A method for simultaneously determining 14 plant-derived stimulants in cosmetics

Abstract

The application relates to the technical field of detection, and proposes a detection method for simultaneously detecting 14 plant-derived stimulants in cosmetics, which adopts ultra-high performance liquid chromatography tandem mass spectrometry to detect 14 plant-derived stimulants in cosmetics. Through the technical scheme, the problems of simultaneously detecting 14 plant-derived stimulants, i.e. norlaudanosine, cannabin, cannabidiol, morphine, papaverine hydrochloride, narcotine, cocaine, norephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, caythine and strychnine in cosmetics are solved.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method for simultaneously determining 14 plant-derived stimulants in cosmetics. Background Technology

[0002] Plant-derived stimulants are compounds that have stimulating effects and are naturally found in plants or plant extracts, or obtained through chemical purification or synthesis. They are prohibited substances explicitly banned by the International Olympic Committee and the World Anti-Doping Agency (WADA). These substances are diverse, with typical examples including amphetamine derivatives such as ephedrine and pseudoephedrine extracted and processed from ephedra plants; caffeine found in coffee beans, tea, and chocolate; and cannabinoids found in cannabis and its homologues. Additionally, norcodonine, found in traditional Chinese medicinal herbs such as Sichuan pepper, lotus seeds, aconite, and Lindera root, is also included on the WADA's prohibited list due to its significant ability to enhance athletes' physical performance.

[0003] If plant-derived stimulants are illegally added to cosmetics and come into contact with or are absorbed by the human body, excessive intake can lead to a series of health risks: ephedrine-like substances can easily cause headaches, palpitations, and insomnia, while cannabinoid compounds are highly toxic and addictive. The illegal presence of these substances seriously threatens consumers' health and also poses a severe challenge to the safety supervision of the cosmetics industry.

[0004] To curb the illegal addition of plant-derived stimulants to cosmetics, establishing efficient and accurate detection methods is crucial. Currently, detection techniques for stimulants mainly include thin-layer chromatography (TLC), capillary electrophoresis, enzyme-linked immunosorbent assay (ELISA), gas chromatography, gas chromatography-tandem mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and GC-MS. However, existing methods have significant limitations in detecting cosmetic matrices: TLC has poor separation efficiency and cannot meet the detection requirements of complex matrices; ELISA is prone to false positives and has a single detection target, making simultaneous analysis of multiple residues impossible; gas chromatography and GC-MS are only applicable to volatile substances, and most plant-derived stimulants require derivatization, which is cumbersome and limits the detection range.

[0005] Among various detection technologies, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) has become the mainstream technology for doping detection due to its advantages of high sensitivity and high qualitative and quantitative accuracy. Sample pretreatment, as a core step in the detection process, directly determines the efficiency and reliability of the detection method. Traditional pretreatment methods, such as organic solvent extraction, solid-phase extraction, and liquid-liquid extraction, generally suffer from problems such as large amounts of organic solvents, cumbersome operation steps, and long pretreatment cycles.

[0006] In recent years, QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) purification technology has been widely used in the field of foodborne stimulant detection due to its outstanding characteristics of being rapid, simple, low-cost, efficient, stable, and safe. However, currently, there is no established method for the simultaneous detection of multiple plant-derived stimulants in cosmetic matrices, and existing technologies cannot meet the requirements for rapid screening and accurate quantification of 14 plant-derived stimulants in various dosage forms of cosmetics, including aqueous solutions, emulsions, and creams. Therefore, it is necessary to propose a detection method based on QuEChERS pretreatment and ultra-high performance liquid chromatography-tandem mass spectrometry. Summary of the Invention

[0007] This invention proposes a method for simultaneously determining 14 plant-derived stimulants in cosmetics, solving the problem in related technologies of simultaneously detecting these 14 plant-derived stimulants in cosmetics, namely norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine.

