CN107525858B - Screening method for chemical risk substances of glucocorticoid in washing and caring products - Google Patents

Abstract

The invention discloses a method for screening chemical risk substances of glucocorticoid in washing and caring products, which comprises the following steps: (1) establishing an accurate mass database and a mass spectrum library of the compound to be detected; (2) pretreatment and detection of an actual sample: performing pretreatment steps such as extraction, centrifugation, purification, filtration and the like on a washing and protecting article sample to obtain a sample solution, and performing ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection; (3) and (3) comparing and analyzing the test result obtained in the step (2) with the accurate mass database and the mass spectrum library established in the step (1), and screening chemical risk substances of the glucocorticoid. The screening method for chemical risk substances of glucocorticoid in the washing and nursing products, which is established by the invention, has the advantages of high accuracy, strong specificity and high sensitivity, and can be suitable for daily detection and quality control of the washing and nursing products.

Description

Screening method for chemical risk substances of glucocorticoid in washing and caring products

Technical Field

The invention relates to a screening method of chemical substances, in particular to a screening method of glucocorticoid chemical risk substances in washing products based on an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometry combined technology.

Background

1. Chemical risk substances in the laundry have a potentially detrimental effect on the health of the consumer.

The washware provides guarantee for people to pursue clean, healthy and fashionable life from the beginning of birth, and with the continuous development and progress of society, the pursuit of people to clean, healthy and fashionable life style is continuously upgraded, so that the washware industry is driven to step into a speedway for development and upgrading. At present, the washing product market products are full of scales, and functional, subdivision and specialized products continuously emerge, so that diversified requirements of vast consumers are met.

Nowadays, the washing and nursing articles become necessities of life of people, and the safety of the washing and nursing articles is also paid more and more attention. In 2014, the Hanyi shampoo is detected by a third-party authoritative detection mechanism to contain possible carcinogen acrylamide, wherein two preservatives, namely methylisothiazolinone and methylchloroisothiazolinone, are detected to be approximately two times higher than the standard, so that the skin allergy of a human body is easily caused; infant shampoo, one of the strong-sign products, has the greatest selling point of no eye irritation, but the product is identified in 2009 by the united states as containing a toxic substance dioxane and a quaternary ammonium salt component capable of releasing formaldehyde; in 2010, phthalic acid ester is detected in a certain proportion by a disease prevention and control center in Beijing city in the process of spot check of domestic washing products, wherein the detection rate of perfume is as high as 92.3% (phthalic acid ester is environmental hormone which harms human reproductive capacity); additionally, triclosan in toiletries and antibiotics and hormones in other toiletries and the like are potentially chemically hazardous substances.

The long-term use of the washing and caring product containing the toxic and harmful substances can cause the problems of facial skin black spot, atrophy and thinning, osteoporosis, muscular atrophy, metabolic disorder and the like, and can seriously cause the occurrence of cancers. In order to attract customers to buy for the effect brought by the merchant in a short time, the merchant still adopts a kit combination system, and hormones and other similar active ingredients are added in one or more products, so that the inspection is avoided. Thus, its hazard is not insignificant.

2. The supervision of chemical risk substances in the washing and caring products is relatively lagged, and the prevention capability is insufficient.

According to the statistics of the national electronic injury monitoring system (NEISS), the safety events caused by various washing and caring products in 2001-2009 are accumulated to 7405. The european union rapid warning system for non-food consumer goods (RAPEX), the american consumer goods safety committee (CPSC), and the U.S. Food and Drug Administration (FDA) have also issued risk warnings against illegal addition of chemicals to care products.

In recent years, China has more and more invested financial resources, manpower and material resources in the aspect of safety supervision of washing products, but the people still feel unconscious in the front of more and more serious product safety events. The main problems are that the supervision mode of China mostly takes post-remedy as a main part, the skill in the aspect of early product prevention is still insufficient, and the main performance is that new regulation and rule systems are frequently developed in developed countries, so that the frequency of products in China is frequently recalled, for example, in 2013, the frequency of the products of the China is 48 times recalled for the strongly-grown infant care products; issue mouthwash and other events are recalled in 2011 by Baojie (China) Co.

3. High resolution mass spectrometry has advantages in the screening of chemical risk substances.

At present, for quantitative detection of target compounds, triple quadrupole tandem mass spectrometry is mainly adopted in domestic and international related researches. And a washing and nursing article detection method adopting a high-resolution mass spectrometry technology is not reported.

