A method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics by high performance liquid chromatography-tandem mass spectrometry

Abstract

The application discloses a high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics. The method comprises the following steps: treating the cosmetic sample to be detected by methanol, and then performing high performance liquid chromatography-tandem mass spectrometry analysis and determination by using a mobile phase containing an ammonium acetate solution. The peak shapes of the 33 components of the anti-inflammatory drugs are all good, and the 33 components can be effectively determined. The recovery rate is between 85% and 110%, the RSD value of the recovery rate is less than 10%, the chromatographic system is stable, the specificity is good, the sensitivity is high, the linearity is good, the precision is high, the accuracy is good, the 33 components can be simultaneously qualitatively and quantitatively determined, and the detection efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic analysis and detection, and particularly relates to a high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics. BACKGROUND

[0002] Anti-inflammatory drugs have the effects of fever reduction and chronic dull pain relief, can reduce pain, reduce temperature and relieve redness, swelling, heat, pain and other symptoms caused by inflammation, and are mainly used for treating cold, fever and pain. However, when used in the skin, they may cause adverse reactions such as skin burning, redness and itching, especially for people with sensitive skin or allergic constitution. Long-term addition of these ingredients in cosmetics may cause cumulative damage such as thinning, drying and peeling of the skin keratin layer, and may affect liver and kidney function through skin absorption.

[0003] The highest historical use amount of acetaminophen in the 'Cosmetic Raw Material Directory' (2021 edition) (hereinafter referred to as the directory) is 0.05% for rinsing products. It is also included in the 'Listed Product Raw Material Use Information' (hereinafter referred to as the information), the use site is hair, the use method is rinsing, and the use amount is 0.013333%. The information does not include resident products and use amounts. The information clearly states that "the use amount of raw materials without authoritative agency assessment reports is objectively included, and no systematic evaluation of the safety of the listed raw materials is organized". The detection of acetaminophen in the children's and acne-removing resident products involved suspected use out of range and the possibility of illegal addition. The remaining 32 components currently have no limit value and are not included in the directory and the information.

[0004] At present, the 'Cosmetic Safety Technical Specification' (2015 edition) has not yet collected the test standards for 33 anti-inflammatory drugs such as acetaminophen in cosmetics, and the limit value has not been specified. Therefore, it is impossible to effectively supervise the addition of such drugs in cosmetics, and there is a regulatory blind spot.

[0005] Therefore, an analysis method for simultaneously detecting 33 anti-inflammatory drugs in cosmetics is needed. SUMMARY

[0006] The purpose of the present application is to provide a high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics, which separates by reverse phase chromatographic column and then detects by positive and negative mode mass spectrometry, uses specific mobile phase for gradient elution of the cosmetic sample to be tested, and uses mass spectrometry for qualitative and quantitative analysis to determine whether the cosmetic sample to be tested contains one or more of the 33 anti-inflammatory drugs, solving the problems in the prior art.

[0007] To solve the above technical problems, the present application adopts the following scheme:

[0008] A high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics, wherein the sample to be tested is treated with methanol, and then analyzed by high performance liquid chromatography-tandem mass spectrometry using a mobile phase containing ammonium acetate solution.

[0009] Further, the method comprises the following steps:

[0010] Step one, cosmetic sample pretreatment: weigh the cosmetic sample in a stoppered colorimetric tube, add methanol, vortex, mix, ultrasonic extraction, centrifuge, and then filter the supernatant to obtain the sample solution to be tested;

[0011] Step two, preparation of 33 anti-inflammatory drug single standard stock solutions, multiple mixed standard stock solutions, mixed standard intermediate solution, and multiple matrix mixed standard solutions:

[0012] Weigh each anti-inflammatory drug standard, add methanol or dimethylformamide to obtain multiple single standard stock solutions;

[0013] Measure multiple single standard stock solutions and mix to obtain mixed standard stock solution 1, mixed standard stock solution 2, mixed standard stock solution 3, mixed standard stock solution 4, and mixed standard stock solution 5;

[0014] Mix the mixed standard stock solutions 1-5 and codeine control solution to obtain the mixed standard intermediate solution;

[0015] Weigh the blank cosmetic sample, add the mixed standard intermediate solution, and follow the sample processing operation steps to obtain multiple matrix mixed standard solutions;

[0016] Step three, detection and analysis:

[0017] Determine the sample solution to be tested and the matrix mixed standard solution by high performance liquid chromatography-tandem mass spectrometry, take the series of concentrations of the components to be tested in the matrix mixed standard solution as the abscissa, and the quantitative ion pair chromatographic peak area of the components to be tested in the matrix mixed standard solution as the ordinate, perform linear regression to obtain the standard curve equation of the components to be tested, and calculate the concentration of the components to be tested in the sample solution to be tested according to the standard curve equation of the components to be tested.

