution of 150 stimulant substances added to the blank matrix control sample at 100 ng, the concentration of the mixed standard substance 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: taking solid internal standards higenamine-D4 (Higenamine-D4), clenbuterol-D9 (Clenbuterol-D9), salbutamol-D3 (Salbutamol-D3), furosemide-D5 (Furosemide-D5), and hydrocortisone-D3 (Hydrocortisone-D3) and dissolving them in methanol and diluting them to a stock solution of 100 ng / μL, and then diluting them to 10 ng / μL to obtain an 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 specifically as follows: 5 mL of acetonitrile is added to the sample to be tested, the negative control sample, and the positive control sample to which the internal standard solution is added, ultrasonic extraction is performed for 30 minutes, and centrifugation is performed for 8 minutes at a speed of 4000 rpm / min to obtain a supernatant.

[0016] Cosmetic matrices are categorized into liquid, lotion (oil-in-water), cream (water-in-oil), and oil. Because the liquid matrix is ​​simpler than the other three matrices, consisting solely of water and water-soluble humectants (such as butylene glycol and glycerin), the aqueous matrix is ​​measured using direct ultrasonic extraction.

[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 specifically as follows: 1.2 mL of deionized pure water is added to each of the sample to be tested, the negative control sample and the positive control sample to which the internal standard solution is added for dispersion, and then 4.8 mL of acetonitrile is added and mixed and vortexed for 10 seconds, ultrasonic extraction is performed for 30 minutes, 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 eluate flows into a glass test tube to which 0.3 g of anhydrous MgSO4 has been added. The eluate is centrifuged for 8 minutes at a speed of 4000 rpm / min to obtain a supernatant after centrifugation.

[0018] Lotions, creams, and oils have complex matrices, often containing solvents, moisturizers, emollients, thickeners, and antioxidants. These three matrices were analyzed using a combination of solid-phase extraction and QuEChERS. After the aforementioned pretreatment, all four matrices met the requirements for UPLC-MS / MS detection in terms of fluidity and turbidity.

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

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

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

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

[0023] The ammonium formate-formic acid buffer contains ammonium formate, formic acid and water, and the concentration of ammonium formate is 10 mmol·L -1 , the volume percentage of formic acid in water is 0.05%;

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

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

[0026] The injection volume was 5 μL.

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

[0028] The mode ionization mode of mass spectrometry analysis was electrospray ionization, the ion source was Agilent AJS ESI, and dynamic multiple reaction monitoring scanning was adopted. 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-voltage iFunnel RF was 150 V(+) / 90 V(-), and the low-voltage iFunnel RF was 60 V(+) / 60 V(-).

