Method for non-targeting screening of hydroxyl esterification-derived glucocorticoids in cosmetics by using low-resolution mass spectrometry

Abstract

The invention discloses a method for non-targeting screening of hydroxyl esterification-derived glucocorticoid in cosmetics by using a low-resolution mass spectrum, which specifically comprises the following steps: adding sodium hydroxide with the concentration of 10 mg / mL into an acetonitrile solvent system under an alkaline condition, and adding a quantitative stabilizer consisting of EDTA (Ethylene Diamine Tetraacetic Acid) and sodium ascorbate into a reaction system, the method comprises the following steps: controlling the reaction time at room temperature to be 1 hour, adding an acetonitrile aqueous solution containing acetic acid to adjust the reaction system to be in an acidic environment and metering the volume, performing hydrolysis deesterification on glucocorticoids derived from different esterification of hydroxyl groups at 21 site and (or) 17 site of a D ring, returning to the state before esterification and derivation, and enabling the product to be stable; furthermore, only one kind of hydroxyl esterification-free glucocorticoid is adopted as a standard substance, and the hydroxyl esterification-derived glucocorticoid in cosmetics can be screened in a non-targeted manner through high performance liquid chromatography-tandem triple quadrupole mass spectrometry.

Description

Technical Field

[0001] The present invention relates to a method for detecting glucocorticoids in cosmetics, in particular to a method for non-targeted screening of glucocorticoids derived from hydroxyl esterification in cosmetics using low-resolution mass spectrometry. Background Art

[0002] Glucocorticoids can reduce the permeability of skin capillaries, reduce exudation and cell infiltration, and have anti-inflammatory, anti-allergic, immunosuppressive, and anti-proliferative effects. They are widely used clinically and are commonly used drugs for the treatment of skin diseases such as eczema and atopic dermatitis. Adding glucocorticoids to cosmetics for a short period of time can make the skin appear smooth and fair. However, when glucocorticoids are used for more than four weeks, a skin dependency reaction will occur, and the skin will become very sensitive. Once discontinued, it will become dehydrated, flaky, and capillaries exposed. It may even lead to pigmentation and an increased rate of fungal infection, which can lead to secondary inflammatory skin diseases. This "dependence" requires at least six months to treat. The addition of glucocorticoids remains one of the most commonly found illegal practices in the current cosmetics regulatory field, and it poses a serious threat to consumer health.

[0003] The "Safety Technical Specifications for Cosmetics" (2015 edition) lists glucocorticoids as prohibited ingredients in cosmetics. In 2019, the National Medical Products Administration (NMPA) incorporated the "Test Method for Hormonal Ingredients in Cosmetics," which utilizes high-performance liquid chromatography-mass spectrometry, into the "Safety Technical Specifications for Cosmetics (2015 edition)." This includes methods for detecting 50 glucocorticoids. Other standards include GB / T 24800.2-2009, "Determination of 41 Glucocorticoids in Cosmetics - Liquid Chromatography / Tandem Mass Spectrometry and Thin-Layer Chromatography," and GB / T 40145-2021, "Determination of Eleven Glucocorticoids, Including Desonide, in Cosmetics - Liquid Chromatography / Tandem Mass Spectrometry." Existing standards and literature describe methods for detecting glucocorticoids, including thin-layer chromatography, liquid chromatography, liquid chromatography-tandem mass spectrometry (LC-MS / MS), and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Sample pretreatment typically utilizes solvent extraction, solid-phase extraction, liquid-liquid extraction, and QuEChERs cleanup techniques.

[0004] Most of the glucocorticoid detection methods reported in existing literature use liquid chromatography-tandem mass spectrometry for targeted detection, using the retention time, quantitative ions, and qualitative ions that are consistent with known standard substances as the qualitative basis. This has problems such as low detection efficiency. The development of non-targeted detection technology for glucocorticoids is a technology urgently needed for the supervision of the cosmetics industry. Currently, non-targeted screening technology is mainly used in the food and drug fields, and is also used in the cosmetics field. However, non-targeted screening is usually highly associated with high-resolution mass spectrometry. Its principle is based on high-resolution mass spectrometry technology, using the accurate mass number, isotope pattern, fragment ion and other information it provides to infer the molecular formula and structural formula of the compound. However, high-resolution mass spectrometry is expensive and requires inspectors to have high mass spectrometry analysis capabilities or spectrum analysis software, making existing non-targeted screening not only complex to operate but also costly.