[0018] Further, in step one, methanol is used as the extraction solvent, the vortexing time is 60 seconds, the ultrasonic extraction time is 20 minutes, and the centrifugation speed is 4000 r / min for 5 minutes.

[0019] Further, weigh out the following ingredients respectively: acetaminophen, lornoxicam, ellamod, diflunisal, loxoprofen, nimesulide, acemetrazine, fenofosine calcium, flurbiprofen, diclofenac sodium, indomethacin, etodoxacin, ibuprofen, mefenamic acid, piroxicam, meloxicam, benorilate, rotundine, sulindac, etoricoxib, ketoprofen, taproxen, etoricoxib, fenbufen, aceclofenac, guacitelsal, oxapazine, and nabumethoxazole. Approximately 10 mg each of ketones, capsaicin, celecoxib, etofenadine, and meclofenamic acid standards were placed in separate 10 mL volumetric flasks. Menoxicam, lornoxicam, and ellamod were dissolved in dimethylformamide and diluted to the mark, while the remaining components were dissolved in methanol and diluted to the mark. The solutions were mixed to obtain single-standard stock solutions with a mass concentration of 1000 μg / mL. Codeine reference standard was prepared as a solution with a concentration of 100 μg / mL.

[0020] Further, 0.01 mL of each of the single standard stock solutions of lornoxicam, etordoxacin, meloxicam, rotundine, etoricoxib, oxapazine and etoricoxib were measured and placed in the same 100 mL brown volumetric flask, dissolved in methanol and diluted to the mark, and shaken well to prepare mixed standard stock solution 1.

[0021] Take 0.04 mL of each of the single standard stock solutions of ellamod, nimesulide, piroxicam, benorilate, sulindac, etoricoxib, guacitelsal, and nabumetone and place them in the same 100 mL brown volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to prepare mixed standard stock solution 2.

[0022] Take 0.1 mL of each of the single standard stock solutions of acetaminophen, ketoprofen, fenbufen, capsaicin and celecoxib and place them in the same 100 mL brown volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to prepare mixed standard stock solution 3.

[0023] Take 0.4 mL of each of the single standard stock solutions of fenofol calcium, diclofenac sodium, indomethacin, mefenamic acid and taproxen and place them in the same 100 mL brown volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to prepare mixed standard stock solution 4.

[0024] Take 0.1 mL of each of the single standard stock solutions of diflunisal, meclofenamic acid, and aceclofenac, 0.2 mL of each of the single standard stock solutions of loxoprofen, acemetrazine, and ibuprofen, and 1 mL of flurbiprofen single standard stock solution and place them in the same 10 mL brown volumetric flask. Dissolve them in methanol and dilute to the mark. Shake well to prepare a mixed standard stock solution 5.

[0025] Further, 1 mL of each of the mixed standard stock solutions 1-5 and 0.04 mL of the codeine control sample solution were measured respectively, and were placed in the same 10 mL brown volumetric flask, dissolved with methanol and diluted to the scale, shaken uniformly to prepare the mixed standard intermediate solution.

[0026] Further, in the step three, the chromatographic conditions are as follows:

[0027] Chromatographic column: Agilent ZORBAX Plus C 18 2.1x100mm, 1.8um; flow rate: 0.3mL / min; column temperature: 40 DEG C; injection volume: 1ul; mobile phase A is 2mmol / L ammonium acetate solution, mobile phase B is 2mmol / L ammonium acetate methanol solution, and gradient elution is adopted.

[0028] Further, in the step three, the mass spectrometry conditions are as follows:

[0029] Electrospray ion source, positive and negative ion multiple reaction monitoring MRM mode; electrospray voltage: 4500V-5500V; gas curtain pressure: 20psi; auxiliary heating temperature: 550 DEG C; ion source gas 1: 55psi ion source gas 2: 55psi; collision gas pressure: 7psi.