[0029] As a preferred embodiment of the detection method of the present invention, the 150 stimulant substances include medostatin, acadesin, metaproterenol, salbutamol, terbutaline, atenolol, cimaterol, amiloride, sotalol, procaterol, acetazolamide, dorzolamide, higenamine, 4-amino-6-chloro-1,3-benzenedisulfonamide, fenoterol, chlorothiazide, ritodrine, hydrochlorothiazide, trimetazidine, carteolol, nadolol, pindolol, triamterene, flumethiazide, tritoquinol, ractopamine, mepindolol, hydroflumethiazide, brinzolamide, timolol, acebutolol, metoprolol, tolbutrol, clenbuterol, chlorpromazine ... terol, formoterol, arformoterol, benfurol, aminoglutethimide, triamcinolone, levobunotolol, penbutolol, olodaterol, esmolol, chlorthalidone, celiprolol, bambuterol, isoxsuprine, oxprenolol, mabuterol, carazolol, labetalol, bisoprolol, torasemide, metipranolol, clopamide, propranolol, trichlorothiazide, alprenolol, clorazanide, bronolol, mozaffarol, methylchlorothiazide, prednisolone, betaxolol, fluprednisolone, isoflurane, prednisone, hydrocortisone, furosemide, zilpaterol, bufurolol, camporeline, raloxifene, indacaterol, metolazone, althiazide , azosemide, methylprednisolone, buthiazide, dexamethasone / betamethasone, carvedilol, eplerenone, MK677, bazedoxifene, flumethasone, epithiazide, letrozole, anastrozole, benzylhydrochlorothiazide, conivaptan, indapamide, beclomethasone, vilanterol, nebivolol, benzthiazide, dexamethasone acetate, flurandrenolide, triamcinolone acetonide, desonide, flunisolide, ethacrynic acid, cyclothiazide, piretanide, prednisolone acetate, fluorometholone, salmeterol, methylprednisolone succinate, fluocortolone, hydrocortisone acetate, fludrocortisone acetate, polythiazide, prednisone acetate, androtrienedione, bendroflumethiazide, deflazacort, cyclopenthiazide Azine, Zelenox, Xipamide, Probenecid, Cortisone Acetate, Bumetanide, Andarine, Mebutiazide, Deoxycorticosterone, Budesonide, Hydrocortisone Butyrate, Exemestane, Spironolactone, Canrenone, Tolvaptan, Triamcinolone Acetate, Mometasone, Fluticasone, Ostarine, Fluocinolone Acetate, Clomiphene Citrate, Toremifene, Arimistane, Tamoxifen Citrate, Halcinonide, Clobetasol Propionate, Fluticasone Propionate, Beclomethasone Dipropionate, GW-1516, Flucortolone Trimethylacetate, GW-0742, Fulvestrant, SR-9009, Cyclofenil, Ciclesonide.

[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 Agilent 6495C.

[0031] Compared with the prior art, the UPLC-MS / MS detection method for the simultaneous determination of 150 stimulant substances in cosmetics provided by the present 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, the acetonitrile ultrasonic extraction method in the QuECHERS method is adopted, and then the matrix is ​​purified by solid phase extraction. The extraction tube uses a filter 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 balancing, and can be gravity-eluted during the sample purification process. In addition, only 20% pure water needs to be added to the extract to achieve the purpose of activating the filter column. Water can also be used as a dispersant and impurity remover at the same time, dispersing the sample while also adsorbing water-soluble impurities.

[0033] (2) The detection method of the present invention adopts a new matrix purification method, which combines the advantages of the solid phase extraction column method and the QuEChERS method. It can not only ensure the simplicity of the pretreatment method, but also ensure that all 150 stimulant substances have high recovery rates and subsequent UPLC-MS / MS detection results are accurate, effectively improving the sensitivity of the detection and greatly 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 chromatographic column contamination and increasing the service life of the chromatographic column.

[0035] (4) The detection method of the present invention uses dynamic multiple reaction monitoring (dMRM) scanning in the UPLC-MS / MS detection process, which can simultaneously detect 150 stimulant substances. The detection time is shortened from 50 minutes using the multiple reaction monitoring (MRM) mode to 23 minutes, saving detection time and improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The chromatograms are representative of some substances in the aqueous matrix (Liquid).

[0037] Figure 2 The chromatograms are representative of some substances in the emulsion matrix (Lotion).

[0038] Figure 3 The following are chromatograms of some representative substances in cream matrices.

[0039] Figure 4 The chromatogram is a representative substance chromatogram of the oil matrix (Oil).

[0040] Figure 5The figure is a bar graph comparing the response values ​​of 20 representative stimulant substances in the lotion matrix in Example 6 under conditions of different masses of anhydrous magnesium sulfate.

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

[0042] Figure 7 The figure is a bar graph comparing the response values ​​of 20 representative stimulant substances in the oil phase (Oil) matrix under different masses of anhydrous magnesium sulfate in Example 6.

