High performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics

Abstract

The invention discloses a high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics, which comprises the following steps: treating a to-be-detected cosmetic sample with methanol, and carrying out high performance liquid chromatography-tandem mass spectrometry analysis determination by adopting a mobile phase containing an ammonium acetate solution. According to the method, peak shapes of 33 components in the anti-inflammatory drug are good, effective determination can be carried out, the recovery rate ranges from 85% to 110%, the RSD value of the recovery rate is smaller than 10%, a chromatographic system is stable, specificity is good, sensitivity is high, linearity is good, precision is high, accuracy is good, the 33 components are qualitatively and quantitatively determined at the same time, and detection efficiency is high.

Description

Technical Field

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

[0002] Antipyretic, analgesic and anti-inflammatory drugs have the effects of reducing fever and relieving chronic dull pain. They can relieve pain, reduce temperature and relieve symptoms such as redness, swelling, heat and pain caused by inflammation. They are mainly used to treat colds, fever and pain. However, when used on the skin, they may cause adverse reactions such as burning sensation, redness and itching, especially for people with sensitive skin or allergic constitution. Adding these ingredients to cosmetics for a long time may cause cumulative damage such as thinning, drying and peeling of the skin stratum corneum, and may affect liver and kidney function through skin absorption.

[0003] The highest historical usage of acetaminophen in rinse-off products in the "Catalogue of Raw Materials Used in Cosmetics" (2021 Edition) (hereinafter referred to as the Catalogue) is 0.05%. It is also included in the "Information on the Use of Raw Materials of Marketed Products" (hereinafter referred to as the Information), with the application site being the hair, the method of use being rinsing, and the usage amount being 0.013333%. Leave-on products and usage amounts are not included. The "Information" clearly states that "there is no objective collection of raw material usage reports from authoritative organizations, and no systematic evaluation of the safety of the listed raw materials has been organized." The detection of acetaminophen in the children's category and the leave-on products claiming to remove acne are suspected of being used beyond the scope, and the possibility of illegal addition cannot be ruled out. The remaining 32 components currently have no unlimited values ​​and are not included in the "Catalogue" and "Information".

[0004] At present, the "Technical Specifications for Safety of Cosmetics" (2015 edition) has not yet included the inspection standards for 33 antipyretic, analgesic and anti-inflammatory drugs such as acetaminophen in cosmetics, nor has it set limits for them. Therefore, it is impossible to effectively regulate the addition of such drugs in cosmetics, and there is a blind spot in supervision.

[0005] In view of this, an analytical method for the simultaneous detection of 33 anti-inflammatory drugs in cosmetics needs to be proposed. Summary of the invention

[0006] The purpose of the present invention 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 uses a reverse phase chromatographic column for separation and then uses positive and negative mode mass spectrometry detection, adopts a specific mobile phase, performs gradient elution on the cosmetic sample to be tested, and uses mass spectrometry for qualitative and quantitative analysis to analyze whether the cosmetic sample to be tested contains one or more of the 33 anti-inflammatory drugs, thereby solving the problems existing in the prior art.

[0007] In order to solve the above technical problems, the present invention adopts the following solutions:

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

[0009] Further, the following steps are included:

[0010] Step 1, cosmetic sample pretreatment: weigh the cosmetic sample into a stoppered colorimetric tube, add methanol, vortex and oscillate, ultrasonically extract, centrifuge, and filter the supernatant to obtain the sample solution to be tested;

[0011] Step 2: Prepare single standard stock solutions, multiple mixed standard stock solutions, mixed standard intermediate solutions and multiple matrix mixed standard solutions of 33 anti-inflammatory drugs:

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

[0013] Measure a plurality of single-label standard stock solutions and mix them evenly 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 respectively;

[0014] Respectively measure the mixed standard stock solutions 1-5 and the codeine reference solution and mix them to obtain a mixed standard intermediate solution;

[0015] Weigh a blank cosmetic sample, add a mixed standard intermediate solution, and process according to the sample processing steps to obtain a plurality of matrix mixed standard solutions;

[0016] Step 3: Detection and analysis:

[0017] The sample solution to be tested and the matrix mixed standard solution are respectively measured by high performance liquid chromatography-tandem mass spectrometry, and a linear regression is performed with the series concentration of the component to be tested in the matrix mixed standard solution as the horizontal coordinate and the quantitative ion pair chromatographic peak area of ​​the component to be tested in the matrix mixed standard solution as the vertical coordinate to obtain the standard curve equation of the component to be tested, and the sample solution to be tested is calculated according to the standard curve equation of the component to be tested to obtain the concentration of the component to be tested in the sample solution to be tested.