[0008] The technical solution of the present invention is as follows: This invention proposes a method for simultaneously determining 14 plant-derived stimulants in cosmetics. The method employs ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to determine these 14 plant-derived stimulants. In the UHPLC-MS / MS method, the mobile phase includes mobile phase A and mobile phase B; mobile phase A is a 0.1-1% formic acid aqueous solution; and mobile phase B is acetonitrile. The elution method in liquid chromatography is gradient elution, and the gradient elution procedure is as follows: Within 0–10 minutes, the volume of the mobile phase B increased from 3% to 10%. Within 10-12 minutes, the volume of the mobile phase B increased from 10% to 20%. Within 12-15 minutes, the volume of the mobile phase B increased from 20% to 95%. During 15-16 minutes, the mobile phase B remained at 95% constant. Within 16–16.1 min, the mobile phase B decreased from 95% to 3%; During the period of 16.1 to 18 minutes, the mobile phase B was kept constant at 3%. The 14 plant-derived stimulants are norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine.

[0009] As a further technical solution, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the chromatographic column of the liquid chromatography is a BEH C18 column.

[0010] As a further technical solution, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the liquid chromatography conditions are: column temperature of 25~40℃; flow rate of 0.2~0.4mL / min; and injection volume of 1~5μL.

[0011] As a further technical solution, the mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry test are as follows: electrospray ionization, positive ion mode; ion source temperature 300~550℃, nebulizer gas pressure 45~55psi, curtain gas pressure 20~30psi, auxiliary gas pressure 45~55psi, electrospray voltage 3500~5500V; scanning mode is multiple reaction monitoring mode.

[0012] As a further technical solution, before using ultra-high performance liquid chromatography-tandem mass spectrometry to measure 14 plant-derived stimulants in cosmetics, a pretreatment step for the cosmetics to be tested is also included: The cosmetic sample to be tested is obtained by extracting it with an extractant and then purifying it with a purifying agent. The extractant includes an acetonitrile solution containing 0.1% to 2% acetic acid by volume, preferably an acetonitrile solution containing 1% acetic acid by volume; The purifying agent includes anhydrous magnesium sulfate, and also includes at least one of PSA, C18, NH2, Florisil, and GCB.

[0013] As a further technical solution, the extraction is ultrasonic extraction.

[0014] As a further technical solution, the purifying agent is composed of C18 and anhydrous magnesium sulfate in a mass ratio of 0.5 to 2:1, preferably composed of C18 and anhydrous magnesium sulfate in a mass ratio of 0.75:1.

[0015] As a further technical solution, when the cosmetic to be tested is a cream or lotion, the process of ultrasonically extracting the cosmetic to be tested with an extractant further includes a demulsification treatment, wherein the demulsifier used in the demulsification treatment is composed of water and n-hexane in a volume ratio of 2:1.

[0016] As a further technical solution, the determination of 14 plant-derived stimulants in cosmetics using ultra-high performance liquid chromatography-tandem mass spectrometry specifically includes the following steps: S1. Preparation of positive control working solution: Using acetonitrile as solvent, prepare a mixed positive control working solution of 14 plant-derived stimulants; S2. The sample solution of the cosmetic product to be tested and the positive control working solution were tested using ultra-high performance liquid chromatography-tandem mass spectrometry to obtain test spectra and analyze and determine the types of plant-derived stimulants in the cosmetic product.

[0017] As a further technical solution, when the cosmetic is a cream or a liquid cosmetic, the limit of quantitation of the detection method is 0.03~5 ng / mL and the limit of detection is 0.01~2 ng / mL; when the cosmetic is a lotion cosmetic, the limit of quantitation of the detection method is 0.2~5 ng / mL and the limit of detection is 0.05~2 ng / mL.

[0018] The working principle and beneficial effects of this invention are as follows: This invention provides a method for simultaneously determining 14 plant-derived stimulants in cosmetics, including norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine. This method provides strong technical support for cosmetic safety supervision and has significant application value in protecting consumer health and regulating the cosmetic market. Attached Figure Description