[0005] The basic structure of glucocorticoids is a 21-carbon cyclopentaphenanthroline nucleus, consisting of three six-membered rings (A, B, and C) and a five-membered ring (D). The hydroxyl groups at positions 21 and 17 on the D ring are easily esterified, resulting in different glucocorticoids using different ester bonds. Esterification of the hydroxyl group at position 21 is the most common, but simultaneous esterification of the hydroxyl groups at positions 17 and 21 can also occur. Glucocorticoids with esterified hydroxyl groups can be monoesters such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, tert-pentyl, and hexyl esters; or diesters such as dimethyl, diethyl, dipropyl, methylethyl, methylpropyl, ethylpropyl, and propylbutyl. Theoretically, using a glucocorticoid with hydroxyl groups at positions 21 and 17 as the basic structure, over 30 new glucocorticoids can be generated through esterification. For example, dexamethasone acetate, dexamethasone valerate, and dexamethasone isonicotinate are derived from dexamethasone; prednisolone hemisuccinate, prednisolone acetate valerate, prednisolone acetate, prednisolone butyrate, and prednisolone valerate are derived from prednisolone; and hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone succinate, hydrocortisone cypionate, and hydrocortisone valerate are derived from hydrocortisone. Known glucocorticoids have been modified by esterification of hydroxyl groups to become novel glucocorticoids with glucocorticoid functions. Over 100 known glucocorticoids have been reported on the market, of which over 50 are derived from esterification of hydroxyl groups at positions 21 and 17, accounting for nearly half of all existing glucocorticoids. Because hormone structure modification is relatively easy, the variety of glucocorticoids is expected to continue to increase.

[0006] Currently, there are relevant literature reports on the hydrolysis and deesterification of glucocorticoids under alkaline conditions: In her graduate thesis "Research on a New Process for the Synthesis of Dexamethasone", Zhu Cuihong determined that the optimal reaction conditions for the hydrolysis of dexamethasone acetate to produce dexamethasone are: the alkaline reagent is potassium carbonate solution, the optimal reaction temperature is 5-10°C, and the reaction time is 1 hour; at the same time, it was concluded that under the conditions of the same concentration of sodium hydroxide and potassium hydroxide, the reaction yield and purity of dexamethasone are low. It is speculated that the possible reason is that the alkalinity is too strong, resulting in more by-products; and the alkalinity of sodium carbonate is too weak, which will also cause incomplete hydrolysis of dexamethasone acetate. In "Qualitative and Quantitative Analysis of Betamethasone Butapyr in Cosmetics", Hao Bin et al. used 0.1 mol / L sodium hydroxide to hydrolyze betamethasone butapyr into betamethasone, assisted by QTOF high-resolution mass spectrometry. By comparing the precise molecular weights of the parent ion and daughter ion and analyzing the fragmentation patterns, they determined that betamethasone butapyr was added to the sample. This method is essentially a non-targeted detection by high-resolution mass spectrometry, and it is necessary to determine the exact chemical name of the added hormone to determine the illegal fact.

[0007] Experiments have confirmed that different hydroxy-esterified glucocorticoids have different optimal hydrolysis conditions and product stability after hydrolysis due to the differences in the corresponding hydroxy-free glucocorticoids and the derived ester bonds. Some can be hydrolyzed under potassium carbonate conditions with stable products, while others cannot be hydrolyzed at all because the alkalinity under potassium carbonate is too weak. Some can be hydrolyzed under low concentrations of sodium hydroxide and the products are stable, but even under low concentrations of sodium hydroxide, the products are unstable. Some can only be hydrolyzed under high concentrations of sodium hydroxide, but the hydrolysis products are very unstable, resulting in the inability to detect the presence of non-esterified glucocorticoids. This may be because under strong alkaline conditions, the hydroxy-free glucocorticoids generated by hydrolysis are prone to further oxidative degradation or structural rearrangement isomerization, resulting in non-unique hydrolysis products, and even the hydroxy-free glucocorticoids are completely isomerized or completely oxidatively hydrolyzed, making them undetectable. For unknown samples that need to be detected, the ester bond hydrolysis reaction conditions mentioned in the literature have limitations, that is, they lack universality, making non-targeted screening of actual samples impossible to achieve. Therefore, existing detection methods can only use glucocorticoids derived from hydroxyl esterification as standards and detect known hormones through targeted screening corresponding to the standards one by one. Not only are there problems such as high cost of using standards and low screening efficiency, but more importantly, with the continuous emergence of new hormones, targeted detection still has a huge risk of missed detection, which is far from meeting regulatory needs and poses great challenges to supervision.