[0018] Furthermore, in the step 1, methanol is used as the extraction solvent; the vortex oscillation time is 60s; the ultrasonic extraction is performed for 20min; and the centrifugal speed is 4000r / min for 5min.

[0019] Further, acetaminophen, lornoxicam, elamod, diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium, indomethacin, etodolac, ibuprofen, mefenamic acid, piroxicam, meloxicam, benolate, rotundine, sulindac, etoricoxib, ketoprofen, dapoxetine, etoricoxib, fenbufen, aceclofenac, guacitsal, oxaprozin, nabumetone, and nabumetone were weighed. About 10 mg of each of the standard substances of ketone, capsaicin, celecoxib, etofenamate and meclofenamic acid were placed in 10 mL volumetric flasks respectively. Menoxicam, lornoxicam and ilamod were dissolved in dimethylformamide and diluted to the scale. The remaining components were dissolved in methanol and diluted to the scale. The solutions were mixed to obtain the single standard stock solutions with a mass concentration of 1000 μg / mL. The codeine reference substance was a solution with a concentration of 100 μg / mL.

[0020] Further, 0.01 mL of each single standard stock solution of lornoxicam, etodolac, meloxicam, rotundine, ericoxib, oxaprozin, and etofenamate were measured and placed in a 100 mL brown volumetric flask, dissolved with methanol and diluted to the mark, and shaken to prepare a mixed standard stock solution 1;

[0021] Measure 0.04 mL of each single standard stock solution of iguratimod, nimesulide, piroxicam, benolate, sulindac, etoricoxib, guacitsal, and nabumetone respectively and place them in a 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 2;

[0022] Measure 0.1 mL of each single standard stock solution of acetaminophen, ketoprofen, fenbufen, capsaicin, and celecoxib respectively and place them in the same 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 3;

[0023] Separately measure 0.4 mL of each single-label standard stock solution of fenoprofen calcium, diclofenac sodium, indomethacin, mefenamic acid, and taprofen, and place them in a 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 4;

[0024] Separately measure 0.1 mL of each single-label standard stock solution of diflunisal, meclofenamic acid, and aceclofenac, 0.2 mL of each single-label standard stock solution of loxoprofen, acemetacin, and ibuprofen, and 1 mL of single-label standard stock solution of flurbiprofen, and place them in the same 10 mL brown volumetric flask, dissolve them with methanol and make up to the scale, shake well, and prepare a mixed standard stock solution 5.

[0025] Furthermore, 1 mL of each mixed standard stock solution 1-5 and 0.04 mL of codeine reference solution were measured and placed in the same 10 mL brown volumetric flask, dissolved with methanol and diluted to the mark, and shaken to prepare a mixed standard intermediate solution.

[0026] Further, in step 3, the chromatographic conditions are:

[0027] Column: Agilent ZORBAX Plus C 18 , 2.1×100mm, 1.8μm; flow rate: 0.3mL / min; column temperature: 40℃; injection volume: 1μL; mobile phase A was 2mmol / L ammonium acetate solution, mobile phase B was 2mmol / L ammonium acetate methanol solution, and gradient elution was used.

[0028] Further, in step 3, the mass spectrometry conditions are:

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

[0030] Furthermore, the gradient elution program of mobile phase A and mobile phase B is:

[0031]

[0032] The beneficial effects of the present invention are as follows: the analytical method of the present invention is based on a specific mobile phase, a reverse phase chromatographic column, chromatographic conditions and mass spectrometry conditions, and uses a positive and negative ion multiple reaction monitoring mode (MRM). Under this condition, the peak shapes of 33 components in the anti-inflammatory drug are good, and effective determination can be performed. The recovery rates are all between 85% and 110%, and the RSD values ​​of the recovery rates are all less than 10%. The chromatographic system is stable, has good specificity, high sensitivity, good linearity, high precision and good accuracy, and 33 components can be determined simultaneously, and qualitative and quantitative determination can be performed simultaneously, thereby improving the detection efficiency of the 33 components in the anti-inflammatory drug, and can be used for the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The chromatogram of lornoxicam and iguratimod in the blank solution in Example 3 of the present invention;