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 The chromatograms of 14 plant-derived stimulants in a cosmetic containing plant extracts were determined using ultra-high performance liquid chromatography-tandem mass spectrometry. Figure 2 The effects of different types of extractants on extraction efficiency; Figure 3 The effect of acetonitrile solutions containing different volume fractions of acetic acid as extractants on the extraction efficiency; Figure 4 The impact of different types of purification reagents on the purification effect; Figure 5 The effect of purification agents composed of C18 and anhydrous magnesium sulfate in different mass ratios on the purification effect; Figure 6 Chromatograms of 14 plant-derived stimulants are shown below; in the figure: 1. Morphine; 2. Methylephedrine; 3. Norpseudoephedrine; 4. Cathinone; 5. Ephedrine; 6. Pseudoephedrine; 7. Norcodone; 8. Strychnine; 9. Caffeine; 10. Cocaine; 11. Noscapine; 12. Papaverine hydrochloride; 13. Cannabidiol; 14. Cannabinol; Figure 7 The liquid chromatograms of the two chromatographic peaks of ephedrine and pseudoephedrine in the experimental group are shown. Figure 8 The liquid chromatograms of the two chromatographic peaks of ephedrine and pseudoephedrine in control group 1 are shown. Figure 9 The liquid chromatograms of the two chromatographic peaks of ephedrine and pseudoephedrine in control group 2 are shown. Figure 10 To determine the chromatogram of a blank cream-type cosmetic base solution using ultra-high performance liquid chromatography-tandem mass spectrometry; Figure 11 To determine the chromatogram of a blank emulsion-type cosmetic matrix solution using ultra-high performance liquid chromatography-tandem mass spectrometry; Figure 12 This study aims to determine the chromatogram of a blank aqueous cosmetic matrix solution using ultra-high performance liquid chromatography-tandem mass spectrometry. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] In the following examples and comparative examples, the high performance liquid chromatography-tandem mass spectrometer was an ABsciex Q-trap6500. Plant-derived stimulant standards: Norcodine (ChromaDex, 00008218-326); Cannabinol (First Standard, S053695); Cannabidiol (First Standard, S029291); Morphine (First Standard, S050801); Papaverine Hydrochloride (First Standard, S050495); Noscapine (First Standard, S049929); Cocaine (First Standard, S039401); Norpseudoephedrine Hydrochloride (cerilliant, FE12021602); Ephedrine Hydrochloride (National Institutes for Food and Drug Control, 171241-201809); Pseudoephedrine Hydrochloride (National Institutes for Food and Drug Control, 171237-201510); Methylephedrine Hydrochloride (National Institutes for Food and Drug Control, 171247-201502); Cathinone (First Standard, S021887); Strychnine (First Standard, S021887); Standard, S064641); Caffeine (National Institutes for Food and Drug Control, 171215-202013); The samples of cream-type cosmetics, emulsion-type cosmetics, and liquid-type cosmetics were all provided by the Hebei Provincial Institute for Drug and Medical Device Inspection. Purifying agents: Octadecyl bonded silica gel adsorbent (C18) and N-propylethylenediamine (PSA) were purchased from Agilent Technologies, USA; Florisil, graphitized carbon (GCB), and amino bonded silica gel (NH2) were all purchased from Tianjin Bona Agilent Technologies Co., Ltd.; MgSO4 was of analytical grade. Formic acid, acetonitrile, and methanol were all of chromatographic purity.