[0008] To date, there have been no reports on the non-targeted screening of glucocorticoids in cosmetics using high-performance liquid chromatography-tandem low-resolution triple quadrupole mass spectrometry (HPLC-QQQ-MS). Summary of the Invention

[0009] The present invention aims to solve the above-mentioned technical problems existing in the prior art and provides a method for non-targeted screening of hydroxy-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry.

[0010] The technical solution of the present invention is a method for non-targeted screening of hydroxyl-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry. First, a cosmetic sample solution is prepared; a stabilizer and a sodium hydroxide aqueous solution with a concentration of 10 mg / mL are added to the cosmetic sample solution in sequence, and the reaction is carried out at room temperature for 1 hour. Then, an acetonitrile aqueous solution containing acetic acid is added to adjust the reaction system to an acidic environment and constant volume, so that the hydroxyl-esterified glucocorticoid is directionally hydrolyzed into a hydroxyl-free glucocorticoid and is stably present. The filtrate is taken as the sample solution to be tested, and the acetonitrile aqueous solution containing acetic acid is prepared by adding 4% by volume of acetic acid to an acetonitrile aqueous solution with a volume ratio of 1:1; the volume ratio of the cosmetic sample solution, the sodium hydroxide aqueous solution, and the stabilizer is 4.5:2:1, and the stabilizer is an aqueous solution of EDTA and sodium ascorbate, wherein the concentration of EDTA is 1 mg / mL and the concentration of sodium ascorbate is 70 mg / mL, the volume of water in the acetonitrile aqueous solution containing 4% by volume acetic acid is 50%; a standard solution is prepared using a hydroxyl-free esterified glucocorticoid, the standard solution and the sample solution to be tested are respectively subjected to high performance liquid chromatography tandem triple quadrupole mass spectrometry analysis, the target compounds are separated by high performance liquid chromatography gradient elution, and the triple quadrupole mass spectrometry analysis is used for non-targeted qualitative screening of glucocorticoids in the sample solution to be tested.

[0011] The preferred preparation of the cosmetic sample solution is to take 0.25 g of cosmetics, add 5 mL of acetonitrile containing 0.2% acetic acid by volume and 4 mL of saturated sodium chloride solution containing 0.2% acetic acid by volume to form a two-phase extraction system, perform dispersed extraction, take 4 mL of the upper acetonitrile layer, first add 0.25 mL each of 10% potassium ferrocyanide aqueous solution and 20% zinc acetate aqueous solution, and shake by vortex or oscillation.

[0012] Preferably, the triple quadrupole mass spectrometry analysis for non-targeted qualitative screening of glucocorticoids in the test sample solution is to compare the MRM spectrum of the test sample solution with the MRM spectrum of the standard solution. If the extracted ions consistent with the two pairs of qualitative and quantitative ion pairs of the standard appear in the MRM spectrum of the test sample solution and the chromatographic peaks with the retention time consistent with the retention time of the standard appear, and the relative abundance ratio of the two pairs of ions does not exceed the maximum deviation from the relative abundance ratio of the standard solution of equivalent concentration, it can be determined that the cosmetics contain glucocorticoids consistent with the standard and / or glucocorticoids derived from the esterification of the hydroxyl groups of the standard.

[0013] The relative abundance ratio of the two pairs of ions preferably does not exceed the maximum deviation from the relative abundance ratio of the standard solution of equivalent concentration as shown below: .