[0034] Figure 2 The chromatograms of diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium and flurbiprofen in the blank solution in Example 3 of the present invention are shown;

[0035] Figure 3The chromatograms of diclofenac sodium, indomethacin, etodolac, ibuprofen, meclofenamic acid and mefenamic acid in the blank solution in Example 3 of the present invention are shown;

[0036] Figure 4 The chromatograms of acetaminophen, codeine, piroxicam, meloxicam, benolate and rotundine in the blank solution in Example 3 of the present invention are shown in FIG.

[0037] Figure 5 The chromatograms of sulindac, etoricoxib, ketoprofen, taprofen, ericoxib and fenbufen in the blank solution in Example 3 of the present invention are shown in FIG.

[0038] Figure 6 The chromatograms of aceclofenac, guacitirsal, oxaprozin, nabumetone, capsaicin, celecoxib and etofenamate in the blank solution in Example 3 of the present invention are shown in FIG.

[0039] Figure 7 The spectrum of lornoxicam and iguratimod in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention by forty times;

[0040] Figure 8 The spectrum of diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium and flurbiprofen in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention forty times;

[0041] Fig. 9 The spectrum of diclofenac sodium, indomethacin, etodolac, ibuprofen, meclofenamic acid, and mefenamic acid in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention forty times;

[0042] Fig.10 The spectrum of acetaminophen, codeine, piroxicam and meloxicam in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention forty times;

[0043] Fig.11 The spectrum of benolate, rotundine, sulindac, etoricoxib, ketoprofen, dapoxetine, ericoxib and fenbufen in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention forty times;

[0044] Fig.12 The spectrum of aceclofenac, guacitsal, oxaprozin, nabumetone, capsaicin, celecoxib and etofenamate in the mixed standard solution obtained by diluting the mixed standard intermediate solution in Example 2 of the present invention forty times;

[0045] Fig.13 The spectrum of lornoxicam and iguratimod in the blank matrix in Example 3 of the present invention;

[0046] Fig.14 The spectrum of diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium and flurbiprofen in the blank matrix in Example 3 of the present invention;

[0047] Fig.15 The spectrum of diclofenac sodium, indomethacin, etodolac, ibuprofen, meclofenamic acid, and mefenamic acid in the blank matrix in Example 3 of the present invention;

[0048] Fig.16 The spectrum of acetaminophen, codeine, piroxicam, meloxicam, benolate and rotundine in the blank matrix in Example 3 of the present invention;

[0049] Fig.17 The spectrum of sulindac, etoricoxib, ketoprofen, taprofen, ericoxib and fenbufen in the blank matrix in Example 3 of the present invention;

[0050] Fig.18 The spectrum of aceclofenac, guacitirsal, oxaprozin, nabumetone, capsaicin, celecoxib and etofenamate in the blank matrix in Example 3 of the present invention;

[0051] Fig.19 The spectrum of lornoxicam and iguratimod in the matrix mixed standard solution in Example 2 of the present invention;

[0052] Fig. 20 The spectrum of diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium and indomethacin in the matrix mixed standard solution in Example 2 of the present invention;

[0053] Fig.21 The spectrum of etodolac, ibuprofen, meclofenamic acid and mefenamic acid in the matrix mixed standard solution in Example 2 of the present invention;

[0054] Fig. 22 The spectrum of acetaminophen, codeine, piroxicam, meloxicam, benolate and rotundine in the matrix mixed standard solution in Example 2 of the present invention;

[0055] Fig.23 The spectrum of sulindac, etoricoxib, ketoprofen, taprofen, ericoxib and fenbufen in the matrix mixed standard solution in Example 2 of the present invention;

[0056] Fig.24 The spectrum of aceclofenac, guacitsal, oxaprozin, nabumetone, capsaicin, celecoxib and etofenamate in the matrix mixed standard solution in Example 2 of the present invention;

[0057] Fig.25The spectrum of acetaminophen detected in the test sample 1 in Example 4 of the present invention;

[0058] Fig.26 The spectrum of acetaminophen detected in the test sample 2 in Example 4 of the present invention;

[0059] Fig. 27 The spectrum of piroxicam detected in the test sample 3 in Example 4 of the present invention;

[0060] Fig.28 This is the spectrum of diclofenac sodium detected in the test sample 3 in Example 4 of the present invention. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] The relative arrangement of components and steps, the numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

[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 according to the actual proportional relationship.