Disclosure of Invention

With the stricter regulations and the higher amount of chemical risk substances to be detected, the conventional method for detecting by a multi-reaction monitoring mode cannot meet the requirement of high-throughput screening, and the determination of the chemical risk substances should be developed rapidly and at high throughput. The resolution and the quality precision of the high-resolution mass spectrum are obviously superior to those of triple quadrupole mass spectrum, and the high-resolution mass spectrum is a mass spectrum which can be simultaneously qualitative and quantitative; high resolution can be realized in a wide mass range, and accurate molecular weight of the substance can be obtained; obtaining a true isotope distribution; the mass spectrometer has a high-sensitivity tandem mass spectrometry function, and realizes accurate mass measurement of parent ions and daughter ions. Therefore, the technical problem to be solved by the invention is to provide a method for screening chemical risk substances in a washing product, which has high resolution and mass precision, can obtain accurate molecular weight information of the substances in a wide mass range, obtain real isotope distribution, has a high-sensitivity tandem mass spectrometry function and realizes accurate mass measurement of parent ions and ionic ions.

A method for detecting chemical risk substances of glucocorticoids in washing products comprises the following steps:

pretreatment and detection of a sample: performing pretreatment steps such as extraction, centrifugation, purification, filtration and the like on a washing and protecting article sample to obtain a sample solution, and performing ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection; in the ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometry detection, the chromatographic separation conditions are as follows:

a chromatographic column: an ACQUITY UPLC BEH Shield RP18 column with a length of 150mm, an inner diameter of 2.1mm and a particle size of 1.7 μm; column temperature: 40 ℃; flow rate: 0.4mL min^-1(ii) a Sample introduction amount: 10 μ L.

The glucocorticoid comprises triamcinolone, prednisolone, hydrocortisone, prednisone, cortisone, methylprednisolone, betamethasone, dexamethasone, flumethasone, beclomethasone, triamcinolone acetonide, flurandrenolide, triamcinolone diacetate, prednisolone acetate, fluoromethalone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisolone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluoromethalone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetate, diflorasone diacetate, betamethasone valerate, prednisolone kate, halcinonide, alclomethasone dipropionate, amcinonide, clobetasol propionate, fluticasone propionate, betamethasone dipropionate, Beclomethasone dipropionate and clobetasone butyrate.

The invention relates to a method for detecting chemical risk substances of glucocorticoid in washing products, wherein in the ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry detection, a mobile phase and a gradient elution program are shown in a table 1:

TABLE 1 chromatographic mobile phase and gradient elution procedure

In the ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection, the quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection conditions are as follows:

electrospray voltage: positive ion mode 3.4 kV; sheath gas pressure: 55, arbitrary units; auxiliary gas pressure: 6, arbitrary units; ion source temperature: 370 ℃; temperature of transmission metal capillary: 320 ℃; lens radio frequency voltage: 55V; scanning range: mass to charge ratio 100-; first-order mass spectrum full-scan resolution: 70000 full width at half maximum; maximum capacity of the orbitrap: 1X 10⁶(ii) a Maximum injection time of the orbitrap: 80 ms;

data dependent secondary ion full scan resolution: 17500 full width at half maximum; isolating the window: mass-to-charge ratio of +/-2; normalized collision energy: 20,40,60 eV; maximum capacity of orbit trap 1X 10⁵(ii) a Maximum injection time of the orbitrap: 80 ms; dynamic exclusion time: and 6 s.

The invention relates to a method for detecting chemical risk substances of glucocorticoid in washing products, wherein the pretreatment comprises the following steps:

weighing 0.3g of a washing and protecting article sample into a 10mL plastic centrifuge tube, adding 2mL of saturated sodium sulfate solution, performing mixed demulsification by swirling for 30s, then adding 4mL of methanol and 4mL of acetonitrile, performing sufficient swirling for 30s, and performing ultrasonic extraction for 30 min; centrifuging the extractive solution at 12000rpm for 8min, transferring the centrifuged supernatant into another 10mL plastic centrifuge tube, adding 100mg N-propyl ethylenediamine solid phase extraction powder, vortexing for 1min, sucking the supernatant extractive solution, filtering with 0.22 μm microporous membrane, and performing ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry.

