A method for detecting pyrrolizidine alkaloids in cosmetics

Abstract

The present invention belongs to the technical field of cosmetics detection, and specifically discloses a method for detecting pyrrolizidine alkaloids in cosmetics, comprising the following steps: (1) extraction: weigh the cosmetic sample to be tested, add the extract, mix evenly, centrifuge, and take the supernatant; (2) purification: purify the supernatant with a pretreated solid phase extraction column, rinse with ultrapure water and methanol, elute with ammonia methanol solution, and then blow dry the eluent with nitrogen to obtain a sample to be re-dissolved, dissolve the sample to be re-dissolved and filter to obtain a liquid to be tested; (3) detection: detect the pyrrolizidine alkaloids in the liquid to be tested with a liquid chromatography-tandem mass spectrometer. The present invention establishes a standard method for determining pyrrolizidine alkaloids in imported and exported cosmetics for the first time, fills the gap in the detection method of banned components of such cosmetics in the "Technical Specifications for Safety of Cosmetics", and provides technical support for the supervision and control of imported and exported cosmetics to ensure consumer safety.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics detection, and particularly relates to a method for detecting pyrrolizidine alkaloids in cosmetics. Background Art

[0002] Pyrrolizidine alkaloids (PAs) are a class of naturally occurring alkaloids in plants. They are one of the most effective natural plant defense toxins known. They are used to resist insects, herbivores, etc. They are widely distributed in more than 6,000 plant species and can be transmitted through the food chain. PAs are also found in commonly used Chinese herbal medicines such as Aster, Coltsfoot, Lithospermum officinale, Chrysanthemum, and Panax notoginseng, which are included in the Chinese Pharmacopoeia and Shennong's Materia Medica. In recent years, PAs and their corresponding PAN-oxides (PANOs) have been shown to exhibit a variety of toxicological effects, including hepatotoxicity, genotoxicity, and carcinogenicity. my country's "Technical Specifications for Safety of Cosmetics" (2021 edition) stipulates that plants of the genus Senecio in the Asteraceae family are prohibited raw materials for cosmetics. The main risk source of plants of the genus Senecio in the Asteraceae family is PAs, including 10 pyrrolizidine alkaloids such as Seneciophylline, Senecioline, Monocrotaline, Senecioline, Senecioline, Licosamine, Heliotropin, Heliotropin, Senecioline, Sesameine, and Enchilidine. With the development of herbal cosmetics in my country and other countries in the world, some herbs will bring PAs into cosmetics, posing safety risks to consumers.

[0003] However, the existing detection methods of pyrrolizidine alkaloids are mostly used for the detection of pyrrolizidine alkaloids in food and plants, and the detection methods of PAs in cosmetics have not yet been reported. And the current detection standards for pyrrolizidine alkaloids are only standard methods for the detection of pyrrolizidine alkaloids in food and plants. For example, the Shenzhen Analysis and Testing Association Group Standard T / SATA035-2022 "Determination of 21 Alkaloids in Honey by Liquid Chromatography-Tandem Mass Spectrometry" detects 21 alkaloids, among which sennecine and sennecine are pyrrolizidine alkaloids; NY / T2769-2015 "Determination of 15 Alkaloids in Pasture by Liquid Chromatography-Tandem Mass Spectrometry" detects pyrrolizidine alkaloids including nephrine, sennecine, crotonine and sennecine; GH / T 1107-2015 "Determination of five bis-pyrrolidine alkaloids in honey by high performance liquid chromatography-mass spectrometry / mass spectrometry" detected five pyrrolizidine alkaloids in honey; SN / T5450-2022 "Determination of nine bis-pyrrolidine alkaloids in animal-derived foods by liquid chromatography-mass spectrometry / mass spectrometry" is applicable to the determination of nine bis-pyrrolidine alkaloids residues in honey, milk and dairy products, livestock and poultry meat (cattle, pigs, chickens) and their offal, including crotonine, kelp, kelp, kelp, lycosamine, heliotropin, heliotropin, and cyanidin. No standard method for the detection of PAs in cosmetics has been issued.