[0043] Figure 8 The figure is a bar graph comparing the response values ​​of 18 representative stimulant substances after the emulsion matrix in Comparative Example 1 is purified using the method of the present invention and the QuECHERS method. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

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

[0046] (1) Instruments:

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

[0048] (2) Standards and reagents

[0049] All standards were purchased from Tianjin Alta Technology Co., Ltd. Methanol, formic acid, and ammonium formate were chromatographically grade reagents; methanol and formic acid were purchased from Dikma (USA), and ammonium formate was purchased from Sigma-Aldrich (Switzerland). 15 mL polypropylene centrifuge tubes and 10 mL Pyrex glass tubes were purchased from Corning (USA). Captivia EMR-Lipid filter-type solid-phase extraction cartridges (6 mL, 600 mg) were purchased from Agilent (USA). Anhydrous magnesium sulfate (salt-packed) was purchased from Agilent (USA), and PSA and C18 were purchased from CNW Technologies (Germany). 2 mL brown injection vials and 200 μL snap-top microvial vials were purchased from ThermoFisher (USA).

[0050] (3) Mixed standard substance working solution

[0051] Spironolactone, Acebutolol, Methylprednisolone Hemisuccinate, Triamcinolone Acetonide, Flurandrenolide, 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: For these 25 substances, solid standards were dissolved in acetonitrile as the solvent to prepare stock solutions with a concentration of 1 mg / mL; the stock solutions were diluted with the same solvent to a mass concentration of 100 ng / μL (0.1 mg / mL); 100 μL of the above 100 ng / μL solution was added to 2 mL brown injection vials, and diluted with solvent to a total volume of 1 mL to prepare a mixed standard substance working solution (10 ng / μL).

[0052] Except for the above 25 substances, methanol was used as the solvent to dissolve the solid standards of the remaining substances to prepare a stock solution with a concentration of 1 mg / mL; the stock solution was diluted with the same solvent to a solution with a mass concentration of 100 ng / μL, and 100 μL of the above 100 ng / μL solution was added to a 2 mL brown injection bottle and diluted with solvent to a total volume of 1 mL of mixed standard substance working solution (10 ng / μL).

[0053] (4) Preparation of internal standard solution

[0054] Solid Higenamine-D4, Clenbuterol-D9, Salbutamol-D3, Furosemide-D5, and Hydrocortisone-D3 were dissolved in methanol and diluted to a stock solution of 100 ng / μL, and then diluted to 10 ng / μL to obtain an internal standard solution.

[0055] Example 1

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

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

[0058] (2) Sample pretreatment for aqueous 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 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 collected and transferred to a 10 mL glass test tube. The supernatant was blown dry under nitrogen at 65°C and reconstituted with 500 μL of the initial mobile phase. The reconstitution solution was 10 mmol / L sodium hydroxide containing 0.05% formic acid. -1 A mixture of ammonium formate solution and acetonitrile, containing 0.05% formic acid, 10 mmol·L -1 The volume ratio of ammonium formate solution to acetonitrile was 90:10, and 10 mmol·L of ammonium formate solution containing 0.05% formic acid was used. -1 In the ammonium formate solution, the volume percentage of formic acid in water is 0.05%. 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 lotion / cream / oil matrix: After artificially adding 100 ng of internal standard solution and 100 ng of mixed standard working solution of 150 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, centrifugation was performed at 4000 rpm / min for 8 minutes. The supernatant was collected and placed on a filter-type solid-phase extraction column (Agilent Captivia EMR-Lipid, specification: 6 mL, 600 mg). The supernatant passed through the column and entered a 10 mL glass test tube placed below the column (0.3 g of anhydrous magnesium sulfate had been added to the glass tube). The glass tube was then centrifuged at 4000 rpm / min for 8 minutes. After centrifugation, the supernatant was collected and placed into a clean 10 mL glass test tube. The supernatant was blown dry under nitrogen at 65°C and reconstituted with 500 μL of the initial mobile phase. The reconstituted solution was passed through a 0.22 μm aqueous membrane and then subjected to UPLC-MS / MS analysis.