[0023] Example 1 A method for simultaneously determining 14 plant-derived stimulants in cream-type cosmetics uses ultra-high performance liquid chromatography-tandem mass spectrometry to determine 14 plant-derived stimulants in cosmetics, including norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine. For a certain cream-type cosmetic containing plant extracts, 14 plant-derived stimulants were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. The specific steps included: S1. Pretreatment of the cosmetic sample to be tested: Weigh 0.5g of the cosmetic sample to be tested, add 2mL of water and 1mL of n-hexane for demulsification, then add the extraction agent and bring the volume to 10mL. After ultrasonic extraction for 20min, the extracted cosmetic sample is obtained. Measure 1.5mL of the extracted cosmetic sample and transfer it to a centrifuge tube containing the purification agent. Mix well and then perform purification treatment: Centrifuge at 3000r / min for 10min, and filter the supernatant through a 0.22μm microporous membrane to obtain the cosmetic sample solution to be tested. The extraction agent is an acetonitrile solution containing 1% acetic acid by volume. The purification agent consists of 150mg C18 and 200mg anhydrous magnesium sulfate. Prepare positive control working solutions: Weigh 10 mg of each plant-derived stimulant standard and dissolve them in acetonitrile to prepare standard stock solutions with a concentration of 100 μg / mL; dilute the standard stock solutions with acetonitrile to obtain mixed standard intermediate solutions; the concentration of the mixed standard intermediate solutions of norpseudoephedrine, morphine, and cathinone is 1 μg / mL, and the concentration of other components is 0.2 μg / mL; respectively, pipette 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, 1 mL, and 2.5 mL of the mixed standard intermediate solutions and dilute them to 10 mL with 1% acetic acid acetonitrile solution to obtain mixed standard working solutions, wherein the mass concentrations of norpseudoephedrine, morphine, and cathinone are 5, 10, 25, 50, 100, and 250 μg / L, respectively, and the mass concentrations of other compounds are 1, 2, 5, 10, 20, and 50 μg / L, respectively; S2. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to test the sample solution of the cosmetic product and the positive control working solution to obtain the test spectrum and analyze and determine the types of plant-derived stimulants in the cosmetic product. In the ultra-high performance liquid chromatography-tandem mass spectrometry, the chromatographic column used in the liquid chromatography was a BEH C18 column, and the chromatographic conditions were as follows: column temperature 30℃; flow rate 0.3 mL / min; injection volume 1 μL; the mobile phase of the liquid chromatography included mobile phase A and mobile phase B; mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was acetonitrile. The elution method is gradient elution, and the gradient elution procedure is as follows: Within 0–10 min, the volume of mobile phase B increased from 3% to 10%. Within 10–12 minutes, the volume of mobile phase B increased from 10% to 20%. Within 12–15 minutes, the volume of mobile phase B increased from 20% to 95%. During 15-16 minutes, the mobile phase B remained at 95% constant. Between 16 and 16.1 min, the mobile phase B decreased from 95% to 3%; From 16.1 to 18 min, the mobile phase B remained constant at 3%; In ultra-high performance liquid chromatography-tandem mass spectrometry, the mass spectrometry conditions were as follows: electrospray ionization, positive ion mode; ion source temperature 550℃, nebulizer gas pressure 55psi, curtain gas pressure 30psi, auxiliary gas pressure 55psi, electrospray voltage 4000V; scanning mode was multiple reaction monitoring mode. The qualitative and quantitative ion-pair mass spectrometry parameters for 14 plant-derived stimulants are shown in Table 1 below. Table 1. Qualitative and quantitative ion-pair mass spectrometry parameters of 14 plant-derived stimulants (Note: * indicates quantitative ions)

[0024] The detection method described in Example 1 was used to test 30 batches of cream-type cosmetics containing plant extracts. The test results showed that one batch of cosmetics contained cannabinol and cannabidiol, with contents of 0.521 μg / g and 53.318 μg / g, respectively. Figure 1 As shown.

[0025] Example 2 A method for simultaneously determining 14 plant-derived stimulants in emulsion cosmetics, using ultra-high performance liquid chromatography-tandem mass spectrometry to determine 14 plant-derived stimulants in cosmetics, including norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine; For a certain emulsion-type cosmetic containing plant extracts, 14 plant-derived stimulants were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. The specific steps included: S1. Pretreatment of the cosmetic sample to be tested: Weigh 0.5g of the cosmetic sample to be tested, add 2mL of water and 1mL of n-hexane for demulsification, then add the extraction agent and bring the volume to 10mL. After ultrasonic extraction for 20min, the extracted cosmetic sample is obtained. Measure 1.5mL of the extracted cosmetic sample and transfer it to a centrifuge tube containing the purification agent. Mix well and then perform purification treatment: Centrifuge at 3000r / min for 10min, and filter the supernatant through a 0.22μm microporous membrane to obtain the cosmetic sample solution to be tested. The extraction agent is an acetonitrile solution containing 1% acetic acid by volume. The purification agent consists of 150mg C18 and 200mg anhydrous magnesium sulfate. Prepare positive control working solutions: Weigh 10 mg of each plant-derived stimulant standard and dissolve them in acetonitrile to prepare standard stock solutions with a concentration of 100 μg / mL; dilute the standard stock solutions with acetonitrile to obtain mixed standard intermediate solutions; the concentration of the mixed standard intermediate solutions of norpseudoephedrine, morphine, and cathinone is 1 μg / mL, and the concentration of other components is 0.2 μg / mL; respectively, pipette 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, 1 mL, and 2.5 mL of the mixed standard intermediate solutions and dilute them to 10 mL with 1% acetic acid acetonitrile solution to obtain mixed standard working solutions, wherein the mass concentrations of norpseudoephedrine, morphine, and cathinone are 5, 10, 25, 50, 100, and 250 μg / L, respectively, and the mass concentrations of other compounds are 1, 2, 5, 10, 20, and 50 μg / L, respectively; S2. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to test the sample solution of the cosmetic product and the positive control working solution to obtain the test spectrum and analyze and determine the types of plant-derived stimulants in the cosmetic product. In the ultra-high performance liquid chromatography-tandem mass spectrometry, the conditions for liquid chromatography and mass spectrometry were the same as in Example 1; The detection method of Example 2 was used to test 30 batches of lotion cosmetics containing plant extracts, and no plant-derived stimulants were detected.