[0004] Therefore, obtaining a detection method for pyrrolizidine alkaloids in cosmetics is one of the research directions that urgently needs to be studied in the field of cosmetics testing. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting pyrrolizidine alkaloids in cosmetics. The method realizes the detection of pyrrolizidine alkaloids in various matrix cosmetics for the first time, and the method has the advantages of good separation, high sensitivity, accuracy and precision.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting pyrrolizidine alkaloids in cosmetics, comprising the following steps:

[0008] (1) Extraction: Weigh the cosmetic sample to be tested, add the extract, mix well, centrifuge, and take the supernatant;

[0009] (2) Purification: The supernatant is purified using a pretreated solid phase extraction column, eluted with ultrapure water and methanol, eluted with an ammonia methanol solution, and then the eluent is blown dry with nitrogen to obtain a sample to be reconstituted, and the sample to be reconstituted is dissolved and filtered to obtain a test solution;

[0010] (3) Detection: Detect the pyrrolizidine alkaloids in the test solution using liquid chromatography-tandem mass spectrometry.

[0011] The present invention provides a method for detecting pyrrolizidine alkaloids (seneciophylline, seneciopine, crotonine, clenepine, seneciophylline, ricosamine, heliotropin, pilocarpine, heliotropin, cynaropicrin, and echinocillin) in cosmetics. The sample is extracted with an extracting solution, purified by a solid phase extraction column, and then post-processed to obtain a test solution, which is then tested on a machine. The statistical results of the present invention show that the average recovery rate of the detection method is between 75% and 110%, and the relative standard deviation (RSD) standard deviation is less than 10%, which meets the requirements of SN / T0001-2016, and the detection limits (LODs) of 10 pyrrolizidine alkaloids, including seneciophylline, seneciopine, crotonine, clepsydra alkaloids, seneciophylline, licosamine, heliotropin, pilocarpine, heliotropin, and echinocypromine, are all 0.007 mg / kg, and the quantification limits (LOQs) are all 0.02 mg / kg.

[0012] The present invention establishes for the first time a standard method for determining pyrrolizidine alkaloids in various matrix cosmetics for import and export, filling the gap in the detection method for banned components of such cosmetics in the "Technical Specifications for Safety of Cosmetics", and provides technical support for the supervision and control of imported and exported cosmetics to ensure consumer safety.

[0013] The method provided by the present invention has good separation for the detection method of pyrrolizidine alkaloids, high sensitivity, accuracy and precision, and the pretreatment method is simple and rapid. The method can be used for risk assessment and screening monitoring of pyrrolizidine alkaloids in cosmetics, and has broad application prospects.

[0014] Furthermore, the solid phase extraction column is a mixed cationic solid phase extraction column (MCX, 3mL / 60mg).

[0015] In the present invention, when a mixed cationic solid phase extraction column (MCX, 3 mL / 60 mg) is used for purification, the recovery rates of pyrrolizidine alkaloids in cosmetics with different bases are between 75.5% and 106%, and the recovery effect is good.

[0016] Furthermore, when the cosmetic sample is a non-cream sample, the specific extraction steps of step (1) are: weigh the cosmetic sample to be tested, add 0.5% by volume of formic acid and acetonitrile, oscillate for 2 min-2.5 min to fully mix the sample and the extract, perform ultrasonic extraction for 10-20 min, centrifuge at 9000-11000 r / min for 5 min, and take the supernatant.

[0017] Furthermore, when the cosmetic sample is a cream sample, the specific extraction steps of step (1) are: weigh the cosmetic sample to be tested, add 0.5% by volume of formic acid and acetonitrile, and at the same time add 1g of sodium chloride, oscillate for 2min-2.5min to fully mix the sample and the extract, perform ultrasonic extraction for 10-20min, centrifuge at 9000-11000r / min for 5min, and take the supernatant.

[0018] When the sample is a cream sample, simply using the extracting solution may not be able to fully disperse the sample, and cream samples are easily emulsified, resulting in a decrease in the recovery rate. The dispersant has a stronger penetration ability, which will make the sample more uniform and can break the emulsion. Sodium chloride is selected to disperse the solution, and then the extraction solution is used for extraction. This method can optimize the recovery rate of 10 compounds in cream cosmetics.

[0019] Furthermore, the specific extraction steps of step (1) are as follows: weigh 1.0 g (accurate to 0.01 g) of the cosmetic sample to be tested and place it in a 50 mL centrifuge tube, add 10 mL of 0.5% by volume formic acid acetonitrile (cream samples need to add 1 g of sodium chloride), vortex for 2 min, ultrasonically extract for 20 min, centrifuge at 10000 r / min for 5 min, take the supernatant, and wait for purification.

[0020] Furthermore, the solid phase extraction column pretreated in step (2) is a solid phase extraction column activated sequentially with methanol and 0.1 mol / L hydrochloric acid.