[0030] Further, the gradient elution procedure of the mobile phase A and the mobile phase B is as follows:

[0031]

[0032] The analysis method of the present application is based on specific mobile phase, reversed phase chromatographic column, chromatographic conditions and mass spectrometry conditions, and under the conditions, the peak shape of 33 components in the anti-inflammatory drug is good, effective determination can be carried out, the recovery rate is between 85% and 110%, the RSD value of the recovery rate is less than 10%, the chromatographic system is stable, the specificity is good, the sensitivity is high, the linearity is good, the precision is high, the accuracy is good, 33 components can be determined at the same time, and qualitative and quantitative determination can be carried out at the same time, the detection efficiency of 33 components in the anti-inflammatory drug is improved, and the actual sample detection can be carried out. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The chromatogram of lornoxicam and imatinib in the blank solution in example 3 of the present application;

[0034] Figure 2 The chromatogram of diflunisal, loxoprofen, nimesulide, acemetacin, calcium fenoprofen and flurbiprofen in the blank solution in example 3 of the present application;

[0035] Figure 3Chromatogram of Diclofenac Sodium, Indometacin, Etoricoxib, Ketoprofen, Diclofenac, Fenbufen in the blank solution in Example 3 of the present application;

[0036] Figure 4 Chromatogram of Acetaminophen, Codeine, Piroxicam, Meloxicam, Benorilate, Droxicam in the blank solution in Example 3 of the present application;

[0037] Figure 5 Chromatogram of Sulindac, Etoricoxib, Ketoprofen, Diclofenac, Fenbufen in the blank solution in Example 3 of the present application;

[0038] Figure 6 Chromatogram of Aclofenac, Pivoxicam, Oxaprozin, Nalidixic Acid, Capsaicin, Celecoxib, Etofenamate in the blank solution in Example 3 of the present application;

[0039] Figure 7 Chromatogram of Lornoxicam, Iralukast in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0040] Figure 8 Chromatogram of Diflunisal, Loxoprofen, Nimesulide, Acemetacin, Fenoprofen Calcium, Flurbiprofen in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0041] Figure 9 Chromatogram of Diclofenac Sodium, Indometacin, Etoricoxib, Ketoprofen, Diclofenac, Fenbufen in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0042] Figure 10 Chromatogram of Acetaminophen, Codeine, Piroxicam, Meloxicam in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0043] Figure 11 Chromatogram of Benorilate, Droxicam, Sulindac, Etoricoxib, Ketoprofen, Diclofenac, Fenbufen in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0044] Figure 12 Chromatogram of Aclofenac, Pivoxicam, Oxaprozin, Nalidixic Acid, Capsaicin, Celecoxib, Etofenamate in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present application by forty times;

[0045] Figure 13 Chromatogram of Lornoxicam, Iralukast in the blank solution in Example 3 of the present application;

[0046] Figure 14 The spectrum of diflunisal, loxoprofen, nimesulide, acemetacin, calcium fenoprofen, flurbiprofen in the blank matrix in Example 3 of the present application;

[0047] Figure 15 The spectrum of diclofenac sodium, indometacin, etodolac, ibuprofen, meclofenamate, mefenamic acid in the blank matrix in Example 3 of the present application;

[0048] Figure 16 The spectrum of acetaminophen, codeine, piroxicam, meloxicam, benorylate, rotundine in the blank matrix in Example 3 of the present application;

[0049] Figure 17 The spectrum of sulindac, etoricoxib, ketoprofen, naproxen, irisecam, fenbufen in the blank matrix in Example 3 of the present application;

[0050] Figure 18 The spectrum of aceclofenac, alclofenac, oxaprozin, nalfon, capsaicin, celecoxib, etofenamate in the blank matrix in Example 3 of the present application;

[0051] Figure 19 The spectrum of lornoxicam, iraemod in the matrix mixed standard solution in Example 2 of the present application;

[0052] Figure 20 The spectrum of diflunisal, loxoprofen, nimesulide, acemetacin, calcium fenoprofen, flurbiprofen, diclofenac sodium, indometacin in the matrix mixed standard solution in Example 2 of the present application;

[0053] Figure 21 The spectrum of etodolac, ibuprofen, meclofenamate, mefenamic acid in the matrix mixed standard solution in Example 2 of the present application;

[0054] Figure 22 The spectrum of acetaminophen, codeine, piroxicam, meloxicam, benorylate, rotundine in the matrix mixed standard solution in Example 2 of the present application;

[0055] Figure 23 The spectrum of sulindac, etoricoxib, ketoprofen, naproxen, irisecam, fenbufen in the matrix mixed standard solution in Example 2 of the present application;

[0056] Figure 24 The spectrum of aceclofenac, alclofenac, oxaprozin, nalfon, capsaicin, celecoxib, etofenamate in the matrix mixed standard solution in Example 2 of the present application;

[0057] Figure 25The spectrum of acetaminophen detected in the test sample 1 in Example 4 of the present application;

[0058] Figure 26 The spectrum of acetaminophen detected in the test sample 2 in Example 4 of the present application;

[0059] Figure 27 The spectrum of piroxicam detected in the test sample 3 in Example 4 of the present application;

[0060] Figure 28 The spectrum of diclofenac sodium detected in the test sample 3 in Example 4 of the present application. DETAILED DESCRIPTION

[0061] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the described embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of the present application.