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

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

[0062] The liquid chromatography column was an Agilent ZORBAX Eclipse Plus C18 column with a size of 2.1×100 mm, a filler 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 is 10mmol·L -1 Ammonium formate aqueous solution (the volume percentage of formic acid in water is 0.05%); mobile phase B is acetonitrile solution.

[0064] The gradient elution program of the liquid chromatography was as follows: from 0.0 to 12.0 min, the volume of the mobile phase B increased from 10% to 50%, with a flow rate of 0.25 mL / min; from 12.0 to 12.01 min, the volume of the mobile phase B remained unchanged at 50%, and the flow rate 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 remained unchanged at 50%, with a flow rate of 0.4 mL / min; from 16.0 to At 17.0 min, the volume of mobile phase B was increased from 50% to 95% at a flow rate of 0.4 mL / min. From 17.0 to 18.0 min, the volume of mobile phase B remained constant at 95% at a flow rate of 0.4 mL / min. From 18.0 to 18.1 min, the volume of mobile phase B was decreased from 95% to 10% at a flow rate of 0.25 mL / min. From 18.1 to 20.0 min, the volume of mobile phase B remained constant 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 mode ionization of the mass spectrometry analysis is electrospray ionization, the ion source is Agilent AJS ESI, dynamic multiple reaction monitoring (dMRM) scanning is adopted, 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°C, the sheath gas temperature is 250°C, the nozzle voltage is 500 V(+) / 500 V(-), the capillary voltage is 4000°C(+) / 3500°C(-), the high-voltage iFunnel RF is 150 V(+) / 90 V(-), and the low-voltage iFunnel RF is 60 V(+) / 60 V(-).

[0066] The dMRM mode parameters for 150 stimulant substances are shown in Table 1.

[0067] Table 1 dMRM mode parameters for 150 stimulant substances

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Example 2

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

[0076] In this example, four matrices were pre-treated according to the specific method of Example 1 to obtain four blank matrix control samples. The four blank matrix control samples were then used to dilute and mix the standard substance working solutions to obtain test solutions. The test solutions were then subjected to UPLC-MS / MS detection according to the method of Example 1 to obtain chromatograms of 150 stimulant substances. The chromatograms of some representative substances in the aqueous matrix (Liquid) are shown in FIG. Figure 1 As shown, the chromatograms of some representative substances of the emulsion matrix (Lotion) are as follows Figure 2 As shown in the figure, the chromatograms of some representative substances of cream matrix (Cream) are as follows Figure 3 As shown, the chromatogram of some representative substances of the oil matrix (Oil) is as follows Figure 4 As shown, ND indicates that the substance was not detected. Based on the chromatogram, each stimulant substance can be qualitatively analyzed. 23 stimulant substances were selected as representative substances, and the internal standard normalized peak area was calculated according to the following formulas 1 and 2, and the matrix effect (ME) was evaluated:

[0077] Formula 1: Normalized peak area = internal standard peak area / target compound peak area;

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

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

[0080] The calculation results are shown in Table 2. The results show that among the 23 stimulant aqueous matrices (Liquid) and oily matrices (Oil), two substances each had an ME < 80%, while the MEs of the remaining substances were all between 80-120%. In the lotion matrix (Lotion), four substances had an ME < 80%, and five substances had an ME > 120%. In the cream matrix (Cream), four substances had an ME < 80%, and one substance had an ME > 120%. The above results indicate that the above four matrices still have certain matrix inhibition and enhancement effects on the detection of stimulant substances. In order to eliminate the influence of the matrix and ensure the accuracy of the analysis results, the method of the present 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 matching standard curve and internal standard method for quantitative calculation.

[0081] Table 2 Matrix effects of 23 representative stimulant substances in four matrices

[0082]

[0083] Example 3

[0084] This example confirms the linear range and limit of detection (LOD) of the specific method of Example 1.