A screening method for chemical risk substances of glucocorticoid in washing products comprises the following steps:

(1) establishing an accurate mass database and a mass spectrum library of a compound to be detected, wherein the accurate mass database comprises the name, molecular formula and chromatographic retention time of the compound, and accurate mass number information of a precursor ion and two characteristic fragment ions, and the mass spectrum library comprises secondary mass spectrograms generated after different collision energies are respectively applied to the compound to be detected;

(2) the invention relates to a sample pretreatment and detection method;

(3) and comparing and analyzing the test result obtained by the sample pretreatment and detection method with the established accurate mass database and mass spectrum library of the compound to be detected, and determining that the compound to be detected is detected in the actual sample only when the accurate mass number, the chromatographic retention time, the isotope peak distribution and the secondary mass spectrum of the precursor ions and the information of the accurate mass database and the mass spectrum library of the two characteristic fragment ions are all matched.

The invention relates to a screening method of glucocorticoid chemical risk substances in washing products, wherein the method for establishing an accurate mass database and a mass spectrum library of a compound to be detected specifically comprises the following steps:

accurate mass database: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mug/L, directly injecting samples by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, respectively carrying out analysis and detection in a positive ion mode and a negative ion mode, and determining the accurate mass number of precursor ions of the corresponding compounds to be detected;

applying collision energy to each compound to be detected to obtain fragment ions of each compound, and selecting two fragment ions with higher response intensity as characteristic fragment ions;

in the process, mass spectrum key parameters such as electrospray voltage, ion source temperature, sheath gas pressure, resolution ratio and the like are optimized respectively;

preparing a mixed standard solution of the compounds to be detected with the concentration of 100 mu g/L, and optimizing the separation condition of the ultrahigh pressure liquid chromatography to obtain the chromatographic retention time of each compound;

establishing an accurate quality database: respectively inputting the name, molecular formula, chemical abstract number, precursor ion accurate mass number, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window of each compound, and additionally inputting a response threshold of the compound to be detected, and when the signal response of the compound to be detected exceeds the threshold, further performing secondary mass spectrometry on the corresponding precursor ions; the accurate mass number information of the compound to be tested, the precursor ions and the fragment ions thereof is shown in Table 2;

TABLE 2 accurate mass number information for the test compounds and their precursor and fragment ions

Establishing a mass spectrum library: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mu g/L, directly injecting and analyzing by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, setting a series of different collision energies, and crushing target compounds to obtain a secondary mass spectrogram of each compound; and inputting and storing all secondary mass spectrograms to obtain a mass spectrographic library of all compounds to be detected.

The mass spectrum of the chemical risk substance of glucocorticoid in the invention is shown in figure 1, wherein each code number refers to the following relationship: 1. triamcinolone acetonide; 2. prednisolone; 3. hydrocortisone; 4. prednisone; 5. cortisone; 6. methylprednisolone; 7. betamethasone; 8. dexamethasone; 9. flumethasone; 10. beclomethasone; 11. triamcinolone acetonide; 12. fluoro-hydrogen shrinkage; 13. triamcinolone diacetate; 14. prednisolone acetate; 15. fluorometholone; 16. hydrocortisone acetate; 17. deflazacort; 18. fludrocortisone acetate; 19. prednisone acetate; 20. cortisone acetate; 21. methylprednisolone acetate; 22. betamethasone acetate; 23. budesonide; 24. hydrocortisone butyrate; 25. dexamethasone acetate; 26. fluorometholone acetate; 27. hydrocortisone valerate; 28. triamcinolone acetonide acetate; 29. fluocinolone acetonide acetate; 30. diflorasone diacetate; 31. betamethasone valerate; 32. prednisolone ester; 33. halcinonide; 34. alclometasone dipropionate; 35. amcinonide; 36. clobetasol propionate; 37. fluticasone propionate; 38. betamethasone dipropionate; 39. beclomethasone dipropionate; 40. clobetasone butyrate.

The invention relates to a screening method of glucocorticoid chemical risk substances in washing products, wherein a compound to be detected is subjected to mass spectrometry in a positive ion mode;

the preparation of the standard solution comprises the following steps: 500-1000. mu.g mL of the mixture was prepared^-1The standard stock solution is stored in the dark at the temperature of 4 ℃; 10. mu.g mL of the composition^-1The mixed standard solution is diluted by methanol to prepare a series of matrix matching standard solutions with different concentrations;

the standard substance of glucocorticoid is dissolved by methanol solvent.