[0021] Furthermore, in step (2), the ammoniated methanol is a mixture of aqueous ammonia and methanol in a volume ratio of 5:95.

[0022] Furthermore, in the step (2), the solution used to dissolve the sample to be reconstituted is 1 mL of a 10% methanol aqueous solution by volume.

[0023] Furthermore, the specific purification step of step (2) is: activate the solid phase extraction column with 3 mL of methanol and 3 mL of 0.1 mol / L hydrochloric acid in sequence, then pass all the supernatant through the column, then rinse with 3 mL of water and 3 mL of methanol in sequence, and then elute with 5 mL of ammonia methanol (5:95, V / V), collect the eluate, blow dry the eluate with nitrogen in a 45°C water bath, add 1 mL of 10% methanol aqueous solution to dissolve it, mix well, and pass it through a 0.22 μm filter membrane to obtain a purified liquid.

[0024] Furthermore, the detection conditions of the liquid chromatography-tandem mass spectrometer are as follows: chromatographic conditions are: chromatographic column: C18 chromatographic column (2.70 μm, 150 mm×3.0 mm); mobile phase A: 0.1% formic acid aqueous solution (containing 5 mmol / L ammonium formate), mobile phase B: acetonitrile; elution mode: gradient elution, flow rate 0.5 mL / min; injection volume: 5.0 μL; column temperature: 40°C.

[0025] Furthermore, the mass spectrometry conditions are: electrospray ion source (ESI); positive ion scanning; multiple reaction monitoring mode (MRM) mode; curtain gas pressure: 30psi; nebulizer gas pressure: 50psi; heating auxiliary gas pressure: 50psi; collision gas pressure: electrospray voltage: 5000V; ion source temperature: 500°C.

[0026] Compared with the prior art, the present invention has achieved beneficial technical effects:

[0027] The present invention establishes for the first time a standard method for determining 10 pyrrolizidine alkaloids (PAs) in imported and exported cosmetics, filling the gap in the detection method for prohibited components of such cosmetics in the "Technical Specifications for Cosmetic Safety", and provides technical support for the supervision and control of imported and exported cosmetics to ensure consumer safety. The method provided by the present invention has good separation for the detection method of pyrrolizidine alkaloids, high sensitivity, accuracy and precision, and a simple and rapid pretreatment method. The method can be used for risk assessment and screening monitoring of pyrrolizidine alkaloids in cosmetics with different matrices, and has a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1-5 The separation effect of different chromatographic columns on the separation and response of 10 pyrrolizidine alkaloids ( Figure 1 It is SHIELD RP-C18 column. Figure 2 It is a POROSHELL 120EC-C18 column. Figure 3 The column is ZORBAX SBAQ-C18. Figure 4 X-BRIDGE BEH-C18 column, Figure 5 is ELIPSE XDB-C18 chromatographic column);

[0029] Figure 6-9 The effect of different mobile phases on the separation and response of 10 pyrrolizidine alkaloids (mobile phase: Figure 6 is acetonitrile-water, Figure 7 0.1% formic acid-acetonitrile, Figure 8 5mmol / L ammonium formate solution-acetonitrile; Fig. 9 is a 5mmol / L ammonium formate solution containing 0.1% formic acid-acetonitrile);

[0030] Fig.10 The effect of different extracts on the recovery of 10 pyrrolizidine alkaloids in cosmetics;

[0031] Fig.11 The effect of different purification columns on the recovery of 10 pyrrolizidine alkaloids in cosmetics;

[0032] Fig.12 To investigate the stability of the standard stock solution of senecioide;

[0033] Fig.13 To investigate the stability of the standard intermediate solution of senecioclase;

[0034] Fig.14 To investigate the stability of the monocrotaline standard stock solution;

[0035] Fig.15 To investigate the stability of the monocrotaline standard intermediate solution;

[0036] Fig.16 To investigate the stability of monocrotaline in the matrix;

[0037] Fig.17 To investigate the stability of Senecio quinata in the matrix. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. However, the scope of protection claimed in the present invention is not limited to the following specific embodiments, and the reagents or instruments used in the detection method of the present invention can be purchased from the market.

[0039] The reagents used in the detection method are prepared as follows:

[0040] (1) 0.5% formic acid in acetonitrile: Accurately transfer 5 mL of formic acid into a 1000 mL volumetric flask, dilute to volume with acetonitrile, and shake well.

[0041] (2) 0.1 mol / L hydrochloric acid: Accurately transfer 8.33 mL of hydrochloric acid into a 1000 mL volumetric flask, dilute to the mark with water, and shake well.