[0014] The present invention comprises the following technical solutions: adding a specific concentration of sodium hydroxide to an acetonitrile extraction solution to adjust the reaction system to an alkaline environment, so as to directionally hydrolyze a hydroxyl-esterified glucocorticoid into a hydroxyl-free glucocorticoid; adding a certain amount of a stabilizer composed of EDTA and sodium ascorbate to the reaction system, terminating the alkaline environment by adding acid, and controlling the reaction temperature to room temperature for 1 hour. Thus, the glucocorticoids derived from different esterifications at the 21st and (or) 17th positions of the D ring can be hydrolyzed and deesterified, thereby returning to the state before derivatization and achieving stability of the product. The conversion rate of the final product reaches 80%. % or more, overcoming the defects of the existing hydrolysis technology such as the lack of universality, and then using only one hydroxyl-free esterified glucocorticoid as a standard, by high performance liquid chromatography-tandem triple quadrupole mass spectrometry, it can be determined whether the cosmetics have added this glucocorticoid and / or any ester derivative of this glucocorticoid derived from hydroxyl esterification on the basis of this hormone, including the methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, amyl ester, tert-amyl ester, hexyl ester, heptyl ester, octyl ester and other monoester derivatives of this hormone, as well as the dimethyl ester, diethyl ester, dipropyl ester, methyl ethyl ester, ethyl propyl ester, methyl propyl ester and other diester derivatives of this hormone. The present invention ensures the universality of the glucocorticoid deesterification reaction, and can detect more than one hundred or even hundreds of new glucocorticoids in a non-targeted manner, greatly improving the detection range of glucocorticoid-positive samples, reducing the missed detection rate, and greatly saving hundreds or even hundreds of expensive standard products. The present invention utilizes a relatively inexpensive liquid chromatography-tandem low-resolution triple quadrupole mass spectrometer. Simply by comparing the MRM spectra of a sample solution hydrolyzed under specific alkaline conditions with the MRM spectra of a limited number of hydroxyl-free, non-esterified glucocorticoid standard solutions, non-targeted screening can be performed for hundreds of novel glucocorticoids derived from these glucocorticoids through esterification. This provides an ultimate solution for combating illegal additives that evade regulation by generating novel glucocorticoids through esterification. Because it eliminates the need for expensive high-resolution mass spectrometry to obtain the precise molecular weight of the target, and does not require the inspector to possess advanced mass spectrometry analysis capabilities or spectrum analysis software, the method offers advantages such as simple operation, low cost, high efficiency, wide applicability, and ease of application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the MRM spectrum obtained in Example 1 of the present invention.

[0016] Figure 2 This is the MRM spectrum obtained in Example 2 of the present invention.

[0017] Figure 3 This is the MRM spectrum obtained in Example 3 of the present invention. DETAILED DESCRIPTION Example 1