[0026] Example 3 A method for simultaneously determining 14 plant-derived stimulants in aqueous cosmetics, using ultra-high performance liquid chromatography-tandem mass spectrometry to determine the 14 plant-derived stimulants in cosmetics, including norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine; For a certain aqueous cosmetic containing plant extracts, ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine 14 plant-derived stimulants in the cosmetic. The specific steps included: S1. Pretreatment of the cosmetic sample to be tested: Weigh 0.5g of the cosmetic sample to be tested, add the extractant and make up to 10mL, and extract by sonication for 20min to obtain the extracted cosmetic sample; Measure 1.5mL of the extracted cosmetic sample and transfer it to a centrifuge tube containing the purification agent, mix well and then perform purification treatment: Centrifuge at 3000r / min for 10min, take the supernatant and filter it through a 0.22μm microporous membrane to obtain the cosmetic sample solution to be tested; wherein, the extractant is an acetonitrile solution containing 1% acetic acid by volume; the purification agent consists of 150mg C18 and 200mg anhydrous magnesium sulfate; Prepare positive control working solutions: Weigh 10 mg of each plant-derived stimulant standard and dissolve them in acetonitrile to prepare standard stock solutions with a concentration of 100 μg / mL; dilute the standard stock solutions with acetonitrile to obtain mixed standard intermediate solutions; the concentration of the mixed standard intermediate solutions of norpseudoephedrine, morphine, and cathinone is 1 μg / mL, and the concentration of other components is 0.2 μg / mL; respectively, pipette 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, 1 mL, and 2.5 mL of the mixed standard intermediate solutions and dilute them to 10 mL with 1% acetic acid acetonitrile solution to obtain mixed standard working solutions, wherein the mass concentrations of norpseudoephedrine, morphine, and cathinone are 5, 10, 25, 50, 100, and 250 μg / L, respectively, and the mass concentrations of other compounds are 1, 2, 5, 10, 20, and 50 μg / L, respectively; S2. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to test the sample solution of the cosmetic product and the positive control working solution to obtain the test spectrum and analyze and determine the types of plant-derived stimulants in the cosmetic product. In the ultra-high performance liquid chromatography-tandem mass spectrometry, the conditions for liquid chromatography and mass spectrometry were the same as in Example 1; The detection method of Example 3 was used to test 30 batches of aqueous cosmetics containing plant extracts, and no plant-derived stimulants were detected.

[0027] The detection methods in Examples 1-3 were optimized through the following experiments: Experiment 1: Extractant Optimization Experiment First, the types of extractants (methanol, acetonitrile, ethanol) were optimized. The extraction efficiency of the extractants was judged by comparing the peak areas of the target analytes. The results are as follows: Figure 2 As shown in the figure. The results indicate that when acetonitrile is used as the extraction reagent, the peak areas of eight substances, including strychnine, noscapine, and morphine, are relatively high, indicating good extraction efficiency. The target analytes are alkaloids; adding a certain amount of acetic acid helps improve the ionization efficiency of the analytes and also improves the peak shape.

[0028] In addition, the effect of adding different concentrations of acetic acid to the extractant on the extraction efficiency was investigated, and the results are as follows: Figure 3 As shown, within the acetic acid concentration range of 0.5% to 2%, the peak area of ​​most substances does not change much. However, for substances such as cathinone, caffeine, and norcodone, the peak area is relatively high and the response is better when the acetic acid concentration is 1%.