[0042] (3) 5% ammonia methanol: Accurately transfer 5 mL of ammonia into a 100 mL volumetric flask, dilute to volume with methanol, and shake well.

[0043] (4) 10% methanol in water: Accurately transfer 100 mL of methanol into a 1000 mL volumetric flask, dilute to the mark with water, and shake well.

[0044] The specific implementation process of the detection method of the present invention is described as follows:

[0045] S1. Preparation of standard solution: Use 10 pyrrolizidine alkaloid (PAs) solid standards to prepare standard stock solution and mixed standard intermediate solution, then use the mixed standard intermediate solution and blank matrix cosmetics to prepare standard working solution.

[0046] (1) Preparation of standard solution

[0047] Weigh 25 mg of 10 PAs solid standards respectively into a 25 mL volumetric flask, dissolve and dilute with methanol to prepare a standard stock solution with a concentration of 1.0 mg / mL, store at -20°C away from light, and the validity period is 6 months.

[0048] (2) Preparation of mixed standard intermediate solution:

[0049] Pipette 0.25 mL of 10 PAs standard stock solutions into 25 mL volumetric flasks respectively, dilute to volume with methanol, prepare a mixed standard intermediate solution with a concentration of 10.0 μg / mL, store at -20°C away from light, and the validity period is 1 month.

[0050] (3) Preparation of standard working fluid

[0051] Accurately transfer a certain volume of standard intermediate solution and dilute it with blank matrix solution (the test solution that does not contain pyrrolizidine alkaloid cosmetics after extraction and purification) into a series of standard working solutions of concentrations of 5.0μg / L, 10.0μg / L, 20.0μg / L, 50μg / L, 100μg / L, and 200μg / L, and prepare them before use.

[0052] S2. Sample processing and testing

[0053] (1) Extraction: Weigh 1.0 g (accurate to 0.01 g) of the cosmetic sample to be tested into a 50 mL centrifuge tube, add 10 mL of 0.5% formic acid acetonitrile (cream samples require the addition of 1 g of sodium chloride), vortex for 2 min, perform ultrasonic extraction for 20 min, centrifuge at 10,000 r / min for 5 min, and collect the supernatant;

[0054] (2) Purification: First, an MCX solid phase extraction column (60 mg, 3 mL) was activated with 3 mL of methanol and 3 mL of 0.1 mol / L hydrochloric acid, respectively. Then, all the supernatant was passed through the column, rinsed with 3 mL of water and 3 mL of methanol, and then eluted with 5 mL of 5% ammonia methanol. The eluted liquid was blown to dryness with liquid nitrogen, and 1 mL of 10% methanol aqueous solution was added for re-dissolution. The mixture was vortexed for 2 min and filtered through a 0.22 μm filter to obtain the test solution.

[0055] (3) Detection: Detect the pyrrolizidine alkaloids in the test solution using liquid chromatography-tandem mass spectrometry.

[0056] Among them, the liquid chromatography conditions are:

[0057] Chromatographic column: C18 chromatographic column (2.70 μm, 150 mm×3.0 mm); mobile phase A: 0.1% formic acid aqueous solution (containing 5 mmol / L ammonium formate), mobile phase B: acetonitrile; elution mode: gradient elution, flow rate 0.5 mL / min; injection volume: 5.0 μL; column temperature: 40°C.

[0058] The gradient elution program is shown in Table 1:

[0059] Table 1 Gradient elution program

[0060] Time A(％) B(％) 0.00 90 10 2.00 90 10 7.00 5 95 9.00 5 95 10.00 90 10 13.00 90 10

[0061] Mass spectrometry conditions:

[0062] Electrospray ion source (ESI); positive ion scanning; multiple reaction monitoring mode (MRM) mode; curtain gas pressure: 30 psi; nebulizer gas pressure: 50 psi; heating auxiliary gas pressure: 50 psi; collision gas pressure: electrospray voltage: 5000 V; ion source temperature: 500°C.

[0063] The mass spectrometry parameters of the 10 pyrrolizidine alkaloids are shown in Table 2 below.

[0064] Table 2 Mass spectrometry parameters of 10 pyrrolizidine alkaloids

[0065]

[0066]

[0067] Among them, “*” is the quantitative ion.

[0068] S3. Result calculation

[0069] The result of step S2 is calculated by using the chromatographic data of the standard working solution obtained in step S2 using MSD ChemStation 02.02 chromatographic data processing software to obtain a standard curve, and then calculating the content of the pyrrolizidine alkaloid in the sample; or calculating the content of the pyrrolizidine alkaloid in the sample according to the following formula (I), but the chromatographic data of the blank experiment needs to be deducted.