[0085] The linear range refers to the ability of an experimental result to exhibit a linear relationship with the concentration of the analyte in the sample within a certain concentration range. Within this range, signal-to-concentration conversion can be performed using a linear relationship. This example used a blank matrix control sample to dilute a mixed standard substance working solution to examine the linear relationship of the target substance. A 10 ng / μl mixed standard substance working solution was diluted proportionally 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 testing on the instrument. Testing showed that most stimulant substances exhibited good linear relationships within the range of 2-400 ng / ml in aqueous, emulsion, cream, and oil matrices (results shown in Tables 3-6).

[0086] The limit of detection (LOD) refers to the lowest concentration or amount at which a target substance can be reliably detected after pretreatment. It is the threshold value achieved when the target substance's signal-to-noise ratio (S / N ≥ 3) is met. The results are shown in Table 3-6. Testing in aqueous matrices revealed that, with the exception of thiazides and azosemide, the LODs for highly polar and moderately polar analytes were 0.02-2 μg / kg. However, weakly polar analytes (such as beclomethasone dipropionate and ciclosonide) were not detectable. In lotion and cream matrices, the LODs for moderately polar analytes also reached 0.02-2 μg / kg. However, the LODs for highly 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. However, since the fragment ion mass number of Toremifene is relatively small and easily interfered by the background of complex matrices, its LOD reaches 400ug / kg. In the oil phase matrix, the LOD of most medium-polar analytes can reach 0.2-50μg / kg. However, the LOD of strong polarity, weak polarity, Vilanterol, and Salmeterol is 400μg / kg or even cannot be detected. According to the analysis, the reasons may be as follows: 1. Due to the high oil content in the oil-based sample, the oil may not be completely adsorbed by the EMR column, and the residual oil is prone to produce an ion suppression effect on weakly polar substances, so the weakly polar analytes are greatly affected by the matrix. 2. Since the chemical structure of Vilanterol and Salmeterol contains long-chain alkyl groups, their LogP are approximately 4.2 and 3.8 respectively, and they are both extremely fat-soluble, resulting in extremely low extraction recovery rates during the matrix purification and oil removal process. 3. Since oil-based samples may form microemulsion particles in the extraction solvent (acetonitrile water), they are easy to encapsulate highly polar substances and prevent solvent contact, resulting in extremely low extraction recovery.

[0087] Table 3. Linearity and Limits of Detection (LOD) of 150 Stimulant Substances in Liquid Matrix

[0088]

[0089]

[0090]

[0091]

[0092] ND means the substance was not detectable.

[0093] Table 4. Linearity and Limits of Detection (LOD) of 150 Stimulant Substances in Lotion

[0094]

[0095]

[0096]

[0097]

[0098] ND means the substance was not detectable.

[0099] Table 5. Linearity and limits of detection (LOD) of 150 stimulant substances in cream matrix

[0100]

[0101]

[0102]

[0103]

[0104] ND means the substance was not detectable.

[0105] Table 6. Linearity and limits of detection (LOD) of 150 stimulant substances in oil matrix (Oil)

[0106]

[0107]

[0108]

[0109]

[0110] ND means the substance was not detectable.

[0111] Example 4

[0112] This example 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 substance working solutions were added to four blank matrix control samples. Six parallel tests were performed at each concentration. The "three-point calibration" method was used to verify the recovery rate and relative standard deviation (RSD) of 150 stimulant substances in each matrix under this detection method.

[0113] The experimental results showed that, excluding compounds that were undetectable or 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 lotion matrices ranged from 60.36% to 119.61%, with precision ranging from 1.29% to 19.87%. The recoveries of 148 compounds in cream matrices ranged from 71.71% to 119.84%, with precision ranging from 0.94% to 19.88%. 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%. Detailed data are shown in Tables 7 to 10.

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

[0115] Formula 2: Recovery = (measured value / spike amount) × 100%;

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

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

[0118]

[0119]

[0120]

[0121] NA means the concentration is below the integration limit.