[0029] Experiment 2: Purifying Agent Optimization Experiment Purifying agents include purifying reagents and desiccant. This experiment first screened five purifying reagents: PSA, C18, NH2, Florisil, and GCB. The experimental results are as follows: Figure 4 As shown, when using GCB and Florisil as purification reagents, the peak areas of most substances were low, possibly due to their strong adsorption, which also interfered with the target substances. The other three purification reagents all met the requirements for removing interfering substances. When NH2 was used as the adsorbent, five target substances, including noscapine, cannabinol, and papaverine hydrochloride, had higher peak areas; when C18 was used as the purification reagent, eight target substances, including cocaine and ephedrine, had higher peak areas and better response.

[0030] Next, this experiment investigated the effect of the mass ratio of the desiccant anhydrous magnesium sulfate and the purifying reagent C18 on the purification effect. The experimental results are as follows: Figure 5 As shown, when the purifier consists of 150 mg C18 and 200 mg anhydrous magnesium sulfate, the peak area is higher and the response is better.

[0031] Experiment 3: Elution Program Selection Experiment Of the 14 plant-derived stimulants in this invention, one pair is isomers (ephedrine and pseudoephedrine), and their quantitative and qualitative ion pairs are completely identical. Therefore, a suitable elution procedure must be employed to ensure good separation of these two isomers. The chromatographic conditions for both the experimental and control groups were the same as in Example 1, with the only difference being the elution procedure, as detailed below: Experimental group: The mobile phase and elution procedure were the same as in Example 1; the liquid chromatogram is shown below. Figure 6 As shown; the liquid chromatograms of the ephedrine and pseudoephedrine peaks are shown below. Figure 7 As shown, the two chromatographic peaks of ephedrine and pseudoephedrine were completely separated.

[0032] The gradient elution program for control group 1 is as follows: Within 0–10 min, the volume of mobile phase B increased from 4% to 10%. Within 10–12 minutes, the volume of mobile phase B increased from 10% to 20%. Within 12–15 minutes, the volume of mobile phase B increased from 20% to 95%. During 15-16 minutes, the mobile phase B remained at 95% constant. Between 16 and 16.1 min, the mobile phase B decreased from 95% to 4%; From 16.1 to 18 min, the mobile phase B remained constant at 4%; The liquid chromatograms of the two peaks of ephedrine and pseudoephedrine are shown below. Figure 8 As shown, the two chromatographic peaks of ephedrine and pseudoephedrine were not completely separated.

[0033] The gradient elution program for control group 2 is as follows: Within 0–10 min, the volume of mobile phase B increased from 5% to 10%. Within 10–12 minutes, the volume of mobile phase B increased from 10% to 20%. Within 12–15 minutes, the volume of mobile phase B increased from 20% to 95%. During 15-16 minutes, the mobile phase B remained at 95% constant. Between 16 and 16.1 min, the mobile phase B decreased from 95% to 5%; From 16.1 to 18 min, the mobile phase B was kept constant at 5%; The liquid chromatograms of the two peaks of ephedrine and pseudoephedrine are shown below. Figure 9 As shown, the chromatographic peaks of ephedrine and pseudoephedrine overlap.