[0070]

[0071] Where: X——the content of pyrrolizidine alkaloids in the cosmetic sample, mg / kg;

[0072] A——Peak area of ​​pyrrolizidine alkaloids in cosmetic sample solution;

[0073] C——the concentration of pyrrolizidine alkaloids in the standard working solution, μg / L;

[0074] V——final volume of cosmetic sample solution, L;

[0075] A S ——Peak area of ​​pyrrolizidine alkaloids in standard working solution;

[0076] m——the amount of sample represented by the final cosmetic sample liquid, g.

[0077] In this embodiment, the content in the cosmetics is quantitatively calculated by the external standard method. Compared with the internal standard method, the external standard method does not add the standard substance to the sample to be tested, but measures it separately under the same chromatographic conditions as the sample to be tested, and compares the obtained chromatographic peak area with the chromatographic peak area of ​​the tested component to obtain the concentration of the pyrrolizidine alkaloid, and then calculates the concentration of the pyrrolizidine alkaloid in the cosmetics, and then calculates the content of the pyrrolizidine alkaloid in the cosmetics.

[0078] Example 1 Optimization of pre-treatment conditions

[0079] 1. Selection of chromatographic column

[0080] In this example, the effects of five types of chromatographic columns, namely SHIELD RP-C18 (4.6×150mm, 3.5um), POROSHELL 120EC-C18 (3.0×150mm, 2.7um), ZORBAX SBAQ-C18 (4.6×100mm, 3.5um), X-BRIDGE BEH-C18 (4.6×100mm, 2.5um) and ELIPSE XDB-C18 (4.6×150mm, 3.5um), on the separation of 10 pyrrolizidine alkaloids were compared. The results showed that the multiple reaction monitoring chromatograms collected by the five chromatographic columns were as shown in FIG. Figure 1-Figure 5 .

[0081] The results showed that the POROSHELL 120EC-C18 (3.0x150mm, 2.7um) chromatographic column can maintain good peak shape and can separate from interferences. Therefore, the POROSHELL 120EC-C18 chromatographic column was selected for this detection method.

[0082] 2. Selection of mobile phase

[0083] The chromatographic column was POROSHELL 120EC-C18, the injection volume was 5.0 μL, and the column temperature was 40°C. Four different aqueous phases (water, 0.1% formic acid solution, 5 mmol / L ammonium formate solution, and 5 mmol / L ammonium formate solution containing 0.1% formic acid) were used to separate and respond to 10 pyrrolizidine alkaloids. The organic phase was acetonitrile. Chromatographic analysis was performed. The results are shown in Figure 6-9 .

[0084] Depend on Figure 6-9 It can be seen that the peak shape of pure water elution is very poor (see Figure 6 ), the addition of 0.1% formic acid can effectively improve the peak shape (see Figure 7 ), adding 5mmol / L ammonium formate can improve the separation (see Figure 8 ), 0.1% formic acid and 5mmol / L ammonium formate have better peak shape and resolution (see Fig. 9 ), therefore, this detection method finally selected 0.1% formic acid and 5mmol / L ammonium formate as the mobile phase elution.

[0085] 3. Extraction solution selection

[0086] 0.05 mL of a mixed standard solution of 10 pyrrolizidine alkaloids at a concentration of 2000 ng / mL was added to a blank cosmetic sample (face cream), and methanol, acetonitrile, water, 0.1 M hydrochloric acid aqueous solution and 0.5% formic acid acetonitrile solution were selected as extracts, respectively. After being treated by the methods (1) and (2) in step S2, a test solution was obtained, and then the test solution was detected and analyzed by liquid chromatography-tandem mass spectrometry, and the recoveries of the 10 pyrrolizidine alkaloids were calculated respectively. The specific results are shown in FIG. Fig.10 shown.

[0087] The results showed that all five extracting solutions had good extraction effects, among which 0.5% formic acid acetonitrile solution had the best extraction effect.

[0088] 4. Selection of purification columns

[0089] 0.05 mL of a mixed standard solution of 10 pyrrolizidine alkaloids at a concentration of 2000 ng / mL was added to a blank cosmetic sample (face cream), and a C18 column (200 mg, 3 mL), an NH2 column (60 mg, 3 mL), a WCX column (60 mg, 3 mL), an HLB column (60 mg, 3 mL), and an MCX column (60 mg, 3 mL) were used for purification, respectively. After being treated by the methods (1) and (2) in step S2, a test solution was obtained, and then liquid chromatography-tandem mass spectrometry was used for detection and analysis, and the recoveries of the 10 pyrrolizidine alkaloids were calculated respectively. The specific results are shown in FIG. Fig.11 shown.