[0122] Table 8. Recoveries and RSDs of 150 stimulant substances in lotion matrix

[0123]

[0124]

[0125]

[0126]

[0127] NA means the concentration is below the integration limit.

[0128] Table 9. Recoveries and RSDs of 150 stimulant substances in cream matrix

[0129]

[0130]

[0131]

[0132]

[0133] NA means the concentration is below the integration limit.

[0134] Table 10. Recoveries and RSDs of 150 stimulant substances in oil matrix (Oil)

[0135]

[0136]

[0137]

[0138]

[0139] NA means the concentration is below the integration limit.

[0140] Example 5

[0141] This example tests and analyzes commercially available lotions (Liquid) and emulsions (Lotion), including the following steps:

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

[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 blank lotion and emulsion matrix to prepare a positive control sample. Blank lotion and emulsion matrix serve as negative control samples.

[0144] (3) Testing of lotion samples: 5 mL of acetonitrile was added to each of the test sample, positive control sample, and negative control sample, mixed, and then ultrasonically extracted for 30 min. After ultrasonic extraction, the samples were centrifuged at 4000 rpm / min for 8 min. The supernatant was collected and transferred to a 10 mL glass tube. The supernatant was dried under nitrogen at 65°C and then reconstituted with 500 μL of the initial mobile phase. The reconstituted solution was passed through a 0.22 aqueous membrane and analyzed by UPLC-MS / MS.

[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 the matrix was dispersed, 4.8 mL of acetonitrile was added. After mixing, ultrasonic extraction was performed for 30 minutes. After ultrasonic extraction, centrifugation was performed at 4000 rpm / min for 8 minutes. After centrifugation, the supernatant was taken and placed on a filter-type solid phase extraction column (Agilent Captivia EMR-Lipid, specification: 6 mL, 600 mg). After passing through the small column, the supernatant entered a 10 mL glass test tube placed below the small column (0.3 g of anhydrous magnesium sulfate was added to the glass tube). The glass tube was then centrifuged at 4000 rpm / min for 8 minutes. After centrifugation, the supernatant was taken and placed in a clean 10 mL glass test tube. The supernatant was blown dry under nitrogen at 65°C, and 500 μL of the initial mobile phase was added for reconstitution. The reconstituted solution was passed through a 0.22 μm aqueous membrane and then subjected to UPLC-MS / MS analysis.

[0146] (5) Comparison of the analytical results of the test drug, negative control drug, and positive control drug showed that a trace amount of higenamine (concentration of 195 ng / g) was detected in the lotion, and a trace amount of higenamine (concentration of 30.13 ng / g) was detected in the emulsion sample.

[0147] Example 6

[0148] This optimization example compares and optimizes the mass of anhydrous magnesium sulfate used in the pretreatment of lotions, creams, and oil phases.

[0149] The role of anhydrous magnesium sulfate is to act as a dehydrating agent to absorb excess water during the pretreatment process, which is beneficial to the nitrogen blowing process. However, too little anhydrous magnesium sulfate will lead to incomplete water removal, and too much anhydrous magnesium sulfate will also lead to the adsorption of the target substance. Therefore, this optimization example selects 0.1g, 0.3g, 0.5g, 0.7g, and 1g of anhydrous magnesium sulfate as a dehydrating agent for comparative experiments to ensure that the water is completely removed while reducing the adsorption of the target substance. The experimental results are as follows Figures 5 to 7 shown.

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

[0151] (1) As the mass of anhydrous magnesium sulfate in the emulsion matrix increases, the response of each representative stimulant substance decreases. Anhydrous magnesium sulfate may have a certain adsorption effect on the target substance. However, when the mass of anhydrous magnesium sulfate is 0.1g, a small amount of water is still found during the nitrogen blowing process, which leads to an extension of the nitrogen blowing time and an increase in the nitrogen blowing temperature. When the mass of anhydrous magnesium sulfate is 0.3g, no water is found during the nitrogen blowing process, and the response of the target substance is equivalent to that of 0.1g. Therefore, 0.3g of anhydrous magnesium sulfate is selected as the optimal mass.