[0064] Additionally, descriptions of well-known structures, functions, and configurations may be omitted for clarity and conciseness.One of ordinary skill in the art will recognize that various changes and modifications may be made to the examples described herein without departing from the spirit and scope of the present disclosure.

[0065] Technologies, methods, and apparatus known to ordinary technicians in the relevant field may not be discussed in detail, but where appropriate, such technologies, methods, and apparatus should be considered part of the authorization specification.

[0066] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may 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) Six different extraction solvents were used for comparison: methanol; 80% methanol; 50% methanol; acetonitrile; 80% acetonitrile; and 50% acetonitrile.

[0071] The results showed that the extraction recoveries of methanol and acetonitrile were both good, reaching 80% or above. Considering that mobile phase B contained methanol solution, methanol was finally selected as the extraction reagent.

[0072] (2) Screening of ultrasonic extraction time

[0073] After methanol was determined as the extraction reagent, ultrasonic extraction was selected, and the extraction time was 10min, 20min, 30min, and 40min respectively. The results showed that the extraction recovery rate of 20min was the best among the four time periods, ranging from 90% to 110%, and finally ultrasonic extraction time was determined to be 20min.

[0074] 2. Screening of mobile phase

[0075] The following conditions were used to screen the mobile phase:

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

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

[0078] (3) 2mmol / 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) 2mmol / L ammonium acetate solution-2mmol / L ammonium acetate methanol solution.

[0089] The results show that the 33 components have better peak shapes and higher responses when 2mmol / L ammonium acetate solution is used as the aqueous phase (mobile phase A) and 2mmol / L ammonium acetate methanol solution is used as the organic phase (mobile phase B). The response values ​​and peak shapes of the 33 components can be taken into account. Therefore, 2mmol / L ammonium acetate solution and 2mmol / L ammonium acetate methanol solution are selected as the mobile phases, and the gradient elution program is shown in the following table:

[0090] Table 1 Gradient elution program

[0091]

[0092]

[0093] 3. Screening of mass spectrometry conditions

[0094] Using a needle pump with a flow rate set to 7 μL / min, 33 target compound control solutions with a mass concentration of 50 μg / L were continuously injected into the electrospray ion source to examine the responses in positive and negative modes.

[0095] Benormate, guacitsal, etofenamate, tadalafil, fenbufen, ketoprofen, oxaprozin, sulindac, piroxicam, meloxicam, nabumetone, celecoxib, etoricoxib, acetaminophen, capsaicin, ericoxib, aceclofenac, rotundine, codeine in positive ion mode, guacitsal is [M+NH4] + , the rest are [M+H] + .

[0096] The negative ion mode of diflunisal, ibuprofen, fenoprofen calcium, flurbiprofen, loxoprofen, mefenamic acid, meclofenamic acid, indomethacin, acemetacin, diclofenac sodium, lornoxicam, etodolac, nimesulide, and iguratimod are all [MH] - , among which diclofenac sodium has two parent ions, and the other parent ion is an isotope of chlorine.

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

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

[0099] The final mass spectrometry conditions were determined as follows: electrospray ion source, positive and negative ion multiple reaction monitoring MRM mode; electrospray voltage (IS): 5500V~4500V; curtain gas 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 quantitation ions.

[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 comprises the following steps:

[0107] (1) Pretreatment of cosmetic samples: Weigh the cosmetic sample into a stoppered colorimetric tube, add methanol, vortex and mix, perform ultrasonic extraction, centrifuge, and filter the supernatant to obtain the sample solution to be tested.