[0034] The detection methods of Examples 1-3 were validated as follows: Experiment 4 Specificity Verification The preparation methods for blank cosmetic base solutions commonly found in the market, such as blank creams, blank lotions, and blank liquids, are as follows: Weigh out 0.5g of blank cream cosmetics and 0.5g of blank emulsion cosmetics respectively. Add 2mL of water and 1mL of n-hexane to break the emulsion, then add acetonitrile solution containing 1% acetic acid and make up to 10mL. After ultrasonic extraction for 20min, the extracted cosmetic samples are obtained. Measure 1.5mL of the extracted cosmetic samples and transfer them to a centrifuge tube containing 150mg C18 and 200mg anhydrous magnesium sulfate. Mix well and then purify: centrifuge at 3000r / min for 10min, and filter the supernatant through a 0.22μm microporous membrane to obtain blank cream cosmetic matrix solution and blank emulsion cosmetic matrix solution respectively. Weigh 0.5g of blank aqueous cosmetic, add acetonitrile solution containing 1% acetic acid and bring the volume to 10mL. After ultrasonic extraction for 20min, the extracted cosmetic sample is obtained. Measure 1.5mL of the extracted cosmetic sample and transfer it to a centrifuge tube containing 150mg C18 and 200mg anhydrous magnesium sulfate. Mix well and then purify: centrifuge at 3000r / min for 10min, and filter the supernatant through a 0.22μm microporous membrane to obtain the blank aqueous cosmetic matrix solution. The prepared blank cream-type cosmetic matrix solution, blank emulsion-type cosmetic matrix solution, and blank aqueous cosmetic matrix solution were tested using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), respectively, under the same testing conditions as in Example 1. The test results are as follows: Figures 10-12 As shown, the results indicate that no interfering substances were present in the above three blank cosmetic matrix solutions.

[0035] Experiment 5: Linearity, Matrix Effect, and Limit of Detection and Limit of Quantitation Tests Because cosmetic matrices are complex, they can easily interfere with the analytes during detection. During detection, the matrix effect can cause ion enhancement or inhibition of the target compound. This experiment used three blank cosmetic matrix solutions prepared in Experiment 3 to prepare matrix-matched standard solutions and reagent standard solutions ranging from 1 to 250 ng / mL, and calculated the matrix effect (ME) using the following formula: ME = Slope of matrix-matched standard curve / Slope of reagent standard curve × 100%; If ME is in the range of 80% to 120%, it indicates that the matrix effect is not obvious; If ME > 120%, it indicates a significant matrix enhancement effect; If ME < 80%, it indicates a significant matrix inhibition effect; The calculation results are shown in Table 2. In all three types of cosmetic matrix, eight substances had a ME < 80%, indicating a significant matrix inhibition effect. In emulsion-type cosmetics, two target substances had a ME > 120%, indicating a significant matrix enhancement effect. To reduce the influence of the matrix effect, matrix matching curves were used for linearity assessment and content determination.

[0036] Next, 14 plant-derived stimulants were added to the three blank cosmetic matrix solutions prepared in Experiment 3. Standard curves were plotted using the peak areas of the 14 plant-derived stimulants against the mass concentration of the analyte. Based on the instrument's response, the limits of detection and quantitation were determined with S / N≥3 and S / N≥10, respectively. The results are shown in Table 2. In cream and aqueous cosmetics, norpseudoephedrine, morphine, and cathinone showed good linearity in the range of 5–250 ng / mL, while the other 11 substances showed good linearity in the range of 1–50 ng / mL. In emulsion cosmetics, norpseudoephedrine, morphine, and cathinone showed good linearity in the range of 5–250 ng / mL, while cannabinol and cannabidiol showed good linearity in the range of 2–50 ng / mL. The limits of quantitation (LOQ) for cream and aqueous products ranged from 0.03–5 ng / mL, and the limits of detection (LOD) ranged from 0.01–2 ng / mL. For emulsion products, the LQ ranged from 0.2–5 ng / mL, and the LOD ranged from 0.05–2 ng / mL.

[0037] Table 2. Linear range, linear equation, correlation coefficient, ME, limit of detection, and limit of quantitation for 14 plant-derived stimulants.

[0038] Experiment 6: Accuracy and Precision Experiment The blank cream-type cosmetic matrix solution, blank emulsion-type cosmetic matrix solution, and blank aqueous cosmetic matrix solution prepared in Experiment 3 were used to conduct a spiked recovery experiment to examine the accuracy and precision of this detection method.

[0039] The spiking concentrations of cathinone, morphine, and norpseudoephedrine were 5, 50, and 250 ng / mL, respectively. For emulsion cosmetics, the spiking concentrations of cannabinol and cannabidiol were 2, 10, and 50 ng / mL, respectively. The spiking concentrations of other substances were 1, 10, and 50 ng / mL, respectively. The results are shown in Table 3. Table 3. Spike recoveries and precision of 14 plant-derived stimulants (n=3)

[0040] The results in Table 3 show that the recovery rate of this method is 77.41%~115.26%, and the relative standard deviation (RSD) is 0.30%~11.69%, indicating that the method has good accuracy and precision at different concentration levels.