[0062] Unless otherwise specifically stated, the relative arrangements of parts and steps, numerical expressions, and numerical values set forth in the various examples herein are not limiting of the scope of the present application.

[0063] At the same time, it should be understood that, for the convenience of description, the sizes of the various parts shown in the drawings are not drawn in accordance with the actual proportional relationship.

[0064] In addition, for the sake of brevity and clarity, descriptions of well-known structures, functions and configurations are omitted. Those of ordinary skill in the art will recognize that various changes and modifications of the examples described herein can be made without departing from the spirit and scope of the disclosure.

[0065] Techniques, methods, and devices known to those of ordinary skill in the relevant art can not be discussed in detail herein. However, where appropriate, such techniques, methods, and devices should be considered as falling within the scope of the present disclosure.

[0066] In all of the examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as a limitation. Thus, other examples of example embodiments can have different values.

[0067] Example 1

[0068] Example 1 of the present application is the screening of cosmetic sample pretreatment, mobile phase and mass spectrometry conditions.

[0069] 1. Screening of cosmetic sample pretreatment

[0070] (1) Comparison by 6 different extraction solvents: methanol; 80% methanol; 50% methanol; acetonitrile; 80% acetonitrile; 50% acetonitrile.

[0071] The results show that the extraction recovery of methanol and acetonitrile is better, reaching 80% and above. Considering that mobile phase B contains methanol solution, methanol is finally selected as the extraction reagent.

[0072] (2) Selection of ultrasonic extraction time

[0073] After determining methanol as the extraction reagent, ultrasonic extraction is selected, and extraction is performed for 10 min, 20 min, 30 min, and 40 min, respectively. The results show that the extraction recovery of 4 times is optimal at 20 min, between 90% and 110%, and ultrasonic extraction is finally determined for 20 min.

[0074] 2. Selection of mobile phase

[0075] The following conditions are used to select the mobile phase:

[0076] (1) 2 mmol / L ammonium acetate solution-acetonitrile;

[0077] (2) 2 mmol / L ammonium acetate solution-methanol;

[0078] (3) 2 mmol / L ammonium formate solution-acetonitrile;

[0079] (4) 0.01% formic acid solution-methanol;

[0080] (5) 0.01% acetic acid solution-methanol;

[0081] (6) 0.05% acetic acid solution-methanol;

[0082] (7) 0.1% acetic acid solution-methanol;

[0083] (8) 0.01% acetic acid, 2 mmol / L ammonium acetate solution-methanol;

[0084] (9) 5 mmol / L ammonium acetate solution-methanol;

[0085] (10) water-methanol;

[0086] (11) 0.01% acetic acid, 5 mmol / L ammonium acetate solution-methanol;

[0087] (12) 0.05% acetic acid, 2 mmol / L ammonium acetate solution-methanol;

[0088] (13) 2 mmol / L ammonium acetate solution-2 mmol / L ammonium acetate methanol solution.

[0089] The results show that 33 components have better peak shape and higher response when 2 mmol / L ammonium acetate solution is selected as the aqueous phase (mobile phase A) and 2 mmol / L ammonium acetate methanol solution is selected as the organic phase (mobile phase B). Therefore, 2 mmol / L ammonium acetate solution and 2 mmol / L ammonium acetate methanol solution are selected as the mobile phase, and the gradient elution program is shown in the following table:

[0090] Table 1 Gradient elution program

[0091]

[0092]

[0093] 3. Selection of mass spectrometry conditions

[0094] A needle pump is used, and the flow rate is set to 7 μL / min. The 33 target compound control solutions with a mass concentration of 50 μg / L are continuously injected into the electrospray ion source to investigate the response in positive and negative modes.

[0095] Benorilate, aceclofenac, etofenamate, naproxen, fenbufen, ketoprofen, oxaprozin, sulindac, piroxicam, meloxicam, nalfamotum, celecoxib, etoricoxib, acetaminophen, capsaicin, irbesartan, aceclofenac, rotundine, codeine, positive ion mode, aceclofenac [M+NH4] + , the rest are [M+H] + .