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

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

[0110] (21) Weigh about 10 mg of each of acetaminophen, lornoxicam, iguratimod, diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium, indomethacin, etodolac, ibuprofen, mefenamic acid, piroxicam, meloxicam, benolate, rotundine, sulindac, etoricoxib, ketoprofen, taprofen, etoricoxib, fenbufen, aceclofenac, guacitsal, oxaprozin, nabumetone, capsaicin, celecoxib, etofenamate, and meclofenamic acid standards, and place them in 10 mL volumetric flasks. Dissolve menoxicam, lornoxicam, and iguratimod in dimethylformamide and dilute to the mark. Dissolve the remaining components in methanol and dilute to the mark. Mix well to obtain a single standard stock solution with a mass concentration of 1000 μg / mL.

[0111] The codeine reference substance is a solution with a concentration of 100 μg / mL.

[0112] (22) Take 0.01 mL of each single-label standard stock solution of lornoxicam, etodolac, meloxicam, rotundine, etoricoxib, oxaprozin, and etofenamate, respectively, and place them in a 100 mL brown volumetric flask. Dissolve them in methanol and make up to the mark. Shake well to prepare a mixed standard stock solution 1.

[0113] (23) Take 0.04 mL of each single-label standard stock solution of elamod, nimesulide, piroxicam, benolate, sulindac, etoricoxib, guacitsal, and nabumetone and place them in a 100 mL brown volumetric flask, dissolve them in methanol and make up to the mark, shake well, and prepare mixed standard stock solution 2.

[0114] (24) Take 0.1 mL of each single-label standard stock solution of acetaminophen, ketoprofen, fenbufen, capsaicin, and celecoxib and place them in a 100 mL brown volumetric flask. Dissolve them in methanol and make up to the mark. Shake well to prepare mixed standard stock solution 3.

[0115] (25) Take 0.4 mL of each single-label standard stock solution of fenoprofen calcium, diclofenac sodium, indomethacin, mefenamic acid, and taprofen, respectively, and place them in a 100 mL brown volumetric flask. Dissolve them in methanol and make up to the mark. Shake well to prepare mixed standard stock solution 4.

[0116] (26) 0.1 mL of each single-label standard stock solution of diflunisal, meclofenamic acid, and aceclofenac, 0.2 mL of each single-label standard stock solution of loxoprofen, acemetacin, and ibuprofen, and 1 mL of single-label standard stock solution of flurbiprofen were measured and placed in a 10-mL brown volumetric flask, dissolved with methanol and diluted to the mark, and shaken to prepare a mixed standard stock solution 5.

[0117] (27) Take 1 mL of each mixed standard stock solution 1-5 and 0.04 mL of codeine reference solution, place them in a 10 mL brown volumetric flask, dissolve them in methanol and make up to the mark, shake well, and prepare the mixed standard intermediate solution.

[0118] (28) Weigh 6 portions of 0.2 g of blank cosmetic sample and place them in 10 mL stoppered colorimetric tubes. Add appropriate amounts of mixed standard intermediate solution to each sample and process according to the sample processing steps to obtain matrix mixed standard solutions with multiple concentration gradients. The concentrations of the components are shown in the table below:

[0119] Table 3 Concentration of matrix mixed standard solution of 33 components in anti-inflammatory drugs

[0120]

[0121]

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

[0123] (31) Instruments and reagents:

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

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

[0126] (32) Chromatographic conditions:

[0127] Reversed phase column: Agilent ZORBAX Plus C 18 (2.1×100mm, 1.8μm);

[0128] Flow rate: 0.3 mL / min;

[0129] Column temperature: 40°C;

[0130] Injection volume: 1 μL;

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

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

[0133] (33) Mass spectrometry conditions:

[0134] Electrospray ion source, positive and negative ion multiple reaction monitoring MRM mode; electrospray voltage: 4500V-5500V; curtain gas pressure: 20psi; auxiliary heating temperature: 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.

[0135] (34) Linearity and range

[0136] Linear regression was performed with the concentration series of the components to be tested in the matrix mixed standard solution as the horizontal axis and the quantitative ion pair chromatographic peak area of ​​the components to be tested in the matrix mixed standard solution as the vertical axis to obtain the standard curve equation of the components to be tested. The r values ​​all reached above 0.99. The results are shown in Table 4.

[0137] Table 4 Linear equations, linear ranges, and correlation coefficients of 33 components in the matrix mixed standard solution

[0138]

[0139]

[0140] (35) Load the sample for analysis, record the spectrum, and obtain the concentration of the component to be tested in the sample solution.