The screening method of the chemical risk substances in the washing and nursing product is different from the prior art in that:

the method for screening the chemical risk substances of the glucocorticoid in the washing products based on the ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry technology is established, so that the technical problem of screening and confirming the glucocorticoid in the washing products is effectively solved, and the risk evaluation of toxic and harmful substances in the washing products and the establishment of limit standards can be carried out, so that the occurrence probability of the damage events caused by the chemical risk substances to consumers is reduced, and a healthy and good consumption environment is created; the research result can also guide enterprises to avoid using chemical risk substances causing potential safety hazards in the production process of products, so that consumers can more clearly recognize the potential hazards of the chemical risk substances in the washing and protecting products, the overall safe consumption view of the society is improved, and good social benefits are generated.

The related laws and regulations and standards of the washing and caring products are main means and technical bases for measuring and controlling the quality characteristics of the products, and enterprises and the like can effectively promote the health and standard development of the washing and caring products only by knowing the laws and regulations and standards, so that the products obtain good economic benefits. The screening method established by the invention can provide effective technical support for related detection mechanisms, further improve and improve various management and regulation standards of the washing and nursing products, standardize the market economic rules and the operation order of the washing and nursing product industry, improve the technical innovation capability of the washing and nursing product industry, shorten the technical gap with the international washing and nursing product industry, and has important economic significance for accelerating the sustainable health development of related enterprises in China.

The method has high resolution and mass precision, can obtain the accurate molecular weight of the substances in a wide mass range, obtains real isotope distribution, has the function of high-sensitivity tandem mass spectrometry, and realizes the accurate mass measurement of the parent ions and the daughter ions.

The invention establishes a rapid screening and quantitative analysis strategy for glucocorticoid in a washing product based on the combination of ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometry and an accurate mass database and a spectrum library. By investigating the detection limit, the quantitative limit, the linear relation, the stability and the matrix effect of the method and detecting the actual sample, the rapid analysis method established by the invention has high accuracy, strong specificity and high sensitivity, and can be suitable for daily detection and production quality control of washing and caring products.

The screening method for glucocorticoid chemical risk substances in the washing products of the present invention is further described below with reference to the attached drawings.

Drawings

FIG. 1 is a mass spectrum of a chemical risk substance of glucocorticoid in the present invention, wherein each symbol is expressed as follows: 1. triamcinolone acetonide; 2. prednisolone; 3. hydrocortisone; 4. prednisone; 5. cortisone; 6. methylprednisolone; 7. betamethasone; 8. dexamethasone; 9. flumethasone; 10. beclomethasone; 11. triamcinolone acetonide; 12. fluoro-hydrogen shrinkage; 13. triamcinolone diacetate; 14. prednisolone acetate; 15. fluorometholone; 16. hydrocortisone acetate; 17. deflazacort; 18. fludrocortisone acetate; 19. prednisone acetate; 20. cortisone acetate; 21. methylprednisolone acetate; 22. betamethasone acetate; 23. budesonide; 24. hydrocortisone butyrate; 25. dexamethasone acetate; 26. fluorometholone acetate; 27. hydrocortisone valerate; 28. triamcinolone acetonide acetate; 29. fluocinolone acetonide acetate; 30. diflorasone diacetate; 31. betamethasone valerate; 32. prednisolone ester; 33. halcinonide; 34. alclometasone dipropionate; 35. amcinonide; 36. clobetasol propionate; 37. fluticasone propionate; 38. betamethasone dipropionate; 39. beclomethasone dipropionate; 40. clobetasone butyrate.

Detailed Description

Example 1

A method for detecting chemical risk substances of glucocorticoids in washing products comprises the following steps:

performing pretreatment steps such as extraction, centrifugation, purification, filtration and the like on a washing and protecting article sample to obtain a sample solution, and performing ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection; in the ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometry detection, the chromatographic separation conditions are as follows:

a chromatographic column: an ACQUITY UPLC BEH Shield RP18 column with a length of 150mm, an inner diameter of 2.1mm and a particle size of 1.7 μm; column temperature: 40 ℃; flow rate: 0.4mL min^-1(ii) a Sample introduction amount: 10 μ L, mobile phase and gradient elution program are shown in Table 1:

TABLE 1 chromatographic mobile phase and gradient elution procedure

In the ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection, the quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection conditions are as follows:

electrospray voltage: positive ion mode 3.4 kV; sheath gas pressure: 55, arbitrary units; auxiliary gas pressure: 6, arbitrary units; ion source temperature: 370 ℃; temperature of transmission metal capillary: 320 ℃; lens radio frequency voltage: 55V; scanning range: mass to charge ratio 100-; first-order mass spectrum full-scan resolution: 70000 full width at half maximum; maximum capacity of the orbitrap: 1X 10⁶(ii) a Maximum injection time of the orbitrap: 80 ms;

data dependent secondary ion full scan resolution: 17500 full width at half maximum; isolating the window: mass-to-charge ratio of +/-2; normalized collision energy: 20,40,60 eV; maximum capacity of orbit trap 1X 10⁵(ii) a Maximum injection time of the orbitrap: 80 ms; dynamic exclusion time: and 6 s.

The pretreatment comprises the following steps:

weighing 0.3g of a washing and protecting article sample into a 10mL plastic centrifuge tube, adding 2mL of saturated sodium sulfate solution, performing mixed demulsification by swirling for 30s, then adding 4mL of methanol and 4mL of acetonitrile, performing sufficient swirling for 30s, and performing ultrasonic extraction for 30 min; centrifuging the extractive solution at 12000rpm for 8min, transferring the centrifuged supernatant into another 10mL plastic centrifuge tube, adding 100mg N-propyl ethylenediamine solid phase extraction powder, vortexing for 1min, sucking the supernatant extractive solution, filtering with 0.22 μm microporous membrane, and performing ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry.

The glucocorticoid comprises triamcinolone, prednisolone, hydrocortisone, prednisone, cortisone, methylprednisolone, betamethasone, dexamethasone, flumethasone, beclomethasone, triamcinolone acetonide, flurandrenolide, triamcinolone diacetate, prednisolone acetate, fluoromethalone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisolone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluoromethalone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetate, diflorasone diacetate, betamethasone valerate, prednisolone kate, halcinonide, alclomethasone dipropionate, amcinonide, clobetasol propionate, fluticasone propionate, betamethasone dipropionate, Beclomethasone dipropionate and clobetasone butyrate.

55 samples of the washware were tested for glucocorticoid chemical risk substances using the method of example 1. The results show that 2 samples containing dexamethasone (with the content of 127.561mg/kg and 53.463mg/kg respectively) were screened, 1 sample containing betamethasone valerate (with the content of 17.896mg/kg) and 1 sample containing clobetasol propionate (with the content of 53.972 mg/kg).

Example 2

Instrument and reagent

A Dionex Ultimate 3000 rapid high performance liquid chromatography system (Thermo Fisher, USA); qxctive Focus quadrupole-electrostatic field orbitrap high resolution mass spectrometer (Thermo Fisher, usa); Milli-Q ultra pure water instruments (Millipore, USA); Vortex-Genie 2 Vortex shaker (scientific industries, USA).

Methanol, acetonitrile (chromatographically pure, Thermo Fisher, usa); ammonium hydroxide, formic acid (chromatographically pure, dima technology, usa); the laboratory water was deionized water. Eighteen carbon, N-propylethylenediamine and graphitized carbon black (Macherey-Nagel, Germany); multi-walled carbon nanotubes (Miltenyi Biotec GmbH, germany); anhydrous magnesium sulfate, sodium chloride (guangzhou west longgao chemical ltd); 0.22 μm microporous filter membrane (Pall, USA).

500-1000. mu.g mL of the mixture was prepared^-1The standard stock solution of (4) was stored at 4 ℃ in the dark. 10. mu.g mL of the composition^-1The standard solution is mixed and then diluted by methanol to prepare a series of matrix matching standard solutions with different concentrations.

The molecular formula, molecular weight, chemical abstracts number, oil-water distribution coefficient, solvent formulation and other information of the target compound are shown in Table 3.

TABLE 3 information on the name, molecular formula, molecular weight, chemical Abstract number, oil-water partition coefficient, and formulation solvent of the target Compound

Establishing accurate mass database and mass spectrum library

The accurate mass database includes the compound name, molecular formula (used to calculate the isotopic peak distribution), retention time, and the accurate mass numbers of the precursor ion and the two characteristic fragment ions (table 4). The spectral library includes secondary mass spectra generated by analysis of each compound using different collision energies. The spectral library can be regarded as an auxiliary confirmation means after the sampling data is screened by the accurate quality database.