[0152] (2) In cream and oil matrices, as the amount of anhydrous magnesium sulfate increases, the responses of representative stimulants do not show a significant downward trend, and the differences in response values ​​are negligible, indicating that anhydrous magnesium sulfate has a small adsorption effect on the target substances. Therefore, while ensuring complete water adsorption, the amount of anhydrous magnesium sulfate used should theoretically be as small as possible to reduce the adsorption of the target substances. Therefore, 0.3g of anhydrous magnesium sulfate was selected as the optimal mass during the pretreatment of these two matrices.

[0153] Comparative Example 1

[0154] This comparative example uses the traditional QuECHERS method to purify lotion, cream, and oil phase matrices.

[0155] 0.5 g (±0.02 g) of each matrix sample was sampled. After manually adding 100 ng of internal standard solution, 5 mL of acetonitrile was added to the sample, vortexed, and 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 the centrifuge tube. After vortexing, the sample was centrifuged at 4000 rpm / min for 8 min. The supernatant was transferred to a 10 mL glass test tube and blown dry under nitrogen at 65°C. 500 μL of the original mobile phase was added for reconstitution. The reconstituted solution was passed through a 0.22 μm aqueous membrane and analyzed by UPLC-MS / MS.

[0156] The experimental results show that after using this purification method for the oil phase (Oil) matrix, there is still 1-2mL of oil that cannot be blown dry, resulting in the inability to be re-dissolved and tested on the machine. After using this purification method for the cream matrix, flocculent precipitation is produced after re-dissolving with the initial mobile phase and it is also impossible to be tested on the machine. After using this purification method and re-dissolving with the initial mobile phase for the emulsion (Lotion) matrix, it can be tested on the machine normally, but the response values ​​of the target substances (18 representative stimulant substances are selected) are all lower than those of the pre-treatment purification method of the present invention. The results are shown in FIG. Figure 8 .

[0157] Therefore, the pretreatment purification method of the present 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 the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A UPLC-MS / MS method for the simultaneous determination of 150 stimulant substances in cosmetics, characterized in that: The method comprises the following steps: (1) adding internal standard solution to the test sample, negative control sample and positive control sample respectively; (2) The test sample, negative control sample, and positive control sample were pretreated separately, and the supernatant obtained by the pretreatment operation was concentrated by nitrogen blowing, and the reconstitution 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; (3) Comparing the analysis results of the test sample, the negative control sample, and the positive control sample to determine the type of stimulant in the test sample.

2. The detection method according to claim 1, wherein In the step (1), the amount of the test 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 of 150 stimulant substances added with 100 ng 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 detection method according to claim 1, wherein In the step (1), the preparation step of the internal standard solution is as follows: solid higenamine-D4, clenbuterol-D9, salbutamol-D3, furosemide-D5, and hydrocortisone-D3 are dissolved in methanol and diluted to a stock solution of 100 ng / μL, and then diluted to 10 ng / μL to obtain an internal standard solution.

4. The detection method according to claim 1, wherein In the step (2), when the sample to be tested is an aqueous matrix sample, the pretreatment operation is specifically as follows: 5 mL of acetonitrile is added to the sample to be tested, the negative control sample, and the positive control sample to which the internal standard solution is added, ultrasonic extraction is performed for 30 minutes, and centrifugation is performed for 8 minutes at a speed of 4000 rpm / min to obtain a supernatant.

5. The detection method according to claim 1, wherein In the step (2), when the sample to be tested 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 sample to be tested, 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 added and mixed and vortexed for 10 seconds, ultrasonic extraction is performed for 30 minutes, 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 eluate flows into a glass test tube to which 0.3 g of anhydrous MgSO4 has been added. The eluate is centrifuged for 8 minutes at a speed of 4000 rpm / min to obtain a supernatant after centrifugation.