[0090] The results are as follows Fig.11 As shown, the MCX column has the best purification effect and a high recovery rate of 10 pyrrolizidine alkaloids in cosmetics. Therefore, the MCX column (60 mg, 3 mL) was finally selected for purification in this detection method.

[0091] Example 2 Confirmatory analysis of the method of the present invention

[0092] 1. Influence of matrix effect

[0093] Blank samples of lipstick, cream, essential oil and toner without 10 pyrrolizidine alkaloids were selected for matrix effect (ME) investigation experiment. The standard mixed intermediate solution was diluted with blank matrix solution and 10% methanol aqueous solution to obtain matrix standard solution and solvent standard solution with concentrations of 5.0μg / L, 10.0μg / L, 20.0μg / L, 50μg / L, 100μg / L and 200μg / L. The peak area A1 of the matrix standard solution and the peak area A2 of the solvent standard solution were substituted into the calculation formula: (A1 / A2-1)×100% for calculation to obtain the matrix effect ME value. The ME value was used as the investigation index. If the ME is between -10% and 10%, it means that the matrix effect is weak and not obvious. If the ME is less than -10%, it shows a strong negative effect. If the ME is greater than 10%, it shows a strong positive effect. The K values ​​of the 10 pyrrolizidine alkaloids in different samples are shown in Table 3.

[0094] Table 3 Matrix effect K values ​​of 10 pyrrolizidine alkaloids in different samples

[0095] Compound Essential Oils cream Toner Lipstick Facial mask Lotion Thousands of miles of light -3.67 5.64 -10.2 -9.58 -8.88 3.32 Senecio philadelphica 10.0 -18.3 -1.58 -11.7 -10.6 -16.8 Monocrotaline 1.19 -12.1 -12.1 -14.3 -12.9 -9.98 Senecioclase -15.6 -22.5 8.07 -15.2 -13.3 -16.9 Heliotropin -19.1 -23.7 -6.15 -15.7 -12.7 -18.9 Licosamine -8.95 -18.8 -6.07 -9.88 -7.69 -16.8 Senecioide -12.2 -14.8 -5.44 -12.3 -11.6 -13.5 Echinopsin -16.2 -21.4 9.23 -14.3 -13.3 -18.6 Heliotropin -14.5 -33.2 -4.86 -23.0 -20.9 -22.6 Senecio sclerotium -22.2 -44.7 -47.8 -44.5 -42.3 -43.6

[0096] As shown in Table 3, different cosmetic matrices have different degrees of inhibitory effects. In particular, senecioide has obvious inhibitory effects in different matrices. Therefore, after comprehensive consideration, this detection method uniformly uses the blank matrix curve to draw the quantitative standard curve.

[0097] 2. Method performance parameter verification

[0098] 2.1. Investigation of the stability of standard solution

[0099] The standard stock solution and the standard mixed intermediate solution are stored at different temperatures and conditions, and the standard substances are quantitatively determined according to the planned cycle, and the stability of the standard substances is examined based on the quantitative values. Each time, the standard solution with a concentration of 0.1 mg / L is diluted from the standard stock solution and the standard intermediate solution and measured on the machine. Since the compounds detected in this standard belong to a large class of substances with similar chemical structures, two representative compounds, monocrotaline and senecioide, are selected to examine the stability of the standard solution.

[0100] The results show that the standard stock solution and the standard intermediate solution are most stable when stored at -20℃ away from light. Therefore, the storage conditions of the standard stock solution used in this standard are determined as follows: storage at -20℃ away from light, which can be stored for 6 months. The storage conditions of the standard intermediate solution are determined as follows: storage at -20℃ away from light, which can be stored for 1 month. Figure 12-Figure 15 shown.

[0101] 2.2 Specificity

[0102] Representative blank samples such as face cream, toner, lipstick, essential oil, facial mask and lotion were analyzed to check whether there was interference in the area where the target analyte appeared. No interference signals were found when 0.1 mg / kg of heliotropin-N-oxide and sedum-N-oxide were added to the representative blank samples, indicating that the standard method has good specificity.