[0041] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously determining 14 plant-derived stimulants in cosmetics, characterized in that, Fourteen plant-derived stimulants in cosmetics were determined using ultra-high performance liquid chromatography-tandem mass spectrometry. In the ultra-high performance liquid chromatography-tandem mass spectrometry, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B; mobile phase A is a 0.1~1% formic acid aqueous solution; mobile phase B is acetonitrile; The elution method in liquid chromatography is gradient elution, and the gradient elution procedure is as follows: Within 0–10 minutes, the volume of the mobile phase B increased from 3% to 10%. Within 10-12 minutes, the volume of the mobile phase B increased from 10% to 20%. Within 12-15 minutes, the volume of the mobile phase B increased from 20% to 95%. During 15-16 minutes, the mobile phase B remained at 95% constant. Within 16–16.1 min, the mobile phase B decreased from 95% to 3%; During the period of 16.1 to 18 minutes, the mobile phase B was kept constant at 3%. The 14 plant-derived stimulants are norcodonine, cannabinol, cannabidiol, morphine, papaverine hydrochloride, noscapine, cocaine, norpseudoephedrine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, methylephedrine hydrochloride, caffeine, cathinone, and strychnine.

2. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 1, characterized in that, In the ultra-high performance liquid chromatography-tandem mass spectrometry, the chromatographic column used in the liquid chromatography is a BEH C18 column.

3. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 1, characterized in that, In the ultra-high performance liquid chromatography-tandem mass spectrometry, the liquid chromatography conditions are: column temperature of 25~40℃; flow rate of 0.2~0.4mL / min; and injection volume of 1~5μL.

4. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 1, characterized in that, During the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) test, the mass spectrometry conditions were as follows: electrospray ionization, positive ion mode; ion source temperature 300~550℃, nebulizer gas pressure 45~55psi, curtain gas pressure 20~30psi, auxiliary gas pressure 45~55psi, electrospray voltage 3500~5500V; and multiple reaction monitoring (MRM) mode.

5. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 1, characterized in that, Before using ultra-high performance liquid chromatography-tandem mass spectrometry to measure 14 plant-derived stimulants in cosmetics, a pretreatment step for the cosmetics to be tested is also included: The cosmetic sample solution is obtained by extracting the cosmetic with an extractant and then purifying it with a purifying agent. The extractant includes an acetonitrile solution containing 0.1% to 2% acetic acid by volume; The purifying agent includes anhydrous magnesium sulfate, and also includes at least one of PSA, C18, NH2, Florisil, and GCB.

6. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 5, characterized in that, The extraction method is ultrasonic extraction.

7. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 5, characterized in that, The purifying agent is composed of C18 and anhydrous magnesium sulfate in a mass ratio of 0.5 to 2:

1.

8. The detection method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 5, wherein when the cosmetic to be tested is a cream or lotion, the method further includes demulsification treatment before ultrasonic extraction of the cosmetic to be tested with an extractant, wherein the demulsifier used in the demulsification treatment is composed of water and n-hexane in a volume ratio of 2:

1.

9. The method for simultaneously determining 14 plant-derived stimulants in cosmetics according to claim 5, characterized in that, The determination of 14 plant-derived stimulants in cosmetics using ultra-high performance liquid chromatography-tandem mass spectrometry includes the following steps: S1. Preparation of positive control working solution: Using acetonitrile as solvent, prepare a mixed positive control working solution of 14 plant-derived stimulants; S2. The sample solution of the cosmetic product to be tested and the positive control working solution were tested using ultra-high performance liquid chromatography-tandem mass spectrometry to obtain test spectra and analyze and determine the types of plant-derived stimulants in the cosmetic product.

10. A method for simultaneously determining 14 plant-derived stimulants in cosmetics according to any one of claims 1 to 9, characterized in that, When the cosmetic is a cream or liquid cosmetic, the limit of quantitation of the detection method is 0.03~5 ng / mL and the limit of detection is 0.01~2 ng / mL; when the cosmetic is a lotion cosmetic, the limit of quantitation of the detection method is 0.2~5 ng / mL and the limit of detection is 0.05~2 ng / mL.

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