[0018] The present invention provides a method for non-targeted screening of glucocorticoids derived from esterified hydroxyl groups in cosmetics using low-resolution mass spectrometry, which is carried out according to the following steps: (1) Preparation of standard solution: ① Accurately weigh 10 mg of dexamethasone standard into a 10 mL brown volumetric flask, dilute to the mark with methanol, and prepare a 1.0 mg / mL glucocorticoid standard stock solution. Store frozen. ②Pipette the standard stock solution of glucocorticoids into a volumetric flask and dilute to the mark with acetonitrile to prepare a mixed standard intermediate solution with a concentration of 1 μg / mL; ③ Pipette the mixed standard intermediate solution into a volumetric flask and dilute to the mark with 50% acetonitrile water containing 0.2% acetic acid to prepare standard solutions with concentrations of 2.5, 10, 20, 40, 60, and 100 ng / mL, respectively. The 0.2% acetic acid and 50% acetonitrile water are expressed in volume percentages. (2) Preparation of cosmetic sample solution ①Extraction of cosmetic sample solution Accurately weigh 0.25 g (accurate to 0.001 g) of each of the four non-essential oil cosmetic samples into four 10 mL graduated centrifuge tubes with stoppers. First, add 4 mL of saturated sodium chloride solution containing 0.2% (v / v) acetic acid and vortex to mix thoroughly. Then, add 5 mL of acetonitrile containing 0.2% (v / v) acetic acid. Vortex thoroughly for 1 min and centrifuge at 5000 rpm for 10 min. Pipette 4 mL of the upper acetonitrile layer into a 10 mL centrifuge tube, add 0.25 mL each of a 10% aqueous solution of potassium ferrocyanide and a 20% aqueous solution of zinc acetate, and vortex or shake to mix thoroughly. These solutions are used as cosmetic sample solutions. The four non-essential oil cosmetic samples are model samples prepared by adding 100 μL of a 10 mg / L standard solution of dexamethasone, dexamethasone acetate, dexamethasone valerate, or dexamethasone isonicotinate to the non-essential oil cosmetic samples. ②Purification and hydrolysis of sample solution First, add 1 mL of a stabilizer to the cosmetic sample solution. The stabilizer is an aqueous solution of EDTA and sodium ascorbate, wherein the concentration of EDTA is 1 mg / mL and the concentration of sodium ascorbate is 70 mg / mL. Then, add 2 mL of a 10 mg / mL sodium hydroxide aqueous solution. After vortexing or shaking, react at room temperature for 1 hour to allow the hydroxyl esterified dexamethasone-like glucocorticoid to be directionally hydrolyzed into dexamethasone. Then, add an acetonitrile aqueous solution containing acetic acid to adjust the solvent environment to acidic, terminate the hydrolysis reaction, and make the volume to 10 mL. Shake well, centrifuge at 10,000 r / min for 5 minutes, filter through a 0.22 μm filter membrane, discard 2 drops of the initial filtrate, and take the subsequent filtrate as the sample solution to be tested. The acetonitrile aqueous solution containing acetic acid is prepared by adding 4% by volume of acetic acid to an acetonitrile aqueous solution with a volume ratio of 1:1; (3) Liquid chromatography-tandem triple quadrupole mass spectrometry analysis conditions ① Liquid chromatography analysis conditions are as follows: The liquid chromatography reference conditions are as follows: a) Chromatographic column: C18, 3.0 mm × 150 mm, 1.7 μm; b) Column temperature: 40°C; c) Mobile phase: A: 0.2% acetic acid in water; B: 0.2% acetic acid in methanol; d) Flow rate: 0.4 mL / min; e) Injection volume: 5.0 μL; f) Liquid chromatography gradient separation conditions are shown in Table 1: Table 1 Liquid chromatography gradient separation conditions

[0019] ② Mass spectrometry analysis conditions are as follows: Ion source: electrospray ionization (ESI); scan mode: positive ion mode; detection mode: multiple reaction monitoring (MRM); ionization voltage: 5500 V; ion source temperature: 550°C; curtain gas (CUR) pressure: 35 psi; collision gas (CAD) pressure: 9 psi; nebulizer gas pressure: 55 psi; auxiliary heating gas pressure: 60 psi; mass spectrometry parameters for parent ion, product ion, cone voltage, and collision energy were the same as those for dexamethasone without esterification shown in No. 32 in Table 2: Table 2 Mass spectrometry parameters of unesterified glucocorticoids

[0020] (4) Qualitative analysis The dexamethasone standard solution obtained in step (1) and the sample solution to be tested obtained in step (2) were analyzed according to the liquid chromatography-tandem triple quadrupole mass spectrometry analysis conditions described in step (3), and the MRM spectra of the sample solution obtained under the same liquid chromatography-tandem triple quadrupole mass spectrometry conditions were compared with the MRM spectra of the dexamethasone standard solution. Figure 1 Zhijunru Figure 1 The results show that the four model sample solutions all have extracted ion current signals consistent with the qualitative and quantitative ion pairs of the dexamethasone standard. The chromatographic peaks with the retention time consistent with the retention time of the dexamethasone standard are also present in the MRM spectra of the four model sample solutions. The maximum allowable deviation of the relative abundance ratio of the two ion pairs and the relative abundance ratio of the dexamethasone standard solution with equivalent concentration does not exceed the requirements shown in Table 3: Table 3 Maximum allowable deviation of relative abundance ratio

[0021] Therefore, the sample was determined to be a glucocorticoid-positive sample, and dexamethasone and / or dexamethasone derivatives whose hydroxyl groups were esterified were present in the cosmetics.