[0096] Diflunisal, ibuprofen, fenoprofen calcium, flurbiprofen, loxoprofen, mefenamic acid, meclofenamic acid, indomethacin, acemetacin, diclofenac sodium, lornoxicam, etodolac, nimesulide, ira, negative ion mode, [M-H] - , of which diclofenac sodium has two parent ions, and the other parent ion is chlorine isotope.

[0097] According to the parent ions determined by each component, the response stable and high intensity fragment ions are selected as daughter ions through full scan of secondary mass spectrometry, so that the qualitative detection is more reliable.

[0098] Optimize DP (de-clustering voltage) and CE (collision energy). The DP optimization range is 0-300 (V), and the ion response highest section is selected as the selected condition. The CE optimization range is 0-180 (V), and the ion pair response highest section is selected as the selected condition. By optimizing the parameters, the ions are measured under the optimal conditions, which has the corresponding strong advantages.

[0099] The final mass spectrometry conditions are as follows: using an electrospray ion source, positive and negative ion multiple reaction monitoring (MRM) mode; electrospray voltage (IS): 5500V-4500V; gas curtain pressure: 20psi; auxiliary heating temperature (TEM): 550℃; ion source gas 1: 55psi; ion source gas 2: 55psi; collision gas pressure: 7psi. The monitoring ion pairs and related parameter settings are shown in Table 2.

[0100] Table 2 Monitoring ion pairs and related parameter settings of 33 components in anti-inflammatory drugs

[0101]

[0102]

[0103]

[0104] *Recommended quantitative ion.

[0105] Example 2

[0106] A high-performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics includes the following steps:

[0107] (1) Cosmetic sample pretreatment: weigh the cosmetic sample into a stoppered colorimetric tube, add methanol, vortex mix, extract by ultrasonic, centrifuge, and then filter the supernatant to obtain the sample solution to be tested.

[0108] Among them, 0.2g of cosmetic sample was placed in a 10mL stoppered colorimetric tube, 5mL of methanol was added, and the mixture was shaken on a vortex mixer for 60s until the cosmetic sample and methanol were uniformly mixed. Methanol was added to near the scale, ultrasonic extraction was performed for 20min, and the sample was allowed to stand to room temperature. The sample was diluted to the scale with methanol, shaken well, centrifuged at 4000r / min for 5min, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was used as the sample solution to be tested.

[0109] (2) Prepare 33 anti-inflammatory drug single standard stock solutions, multiple mixed standard stock solutions, mixed standard intermediate solutions, and multiple matrix mixed standard solutions.

[0110] (21) Respectively take about 10 mg of paracetamol, lornoxicam, imuran, diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium, indometacin, etodolac, ibuprofen, mefenamic acid, piroxicam, meloxicam, benorylate, rotundine, sulindac, etoricoxib, ketoprofen, naproxen, eribuxib, fenbufen, aceclofenac, alclofenac, oxaprozin, nalfon, capsaicin, celecoxib, etofenamate, meclofenamate standard, respectively, place in a 10 mL volumetric flask, among which meloxicam, lornoxicam and imuran are dissolved with dimethylformamide and constant volume to the scale, the rest of the components are dissolved with methanol and constant volume to the scale, mix well, get each single standard stock solution of 1000 μg / mL.

[0111] Codeine control is a solution, the concentration is 100 μg / mL.

[0112] (22) Respectively take 0.01 mL of lornoxicam, etodolac, meloxicam, rotundine, eribuxib, oxaprozin, etofenamate each single standard stock solution into the same 100 mL brown volumetric flask, dissolved with methanol and constant volume to the scale, shake well, make mixed standard stock solution 1.

[0113] (23) Respectively take 0.04 mL of imuran, nimesulide, piroxicam, benorylate, sulindac, etoricoxib, alclofenac, nalfon each single standard stock solution into the same 100 mL brown volumetric flask, dissolved with methanol and constant volume to the scale, shake well, make mixed standard stock solution 2.

[0114] (24) Respectively take 0.1 mL of paracetamol, ketoprofen, fenbufen, capsaicin, celecoxib each single standard stock solution into the same 100 mL brown volumetric flask, dissolved with methanol and constant volume to the scale, shake well, make mixed standard stock solution 3.

[0115] (25) Respectively take 0.4 mL of fenoprofen calcium, diclofenac sodium, indometacin, mefenamic acid, naproxen each single standard stock solution into the same 100 mL brown volumetric flask, dissolved with methanol and constant volume to the scale, shake well, make mixed standard stock solution 4.