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

[0142] Matrix mixed standard solution spectrum see Figures 19 to 24 .

[0143] (36) Qualitative judgment

[0144] The samples were qualitatively determined by high performance liquid chromatography-tandem mass spectrometry. Under the same experimental conditions, the samples should present chromatographic peaks of quantitative ion pairs and qualitative ion pairs, the retention time of the characteristic ion peak of the measured component was consistent with the retention time of the standard solution, and the deviation of the relative abundance ratio of the monitoring ion pairs selected by the sample and the relative abundance ratio of the monitoring ion pairs of the standard solution of equivalent concentration did not exceed the range specified in Table 5, then the corresponding components could be determined to exist in the samples.

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

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

[0147] This method is based on a specific mobile phase, reversed-phase chromatographic column, chromatographic conditions and mass spectrometry conditions. Under the conditions, the positive and negative ion multiple reaction monitoring mode (MRM) is used. The peak shapes of 33 components in anti-inflammatory drugs are good and can be effectively determined. The recoveries are between 85% and 110%, and the RSD values ​​of the recoveries are less than 10%. The method has good separation and high accuracy.

[0148] Example 3 Confirmatory Analysis

[0149] (1) Specificity analysis

[0150] Take blank solution, matrix blank solution, mixed standard solution, and matrix mixed standard solution and inject them into HPLC-MS / MS to record the chromatogram. The blank solution has no interference. The results are shown in Figures 1 to 6 .

[0151] (2) Detection limit and quantification limit analysis

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

[0153] Table 6 Detection concentrations and minimum quantitative concentrations of 33 anti-inflammatory drug components in matrix samples without anti-inflammatory drug components

[0154]

[0155]

[0156] (3) Recovery and precision analysis

[0157] 18 blank matrix samples were measured, each about 0.2g, and appropriate amounts of mixed standard intermediate solution were added to each sample. The samples were processed according to the sample processing method as the sample recovery experiments at three concentration levels: low, medium and high. Six parallel samples were made for each level, and the sample pretreatment and determination were performed according to the method established in this application, and the recovery rate and RSD value were calculated. The specific data are shown in Table 7.

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

[0159]

[0160]

[0161]

[0162]

[0163] Example 4

[0164] The cosmetics currently sold on the market were tested using the method of Example 2. 1 μL of each sample solution to be tested was measured and injected into a liquid chromatograph. The chromatogram was recorded and the corresponding characteristic ion chromatogram peak area was measured. The test results are shown in Table 8.

[0165] Table 8: Cosmetics 1-4 sold on the market were tested using this method

[0166] Sample name Test results Sample 1 Acetaminophen was detected, 0.66 mg / kg (see Fig.25 ) Sample 2 Acetaminophen was detected, 0.74 mg / kg (see Fig.26 ) Sample 3 Piroxicam was detected, 0.10 mg / kg (see Fig. 27 ) Sample 4 Diclofenac sodium was detected, 2500mg / kg (see Fig.28 )

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

[0168] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics, characterized in that: The cosmetic sample to be tested is treated with methanol and then subjected to high performance liquid chromatography-tandem mass spectrometry analysis using a mobile phase containing ammonium acetate solution.

2. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 1, characterized in that: The following steps are involved: Step 1, cosmetic sample pretreatment: weigh the cosmetic sample into a stoppered colorimetric tube, add methanol, vortex and oscillate, ultrasonically extract, centrifuge, and filter the supernatant to obtain the sample solution to be tested; Step 2: Prepare single standard stock solutions, multiple mixed standard stock solutions, mixed standard intermediate solutions and multiple matrix mixed standard solutions of 33 anti-inflammatory drugs: Weigh each anti-inflammatory drug standard, add methanol or dimethylformamide, and obtain multiple single-label standard stock solutions; Measure a plurality of single-label standard stock solutions and mix them evenly 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 respectively; Respectively measure the mixed standard stock solutions 1-5 and the codeine reference solution and mix them to obtain a mixed standard intermediate solution; Weigh a blank cosmetic sample, add a mixed standard intermediate solution, and process according to the sample processing steps to obtain a plurality of matrix mixed standard solutions; Step 3: Detection and analysis: The sample solution to be tested and the matrix mixed standard solution are respectively measured by high performance liquid chromatography-tandem mass spectrometry, and a linear regression is performed with the series concentration of the component to be tested in the matrix mixed standard solution as the horizontal coordinate and the quantitative ion pair chromatographic peak area of ​​the component to be tested in the matrix mixed standard solution as the vertical coordinate to obtain the standard curve equation of the component to be tested, and the sample solution to be tested is calculated according to the standard curve equation of the component to be tested to obtain the concentration of the component to be tested in the sample solution to be tested.

3. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 2, characterized in that: In the step 1, methanol is used as the extraction solvent; the vortex oscillation time is 60 seconds; the ultrasonic extraction is performed for 20 minutes; and the centrifugal speed is 4000 r / min for 5 minutes.

4. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 2, characterized in that: Weigh acetaminophen, lornoxicam, elamod, diflunisal, loxoprofen, nimesulide, acemetacin, fenoprofen calcium, flurbiprofen, diclofenac sodium, indomethacin, etodolac, ibuprofen, mefenamic acid, piroxicam, meloxicam, benolate, rotundine, sulindac, etoricoxib, ketoprofen, dapoxetine, etoricoxib, fenbufen, aceclofenac, guacitsal, oxaprozin, nabumetone, and spicy About 10 mg of each standard substance of piperine, celecoxib, etofenamate and meclofenamic acid were placed in 10 mL volumetric flasks respectively, among which menoxicam, lornoxicam and ilamod were dissolved in dimethylformamide and diluted to the scale, and the remaining components were dissolved in methanol and diluted to the scale, mixed well, and the mass concentration of each single standard stock solution was 1000 μg / mL; the codeine reference substance was a solution with a concentration of 100 μg / mL.

5. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 4, characterized in that: Measure 0.01 mL of each single standard stock solution of lornoxicam, etodolac, meloxicam, rotundine, etofenamate, oxaprozin, and etofenamate respectively, place in a 100 mL brown volumetric flask, dissolve with methanol and dilute to the mark, shake well, and prepare mixed standard stock solution 1; Measure 0.04 mL of each single standard stock solution of iguratimod, nimesulide, piroxicam, benolate, sulindac, etoricoxib, guacitsal, and nabumetone respectively and place them in a 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 2; Measure 0.1 mL of each single standard stock solution of acetaminophen, ketoprofen, fenbufen, capsaicin, and celecoxib respectively and place them in the same 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 3; Separately measure 0.4 mL of each single-label standard stock solution of fenoprofen calcium, diclofenac sodium, indomethacin, mefenamic acid, and taprofen, and place them in a 100 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare mixed standard stock solution 4; Separately measure 0.1 mL of each single-label standard stock solution of diflunisal, meclofenamic acid, and aceclofenac, 0.2 mL of each single-label standard stock solution of loxoprofen, acemetacin, and ibuprofen, and 1 mL of single-label standard stock solution of flurbiprofen, and place them in the same 10 mL brown volumetric flask, dissolve them with methanol and make up to the scale, shake well, and prepare a mixed standard stock solution 5.

6. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 5, characterized in that: Measure 1 mL of each mixed standard stock solution 1-5 and 0.04 mL of codeine reference solution, place them in the same 10 mL brown volumetric flask, dissolve them with methanol and make up to the mark, shake well, and prepare the mixed standard intermediate solution.

7. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 5, characterized in that: In step 3, the chromatographic conditions are: Column: Agilent ZORBAX Plus C 18 , 2.1×100mm, 1.8μm; flow rate: 0.3mL / min; column temperature: 40℃; injection volume: 1μL; gradient elution was used.

8. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 7, characterized in that: Mobile phase A was 2 mmol / L ammonium acetate solution, and mobile phase B was 2 mmol / L ammonium acetate methanol solution.

9. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 7, characterized in that: In step 3, the mass spectrometry conditions are: An electrospray ion source was used, and the positive and negative ion multiple reaction monitoring (MRM) mode was used; electrospray voltage: 4500V-5500V; curtain gas pressure: 20psi; auxiliary heating temperature: 550°C; ion source gas 1: 55psi ion source gas 2: 55psi; collision gas pressure: 7psi.

10. The high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously detecting 33 components of anti-inflammatory drugs in cosmetics according to claim 7, characterized in that: The gradient elution program of the mobile phase A and the mobile phase B is:

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