[0103] 2.3 Detection Limit and Quantification Limit

[0104] 20 standard spiked samples were tested at three concentration levels: LODR-3SD, LODR, and LODR+3SD. The concentration at which 19 samples could be effectively detected was the detection limit of the method. Table 4 shows the verification data of the detection limit of the LC-MS / MS detection method for 10 pyrrolizidine alkaloids. For the LC-MS / MS detection method, the sensitivity of different models of products varies greatly. In order to facilitate the use of the standard, this standard is formulated with a higher detection limit so that low-end LC-MS / MS instruments can also reproduce the detection limit of this standard. This standard uses a larger method detection limit based on full verification and considering the toxicological data of the target substance. That is, the detection limit of the LC-MS / MS detection method for 10 pyrrolizidine alkaloids is 0.007 mg / kg.

[0105] Table 4 Detection limits and quantification limits of 10 pyrrolizidine alkaloids

[0106]

[0107]

[0108] The confirmation of the quantitative limit of the method in this standard is verified by adding certified reference materials to the samples. The concentration level corresponding to the signal-to-noise ratio (S / N) is 10 times or slightly greater than 10 times, and its accuracy and precision should meet the method and regulatory requirements, then the concentration is determined as the quantitative limit. The verification data is shown in Table 6 for the lowest concentration level. The quantitative limits of the six cosmetic matrices confirmed by this method are all 0.02 mg / kg.

[0109] 2.4 Standard curve and linear range

[0110] A certain volume of standard intermediate solution was accurately transferred and diluted with blank matrix to a series of concentrations of 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50 μg / L, 100 μg / L, and 200 μg / L. The standard working solution series of 10 pyrrolizidine alkaloids ranging from 5 μg / L to 200 μg / L were respectively determined. The peak areas of the 10 pyrrolizidine alkaloids were used as the ordinate and the corresponding mass concentrations were used as the abscissa to draw the standard curve. The results showed that the 10 pyrrolizidine alkaloids were linearly related to the corresponding peak areas in the range of 5 μg / L to 200 μg / L. The linear equation and correlation coefficient (r 2 )See Table 5.

[0111] Table 5 Linear equations and correlation coefficients of 10 pyrrolizidine alkaloids

[0112]

[0113]

[0114]

[0115] 2.5 Precision and recovery

[0116] Six kinds of cosmetics including lipstick, cream, essential oil, toner, mask and lotion that do not contain 10 kinds of pyrrolizidine alkaloids were taken as blank sample matrices, and 10 kinds of pyrrolizidine alkaloid mixed standard solutions with mass concentrations of 20.0 μg / kg, 40.0 μg / kg, mg / kg and 100 μg / kg were added, with 6 parallel samples for each concentration. The samples were processed according to the step S2 to obtain the test solution, and the solution was determined according to the liquid chromatography conditions and mass spectrometry conditions. The external standard method was used for quantification. The average spiked recoveries and relative standard deviations (RSDs) of the 10 kinds of pyrrolizidine alkaloids are shown in Table 6.

[0117] Table 6 Average spiked recoveries and precisions of 10 pyrrolizidine alkaloids in different cosmetics (n=6)

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] As shown in the table, after 6 different matrix cosmetic samples were spiked with 10 pyrrolizidine alkaloids, the recovery ranges of each compound were: lipstick 75.5%-104%, face cream 76.8%-103%, essential oil 76.0%-106%, toner 80.6%-105%, mask 80.5%-103%, lotion 79.5%-98.8%. The RSD ranges were: lipstick 1.81%-8.39%, face cream 1.57%-9.18%, essential oil 1.25%-7.40%, toner 1.77%-9.76%, mask 1.99%-8.04%, lotion 2.77%-8.18%.

[0126] By comparing and analyzing the verification results of various matrices in the laboratory, the calculated relative standard deviations were all less than 10%. The statistical results showed that the verification results of this method met the requirements of GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods" and SN / T 0001-2016 "Basic Provisions for the Preparation of Physical and Chemical Determination Methods for Exported Foods and Cosmetics".

[0127] 2.6 Analyte stability in matrix

[0128] Take appropriate amount of toner and essential oil blank sample, mix them evenly, and divide them into 7 portions. Prepare small volume of aqueous solution of monocrotaline and senecioline, and add them to blank toner and essential oil respectively. Analyze one portion immediately, store the other portions under normal sample storage conditions, and then analyze them after 1 week, 2 weeks, 4 weeks, 8 weeks, 15 weeks and 20 weeks. The results are as follows: Figure 16-17 The results showed that the remaining amount was more than 90% after 20 weeks, indicating that pyrrolizidine alkaloids were relatively stable in toner and essential oil matrices.