[0116] (26) Respectively take 0.1 mL of diflunisal, meclofenamate, aceclofenac each single standard stock solution, 0.2 mL of loxoprofen, acemetacin, ibuprofen each single standard stock solution, 1 mL of flurbiprofen single standard stock solution into the same 10 mL brown volumetric flask, dissolved with methanol and constant volume to the scale, shake well, make mixed standard stock solution 5.

[0117] (27) Pipette 1 mL of each mixed standard stock solution 1-5, 0.04 mL of codeine control solution into the same 10 mL brown volumetric flask, dissolve with methanol and dilute to the mark, shake well to prepare the mixed standard intermediate solution.

[0118] (28) Weigh 0.2 g of blank cosmetic sample in 6 portions, respectively, into 10 mL colorimetric tubes with stoppers, add appropriate amount of mixed standard intermediate solution, and process according to the sample processing procedure to obtain multiple concentration gradient matrix mixed standard solutions. The concentrations of each component are shown in the table:

[0119] Table 3 Matrix mixed standard solution concentration table of 33 components in anti-inflammatory drugs

[0120]

[0121]

[0122] (3) High performance liquid chromatography-tandem mass spectrometry determination and analysis

[0123] (31) Instruments and reagents:

[0124] Instruments: LC30-AB5500 high performance liquid chromatography-tandem mass spectrometer, USA SCIEX company; Milli-Q ultrapure water instrument, Germany Merck company; XS205 analytical balance, Mettler-Toledo Instrument Co., Ltd.; high-speed centrifuge, Thermo Fisher Scientific.

[0125] Reagents: methanol (chromatographic pure); ammonium acetate (chromatographic pure); dimethylformamide (DMF) (analytical pure); water (ultrapure water).

[0126] (32) Chromatographic conditions:

[0127] Reversed phase chromatographic column: Agilent ZORBAX Plus C 18 (2.1x100mm, 1.8μm);

[0128] Flow rate: 0.3 mL / min;

[0129] Column temperature: 40℃;

[0130] Injection volume: 1 μL;

[0131] Mobile phase A: 2 mmol / L ammonium acetate solution, mobile phase B: 2 mmol / L ammonium acetate methanol solution

[0132] Gradient elution was used, and the specific gradient elution program is shown in Table 1.

[0133] (33) Mass spectrometry conditions:

[0134] The electrospray ion source, positive and negative ion multiple reaction monitoring MRM mode; electrospray voltage: 4500V~5500V; gas curtain pressure: 20psi; auxiliary heating temperature: 550℃; ion source gas 1: 55psi ion source gas 2: 55psi; collision gas pressure: 7psi. The monitoring ion pair and related parameter settings are shown in Table 2.

[0135] (34) Linear and range

[0136] The standard curve equation of the component to be tested was obtained by linear regression with the series concentration of the component to be tested in the matrix mixed standard solution as the abscissa and the quantitative ion pair chromatographic peak area of the component to be tested in the matrix mixed standard solution as the ordinate. The r all reached above 0.99, and the results are shown in Table 4.

[0137] Table 4 Linear equation, linear range and correlation coefficient of 33 components to be tested in the matrix mixed standard solution

[0138]

[0139]

[0140] (35) Sample analysis and recording of the spectrum to obtain the concentration of the component to be tested in the sample solution.

[0141] The spectrum of the mixed standard solution obtained by diluting the mixed standard intermediate solution by 40 times is shown in Figures 7 to 12 , and the mixed standard solution is mainly used for positioning;

[0142] The spectrum of the matrix mixed standard solution is shown in Figures 19 to 24 .

[0143] (36) Qualitative determination

[0144] The sample was qualitatively determined by high performance liquid chromatography-tandem mass spectrometry. Under the same test conditions, the sample should present the chromatographic peaks of the quantitative ion pair and the qualitative ion pair. The retention time of the characteristic ion peak of the component to be tested is consistent with the corresponding retention time of the standard solution. The deviation of the relative abundance ratio of the selected monitoring ion pair of the sample from the relative abundance ratio of the monitoring ion pair of the standard solution of the same concentration is not more than the range specified in Table 5, and it can be determined that the corresponding component exists in the sample.