[0022] The mass spectrometric response of the esterified dexamethasone derivative hydrolyzed to dexamethasone was compared with the mass spectrometric response of the dexamethasone standard to determine the recovery rate of dexamethasone produced by the hydrolysis of the esterified dexamethasone derivative, as shown in Table 4.

[0023] Table 4 Recovery of dexamethasone derivatives converted to dexamethasone

[0024] As can be seen from Table 4, under the conditions for preparing the sample solutions to be tested, dexamethasone is basically stable, and the dexamethasone derivatives can stably exist after the ester bond is hydrolyzed into dexamethasone; the dexamethasone acetate, dexamethasone valerate or dexamethasone isonicotinate in the other three samples are all hydrolyzed to form dexamethasone. Although the conversion rates of different derivatives to dexamethasone under these conditions vary, the conversion rates are all higher than 82%. If dexamethasone derivatives are added to the cosmetic sample, a qualitative judgment can be made by detecting dexamethasone, and the content of the added glucocorticoid in terms of dexamethasone can be determined based on the conversion relationship. Example 2

[0025] The present invention provides a non-targeted screening method for glucocorticoids derived from esterified hydroxyl groups in cosmetics, which is carried out according to the following steps: (1) Preparation of standard solution The process is basically the same as Example 1, except that the standard substance is hydrocortisone.

[0026] (2) Preparation of cosmetic sample solution Five model samples were prepared by adding 100 μL of 10 mg / L hydrocortisone, hydrocortisone acetate, hydrocortisone valerate, hydrocortisone butyrate, and hydrocortisone succinate standard solutions to non-essential oil cosmetic samples. Other operating conditions are the same as those in Example 1. The difference is that in Example 2, the hydroxy-esterified hydrocortisone-like glucocorticoid is directed hydrolyzed into hydrocortisone.

[0027] (3) Liquid chromatography-tandem triple quadrupole mass spectrometry analysis conditions The method is basically the same as that of Example 1. The difference from Example 1 is that the mass spectrometry parameters used are the mass spectrometry parameters of non-esterified hydrocortisone as shown in No. 10 in Table 2.

[0028] (4) Qualitative analysis The analysis steps are the same as in Example 1.

[0029] The MRM spectra of all the sample solutions obtained in Example 2 of the present invention and the MRM spectra of the hydrocortisone standard solution Figure 1 Zhijunru Figure 2 The results showed that the five model sample solutions all showed extracted ion current signals consistent with the qualitative and quantitative ion pairs of the hydrocortisone standard in the MRM spectra. The chromatographic peaks with retention times consistent with those of the hydrocortisone standard, and the maximum allowable deviation of the relative abundance ratio of the two ion pairs from that of a hydrocortisone standard solution of equivalent concentration did not exceed the requirements shown in Table 3. Therefore, the samples were determined to be glucocorticoid-positive, indicating the presence of hydrocortisone and / or hydrocortisone derivatives with esterified hydroxyl groups in the cosmetics.

[0030] The mass spectrometric response of the hydrolysis of the esterified derivatized hydrocortisone derivative to hydrocortisone was compared with the mass spectrometric response of the hydrocortisone standard to determine the recovery rate of hydrocortisone produced by the hydrolysis of the esterified derivatized hydrocortisone derivative, as shown in Table 5. Table 5 Hydrocortisone derivatives are converted into the recovery of hydrocortisone

[0031] As can be seen from Table 5, under the conditions for preparing the sample solutions to be tested, hydrocortisone is basically stable, and the ester bond of hydrocortisone derivatives is hydrolyzed to hydrocortisone and can exist stably; hydrocortisone acetate, hydrocortisone valerate, hydrocortisone butyrate, and hydrocortisone succinate in the other four samples are all hydrolyzed to form hydrocortisone. Although the conversion rates of different derivatives to hydrocortisone under these conditions vary, the conversion rates are all higher than 84%. If hydrocortisone derivatives are added to cosmetic samples, a qualitative judgment can be made by detecting hydrocortisone, and the content of the added glucocorticoid in terms of hydrocortisone can be determined based on the conversion relationship. Example 3

[0032] The present invention provides a method for non-targeted screening of glucocorticoids derived from esterified hydroxyl groups in cosmetics using low-resolution mass spectrometry, which is carried out according to the following steps: (1) Preparation of standard solution The method is basically the same as Example 1, except that the standard substance is prednisolone.