[0129] 2.7 Method Robustness

[0130] The robustness of the method is tested by carefully introducing some small changes to the method and checking their impact on the results. The first step in the method robustness test is to determine the variables that need to be evaluated. This standard selects monocrotaline (addition amount: 100μg / kg) as a representative compound, and examines the robustness of the method with five variables: sample weight (0.9g, 1.1g), extract volume (9mL, 11mL), ultrasonic extraction time (18min, 22min), centrifugation time (4.5min, 5.5min), and eluent volume (4.5mL, 5.5mL). The experimental design is shown in Table 7. The statistical results show that the di values ​​are all less than 2 times the standard deviation, indicating that the influence of the five factors is not significant.

[0131] Table 7 Method robustness

[0132]

[0133] The statistical results in Table 7 show that the di values ​​are all less than 2 times the standard deviation, indicating that the influence of the five factors is not significant.

[0134] Example 3 Actual sample analysis

[0135] A total of 30 cosmetic samples purchased from supermarkets and ports in Guangzhou were tested for 10 pyrrolizidine alkaloids, of which one cosmetic sample was found to contain seneciopine at a content of 0.079 mg / kg. No pyrrolizidine alkaloids were detected in other samples.

[0136] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the invention.

Claims

1. A method for detecting pyrrolizidine alkaloids in cosmetics, characterized in that: The following steps are involved: (1) Extraction: Weigh the cosmetic sample to be tested, add the extract, mix well, centrifuge, and take the supernatant; Wherein, when the cosmetic sample is a non-cream sample, the extraction steps are: weigh the cosmetic sample to be tested, add 0.5% by volume of formic acid and acetonitrile, oscillate for 2 min-2.5 min to fully mix the sample and the extract, perform ultrasonic extraction for 10-20 min, centrifuge at 9000-11000 r / min for 5 min, and take the supernatant; When the cosmetic sample is a cream sample, the extraction steps are as follows: weigh the cosmetic sample to be tested, add 0.5% by volume of formic acid acetonitrile, and simultaneously add 1 g of sodium chloride, shake for 2 min-2.5 min to fully mix the sample and the extract, perform ultrasonic extraction for 10-20 min, centrifuge at 9000-11000 r / min for 5 min, and take the supernatant; (2) Purification: purifying the supernatant with a pretreated solid phase extraction column, eluting with ultrapure water and methanol, and then eluting with an ammonia methanol solution, and then blowing the eluent dry with nitrogen to obtain a sample to be reconstituted, dissolving the sample to be reconstituted and filtering to obtain a test solution, wherein the solid phase extraction column is a mixed cationic solid phase extraction column, and the ammonia methanol is a mixture of ammonia and methanol in a volume ratio of 5:95; (3) Detection: Detecting the pyrrolizidine alkaloids in the test solution using a liquid chromatography-tandem mass spectrometer; The detection conditions of the liquid chromatography-tandem mass spectrometer are as follows: Chromatographic conditions are as follows: Chromatographic column: C18 chromatographic column, 2.70 μm, 150 mm×3.0 mm; Mobile phase A: 5 mmol / L ammonium formate solution containing 0.1% formic acid, Mobile phase B: acetonitrile; Elution mode: gradient elution, the gradient elution program is shown in the following table, flow rate 0.5 mL / min; ； Injection volume: 5.0 μL; column temperature: 40 °C; The mass spectrometry conditions were: Electrospray ion source; positive ion scanning; multiple reaction monitoring mode; curtain gas pressure: 30 psi; nebulizer gas pressure: 50 psi; heating auxiliary gas pressure: 50 psi; collision gas pressure: electrospray voltage: 5000 V; ion source temperature: 500 °C; Among them, the pyrrolizidine alkaloids are seneciopine, seneciopine, monocrotaline, seneciopine, senegal, licosamine, heliotropin, pilocarpine, heliotropin and cyanidin.

2. The method for detecting pyrrolizidine alkaloids in cosmetics according to claim 1, characterized in that: The solid phase extraction column pretreated in the step (2) is a solid phase extraction column activated sequentially with methanol and 0.1 mol / L hydrochloric acid.

3. The method for detecting pyrrolizidine alkaloids in cosmetics according to claim 1, characterized in that: In the step (2), the solution used to dissolve the sample to be reconstituted is 1 mL of a 10% methanol aqueous solution.

Images