[0145] Table 5 Maximum allowable deviation of ion relative abundance in qualitative determination

[0146] Relative ion abundance (k) k>50％ 50％≥k>20％ 20％≥k>10％ k≤10％ Maximum allowable deviation ±20％ ±25％ ±30％ ±50％

[0147] The method is based on specific mobile phase, reversed phase chromatographic column, chromatographic conditions and mass spectrometry conditions, and peaks of 33 components in anti-inflammatory drugs are well determined under the conditions of positive and negative ion multiple reaction monitoring mode (MRM), the recovery rate is between 85% and 110%, the RSD value of the recovery rate is less than 10%, the method has good separation degree and high accuracy.

[0148] Example 3 verification analysis

[0149] (1) Specificity analysis

[0150] The blank solution, matrix blank solution, mixed standard solution and matrix mixed standard solution were injected into the high performance liquid chromatography-tandem mass spectrometer, respectively, and the chromatogram was recorded. The blank solution had no interference, and the results are shown in Figures 1 to 6 .

[0151] (2) Analysis of detection limit and quantitative limit

[0152] The sample without 33 anti-inflammatory components was taken as a blank matrix, 0.05 mL of mixed standard intermediate solution was added, and the detection limit was prepared according to the sample pretreatment method. The detection concentration was calculated according to the concentration when the signal-to-noise ratio (S / N) was 3, and the minimum quantitative concentration was calculated according to the concentration when the signal-to-noise ratio (S / N) was 10. See Table 6 for details, and the blank matrix spectrum is shown in Figures 13 to 18 .

[0153] Table 6 Detection concentration and minimum quantitative concentration of 33 anti-inflammatory components in matrix sample without anti-inflammatory components

[0154]

[0155]

[0156] (3) Recovery and precision analysis

[0157] The blank matrix sample was weighed 18 times, about 0.2 g each time, and the mixed standard intermediate solution was added appropriately. According to the sample processing method, 6 parallel samples were prepared for each level, and the sample pretreatment and determination were carried out according to the method established in this application, and the recovery rate and RSD value were calculated. See Table 7 for specific data.

[0158] Table 7 Recovery and precision of 33 anti-inflammatory components in blank matrix sample

[0159]

[0160]

[0161]

[0162]

[0163] Example 4

[0164] The commercially available cosmetic products were detected by the method of Example 2, 1 μL of each sample solution was injected into the liquid chromatograph, and the chromatogram was recorded. The peak area of the corresponding characteristic ion was measured, and the results are shown in Table 8.

[0165] Table 8 Determination of commercially available cosmetic products 1-4 by the method

[0166] Sample name Detection result Sample to be tested 1 Acetaminophen was detected, 0.66 mg / kg (see Figure 25 ) Sample to be tested 2 Acetaminophen was detected, 0.74 mg / kg (see Figure 26 ) Sample to be tested 3 Piroxicam was detected at 0.10 mg / kg (see Figure 27 ) Sample to be tested 4 Dichlofenac sodium was detected, 2500 mg / kg (see Figure 28 )

[0167] The results of the method validation show that the method described in the application has a stable chromatographic system, good specificity, high sensitivity, good linearity, high precision, and good accuracy. The method can simultaneously determine 33 components, and can simultaneously perform qualitative and quantitative determination, thereby improving the detection efficiency of the 33 components in the anti-inflammatory drugs and being suitable for the detection of actual samples.

[0168] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the scope of the technical solution of the present application.