6. The detection method according to claim 1, wherein In the step (2), the temperature of nitrogen blowing and concentration is 65°C, the amount of the reconstitution solution is 500 μL, and the reconstitution solution is 10 mmol·L containing 0.05% formic acid. -1 A mixture of ammonium formate solution and acetonitrile, containing 0.05% formic acid, 10 mmol·L -1 The volume ratio of ammonium formate solution to acetonitrile was 90:10, and 10 mmol·L of ammonium formate solution containing 0.05% formic acid was used. -1 In the ammonium formate solution, the volume percentage of formic acid in water is 0.05%.

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

8. The detection method according to claim 1, wherein In the step (2), in the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry test, the mass spectrometry conditions are: The mode ionization mode of mass spectrometry analysis was electrospray ionization, the ion source was Agilent AJS ESI, and dynamic multiple reaction monitoring scanning was adopted. 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-voltage iFunnel RF was 150 V(+) / 90 V(-), and the low-voltage iFunnel RF was 60 V(+) / 60 V(-).

9. The detection method according to claim 1, wherein The 150 stimulant substances include medostatin, acadesine, metaproterenol, salbutamol, terbutaline, atenolol, cimaterol, amiloride, sotalol, purcaterol, acetazolamide, dorzolamide, higenamine, 4-amino-6-chloro-1,3-benzenedisulfonamide, fenoterol, chlorothiazide, ritodrine, hydrochlorothiazide, trimetazidine, carteolol, nadolol, pindolol, triamterene, flumethiazide, tritoquinol, ractopamine, mepindolol, hydroflumethiazide, brinzolamide, timolol, acebutolol, metoprolol, tolbuterol, clenbuterol, formoterol, arformoterol, and benzfuran. Lol, aminoglutethimide, triamcinolone, levobunotolol, penbutolol, olodaterol, esmolol, chlorthalidone, celiprolol, bambuterol, isoxsuprine, oxprenolol, mabuterol, carazolol, labetalol, bisoprolol, torsemide, metipranolol, clopamide, propranolol, trichlorothiazide, alprenolol, clorazanide, bronolol, mozaffarol, methylchlorothiazide, prednisolone, betaxolol, fluprednisolone, isoflurane, prednisone, hydrocortisone, furosemide, zilpaterol, bufuralol, camporelline, raloxifene, indacaterol, metolazone, althiazide, azosemide, methylprednisolone , buthiazide, dexamethasone / betamethasone, carvedilol, eplerenone, MK677, bazedoxifene, flumethasone, epithiazide, letrozole, anastrozole, benzyl hydrochlorothiazide, conivaptan, indapamide, beclomethasone, vilanterol, nebivolol, benzthiazide, dexamethasone acetate, flurandrenolide, triamcinolone acetonide, desonide, flunisolide, ethacrynic acid, cyclothiazide, piretanide, prednisolone acetate, fluorometholone, salmeterol, methylprednisolone succinate, fluocortolone, hydrocortisone acetate, fludrocortisone acetate, polythiazide, prednisone acetate, androtrienedione, bendroflumethiazide, deflazacort, cyclopenthiazide, zelenoxetine , xipamide, probenecid, cortisone acetate, bumetanide, Andarine, mebutiazide, deoxycorticosterone, budesonide, hydrocortisone butyrate, exemestane, spironolactone, canrenone, tolvaptan, triamcinolone acetonide acetate, mometasone, fluticasone, Ostarine, fluocinolone acetonide, clomiphene citrate, toremifene, Arimistane, tamoxifen citrate, halcinonide, clobetasol propionate, fluticasone propionate, beclomethasone dipropionate, GW-1516, flucortolone trimethylacetate, GW-0742, fulvestrant, SR-9009, cyclofenac, ciclesonide.

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

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