[0033] (2) Preparation of cosmetic sample solution Five model samples were prepared by adding 100 μL of 10 mg / L standard solutions of prednisolone, prednisolone acetate, prednisolone valerate, prednisolone acetate valerate, and prednisolone hemisuccinate to non-essential oil cosmetic samples. Other operating conditions are the same as those in Example 1. The difference is that in Example 3, the hydroxy-esterified prednisolone-derived glucocorticoid is directed hydrolyzed into prednisolone.

[0034] (3) Liquid chromatography-tandem triple quadrupole mass spectrometry analysis conditions The method is basically the same as Example 1. The difference from Example 1 is that the mass spectrometry parameters are the mass spectrometry parameters without esterified prednisolone as shown in No. 11 in Table 2.

[0035] (4) Qualitative analysis The analysis steps are the same as those in Example 1.

[0036] The MRM spectra of all the sample solutions obtained in Example 3 of the present invention and the MRM spectra of the prednisolone standard solution Figure 1 Zhijunru Figure 3The results showed that the five model sample solutions all showed extracted ion current signals consistent with the two pairs of qualitative and quantitative ion pairs of the prednisolone standard in the MRM spectra. The chromatographic peaks with retention times consistent with the retention time of the prednisolone standard, and the maximum allowable deviation of the relative abundance ratio of the two pairs of ions from the relative abundance ratio of prednisolone solutions of equivalent concentrations did not exceed the requirements shown in Table 3. Therefore, the samples were determined to be glucocorticoid-positive samples, indicating the presence of prednisolone and / or prednisolone derivatives with esterified hydroxyl groups in the cosmetics.

[0037] The mass spectrometric response of the esterified prednisolone derivative hydrolyzed to prednisolone was compared with the mass spectrometric response of the prednisolone standard to determine the recovery rate of prednisolone produced by the hydrolysis of the esterified prednisolone derivative, as shown in Table 6. Table 6 Recovery of prednisolone derivatives converted into prednisolone

[0038] As can be seen from Table 6, under the conditions for preparing the sample solutions to be tested, prednisolone is basically stable, and prednisolone derivatives can be stably present after the ester bond is hydrolyzed into prednisolone; prednisolone acetate, prednisolone valerate, prednisolone acetate valerate, and prednisolone hemisuccinate in the other four samples are all hydrolyzed to form prednisolone. Although the conversion rates of different derivatives to prednisolone under these conditions vary, the conversion rates are all higher than 80%. If prednisolone derivatives are added to cosmetic samples, a qualitative judgment can be made by detecting prednisolone. At the same time, the content of the added glucocorticoid in terms of prednisolone can be determined based on the conversion relationship.

[0039] The reaction mechanism of the above embodiment may be that the hydroxy-free esterified glucocorticoid is more likely to undergo structural rearrangement rather than oxidation under alkaline conditions when sodium hydroxide is added to the organic solvent system. Because the test found that the conventional antioxidants BHA and BHT at the same concentration failed to effectively increase the stability of certain hydroxy-free esterified glucocorticoids in an alkaline environment, while sodium ascorbate performed well, indicating that sodium ascorbate, in addition to having the same antioxidant properties as BHA and BHT, may have an inhibitory effect on the structural rearrangement of the product, thereby making the product after the hydrolysis of the ester bond of the hydroxy-esterified glucocorticoid more stable under alkaline conditions; However, the addition of ascorbic acid failed to achieve the effect of sodium ascorbate, possibly because the addition of ascorbic acid affects the alkaline environment of the reaction system and affects the efficiency of the deesterification reaction. EDTA can chelate metal ions in the solution that may act as catalysts to participate in oxidation reactions or structural rearrangement reactions, reducing the possibility of oxidation reactions or structural rearrangement reactions; At the same time, by controlling the reaction temperature to room temperature and regularly adding acid to the reaction system to terminate the alkaline environment of the reaction system, the possibility of oxidation reactions or structural rearrangement reactions is further reduced.