Claims

1. A high-performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics, characterized by, The cosmetic sample to be measured is treated with methanol, and then analyzed by high performance liquid chromatography-tandem mass spectrometry; Cosmetic sample pretreatment: the cosmetic sample is weighed in a stoppered colorimetric tube, methanol is added as an extraction reagent, vortex mixing, ultrasonic extraction, centrifugation, and then the supernatant is filtered to obtain the sample solution to be measured; the vortex mixing time is 60 s; ultrasonic extraction is performed for 20 min; the centrifugal speed is 4000 r / min for 5 min; The 33 components of anti-inflammatory drugs in the cosmetic are: acetaminophen, lornoxicam, imidafenacin, diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium, indometacin, etodolac, ibuprofen, mefenamic acid, piroxicam, meloxicam, benorylate, rotundine, sulindac, etoricoxib, ketoprofen, naproxen, eribulin, fenbufen, aceclofenac, alclofenac, oxaprozin, nalfon, capsaicin, celecoxib, etofenamate, meclofenamate, codeine; The high performance liquid chromatography-tandem mass spectrometry analysis determination conditions are: The chromatographic conditions are: Column: Agilent ZORBAX Plus C 18 2.1 x 100 mm, 1.8 pm; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 1 pL; gradient elution; The mass spectrometry conditions are: An electrospray ion source is used, and the positive and negative ion multiple reaction monitoring MRM mode is used; the electrospray voltage is 4500V-5500V; the gas curtain gas pressure is 20 psi; the auxiliary heating temperature is 550 DEG C; the ion source gas 1 is 55 psi; the ion source gas 2 is 55 psi; the collision gas pressure is 7 psi; The mobile phase A is 2 mmol / L ammonium acetate solution, and the mobile phase B is 2 mmol / L ammonium acetate methanol solution; The gradient elution program of the mobile phase A and the mobile phase B is: Time min Mobile phase A % Mobile phase B % 0.00 90 10； 1.50 60 40； 6.00 10 90； 10.00 10 90； 10.10 90 10； 13.0 90 10。 2. The method according to claim 1, wherein the method is characterized by, The method comprises the following steps: Single standard stock solutions, multiple mixed standard stock solutions, mixed standard intermediate solutions and multiple matrix mixed standard solutions of 33 anti-inflammatory drugs are prepared; The standard samples of the anti-inflammatory drugs are weighed, methanol or dimethylformamide is added, and multiple single standard stock solutions are obtained; The multiple single standard stock solutions are measured and mixed, and mixed standard stock solution 1, mixed standard stock solution 2, mixed standard stock solution 3, mixed standard stock solution 4 and mixed standard stock solution 5 are obtained; The mixed standard stock solutions 1-5 and the codeine control product solution are mixed respectively, and a mixed standard intermediate solution is obtained; The blank cosmetic sample is weighed, the mixed standard intermediate solution is added, and the sample processing operation steps are followed to obtain multiple matrix mixed standard solutions; Detection and analysis: The sample solution to be measured and the matrix mixed standard solution are respectively determined by high performance liquid chromatography-tandem mass spectrometry, the series of concentrations of the components to be measured in the matrix mixed standard solution are taken as the abscissa, the quantitative ion pair chromatographic peak area of the components to be measured in the matrix mixed standard solution is taken as the ordinate, linear regression is performed, the standard curve equation of the components to be measured is obtained, and the concentration of the components to be measured in the sample solution to be measured is calculated according to the standard curve equation of the components to be measured.

3. The method according to claim 2, wherein the method is characterized by, Take about 10 mg of each of the standard substances of paracetamol, lornoxicam, ibuprofen, celecoxib, etodolac, meloxicam, rofecoxib, diclofenac sodium, indometacin, mefenamic acid, ketoprofen, nimesulide, piroxicam, etoricoxib, acemetacin, loxoprofen, naproxen, capsaicin, etofenamate, tolmetin, ibufenac, oxaprozin, lumiracoxib, and place them in 10 mL volumetric flasks. Dissolve the standard substances of meloxicam, lornoxicam and ibuprofen in dimethylformamide and dilute to the mark, and dissolve the standard substances of the other components in methanol and dilute to the mark. Mix well to obtain each single standard stock solution with a concentration of 1000 μg / mL. The control substance of codeine is a solution with a concentration of 100 μg / mL.

4. The method according to claim 3, wherein the method is characterized by, Take 0.01 mL of each of the single standard stock solutions of lornoxicam, etodolac, meloxicam, rofecoxib, ibuprofen, oxaprozin, and etofenamate, and place them in the same 100 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard stock solution 1. Take 0.04 mL of each of the single standard stock solutions of ibuprofen, etoricoxib, piroxicam, rofecoxib, lumiracoxib, and naproxen, and place them in the same 100 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard stock solution 2. Take 0.1 mL of each of the single standard stock solutions of paracetamol, ketoprofen, ibuprofen, capsaicin, and celecoxib, and place them in the same 100 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard stock solution 3. Take 0.4 mL of each of the single standard stock solutions of loxoprofen, diclofenac sodium, indometacin, mefenamic acid, and tolmetin, and place them in the same 100 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard stock solution 4. Take 0.1 mL of each of the single standard stock solutions of diflunisal and meclofenamic acid, and 0.2 mL of each of the single standard stock solutions of loxoprofen, acemetacin, and diclofenac sodium, and 1 mL of the single standard stock solution of flurbiprofen, and place them in the same 10 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard stock solution 5.

5. The method according to claim 4, wherein the 33 components of anti-inflammatory drugs are simultaneously detected by high performance liquid chromatography-tandem mass spectrometry. Take 1 mL of each of the mixed standard stock solutions 1-5, and 0.04 mL of the control substance solution of codeine, and place them in the same 10 mL brown volumetric flask. Dissolve in methanol and dilute to the mark. Shake well to obtain mixed standard intermediate solution.

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