[0040] Examples 1-3 of the present invention demonstrate the ability of the present invention to use low-resolution mass spectrometry for non-targeted screening of glucocorticoids derived from hydroxyl groups in cosmetics. In addition to Examples 1-3, the present invention also demonstrates the universality of non-targeted detection of currently available hydroxyl-esterified glucocorticoids by spiking negative samples with over 100 glucocorticoids. In practical applications, for a specific cosmetic sample, the standard solution should be prepared using all 40 non-hydroxyl-esterified glucocorticoid reference materials listed in Table 2 to expand the detection range. The mass spectrometry acquisition parameters should use the compound parameters listed in Table 2.

Claims

1. A method for non-targeted screening of hydroxy-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry, characterized by: Prepare a cosmetic sample solution; add a stabilizer and a sodium hydroxide aqueous solution with a concentration of 10 mg / mL to the cosmetic sample solution in sequence, react at room temperature for 1 hour, then add an acetonitrile aqueous solution containing acetic acid to adjust the reaction system to an acidic environment and fix the volume, so that the hydroxy esterified glucocorticoid is directionally hydrolyzed into a hydroxy-free esterified glucocorticoid and is stably present, and take the filtrate as the sample solution to be tested, wherein the acetonitrile aqueous solution containing acetic acid is prepared by adding 4% by volume of acetic acid to an acetonitrile aqueous solution with a volume ratio of 1:1; the volume ratio of the cosmetic sample solution, the sodium hydroxide aqueous solution, and the stabilizer is 4.5:2:1, and the stabilizer is an aqueous solution of EDTA and sodium ascorbate, wherein the concentration of EDTA is 1:

1. mg / mL, the concentration of sodium ascorbate is 70 mg / mL, and the volume of water in the acetonitrile aqueous solution containing 4% by volume of acetic acid is 50%; a standard solution is prepared using hydroxyl-free esterified glucocorticoid, and the standard solution and the sample solution to be tested are respectively subjected to high performance liquid chromatography-tandem triple quadrupole mass spectrometry analysis, the target compounds are separated by high performance liquid chromatography gradient elution, and the triple quadrupole mass spectrometry analysis is used for non-targeted qualitative screening of glucocorticoids in the sample solution to be tested.

2. The method for non-targeted screening of hydroxy-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry according to claim 1, characterized in that: To prepare the cosmetic sample solution, 0.25 g of cosmetics was taken and Separately add 5 mL of acetonitrile containing 0.2% acetic acid by volume and 4 mL of saturated sodium chloride solution containing 0.2% acetic acid by volume to form a two-phase extraction system. Perform dispersed extraction. Take 4 mL of the upper acetonitrile layer and first add 0.25 mL each of 10% potassium ferrocyanide aqueous solution and 20% zinc acetate aqueous solution. Vortex or shake to mix well.

3. The method for non-targeted screening of hydroxy-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry according to claim 1 or 2, characterized in that: The triple quadrupole mass spectrometry non-targeted qualitative screening of glucocorticoids in the test sample solution is performed by comparing the MRM spectrum of the test sample solution with the MRM spectrum of the standard solution. If extracted ions consistent with the qualitative and quantitative ion pairs of the standard appear in the MRM spectrum of the test sample solution and the retention time of the chromatographic peak is consistent with the retention time of the standard, and the relative abundance ratio of the two pairs of ions does not exceed the maximum deviation from the relative abundance ratio of the standard solution with equivalent concentration, it can be determined that the cosmetic contains glucocorticoids consistent with the standard and / or glucocorticoids derived from the esterification of the hydroxyl group of the standard.

4. The method for non-targeted screening of hydroxy-esterified glucocorticoids in cosmetics using low-resolution mass spectrometry according to claim 3, characterized in that The relative abundance ratios of the two pairs of ions do not exceed the maximum deviation from the relative abundance ratios of the standard solutions of equivalent concentrations as shown below: 。

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