The method for establishing the accurate mass database and the mass spectrum library of the compound to be detected specifically comprises the following steps:

accurate mass database: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mug/L, directly injecting samples by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, respectively carrying out analysis and detection in a positive ion mode and a negative ion mode, and determining the accurate mass number of precursor ions of the corresponding compounds to be detected;

applying collision energy to each compound to be detected to obtain fragment ions of each compound, and selecting two fragment ions with higher response intensity as characteristic fragment ions;

in the process, mass spectrum key parameters such as electrospray voltage, ion source temperature, sheath gas pressure, resolution ratio and the like are optimized respectively;

preparing a mixed standard solution of the compounds to be detected with the concentration of 100 mu g/L, and optimizing the separation condition of the ultrahigh pressure liquid chromatography to obtain the chromatographic retention time of each compound;

establishing an accurate quality database: respectively inputting the name, molecular formula, chemical abstract number, precursor ion accurate mass number, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window of each compound, and additionally inputting a response threshold of the compound to be detected, and when the signal response of the compound to be detected exceeds the threshold, further performing secondary mass spectrometry on the corresponding precursor ions;

establishing a mass spectrum library: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mu g/L, directly injecting and analyzing by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, setting a series of different collision energies, and crushing target compounds to obtain a secondary mass spectrogram of each compound; and inputting and storing all secondary mass spectrograms to obtain a mass spectrographic library of all compounds to be detected.

The mass spectrum of the chemical risk substance of glucocorticoid in the invention is shown in figure 1, wherein each code number refers to the following relationship: 1. triamcinolone acetonide; 2. prednisolone; 3. hydrocortisone; 4. prednisone; 5. cortisone; 6. methylprednisolone; 7. betamethasone; 8. dexamethasone; 9. flumethasone; 10. beclomethasone; 11. triamcinolone acetonide; 12. fluoro-hydrogen shrinkage; 13. triamcinolone diacetate; 14. prednisolone acetate; 15. fluorometholone; 16. hydrocortisone acetate; 17. deflazacort; 18. fludrocortisone acetate; 19. prednisone acetate; 20. cortisone acetate; 21. methylprednisolone acetate; 22. betamethasone acetate; 23. budesonide; 24. hydrocortisone butyrate; 25. dexamethasone acetate; 26. fluorometholone acetate; 27. hydrocortisone valerate; 28. triamcinolone acetonide acetate; 29. fluocinolone acetonide acetate; 30. diflorasone diacetate; 31. betamethasone valerate; 32. prednisolone ester; 33. halcinonide; 34. alclometasone dipropionate; 35. amcinonide; 36. clobetasol propionate; 37. fluticasone propionate; 38. betamethasone dipropionate; 39. beclomethasone dipropionate; 40. clobetasone butyrate.

Third, sample pretreatment

Weighing 0.3g of a washing and protecting article sample into a 10mL plastic centrifuge tube, adding 2mL of saturated sodium sulfate solution, performing mixed demulsification by swirling for 30s, then adding 4mL of methanol and 4mL of acetonitrile, performing sufficient swirling for 30s, and performing ultrasonic extraction for 30 min; centrifuging the extractive solution at 12000rpm for 8min, transferring the centrifuged supernatant into another 10mL plastic centrifuge tube, adding 100mg N-propyl ethylenediamine solid phase extraction powder, vortexing for 1min, sucking the supernatant extractive solution, filtering with 0.22 μm microporous membrane, and performing ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry.

Fourthly, chromatographic separation conditions

A chromatographic column: an ACQUITY UPLC BEH Shield RP18 column with a length of 150mm, an inner diameter of 2.1mm and a particle size of 1.7 μm; column temperature: 40 ℃; flow rate: 0.4mL min^-1(ii) a Sample introduction amount: 10 μ L, mobile phase and gradient elution program are shown in Table 1:

TABLE 1 chromatographic mobile phase and gradient elution procedure

Fifthly, mass spectrum detection conditions

Electrospray voltage: positive ion mode 3.4 kV; sheath gas pressure: 55, arbitrary units; auxiliary gas pressure: 6, arbitrary units; ion source temperature: 370 ℃; temperature of transmission metal capillary: 320 ℃; lens radio frequency voltage: 55V; scanning range: mass to charge ratio 100-; first-order mass spectrum full-scan resolution: 70000 full width at half maximum; maximum capacity of the orbitrap: 1X 10⁶(ii) a Maximum injection time of the orbitrap: 80 ms;

data dependent secondary ion full scan resolution: 17500 full width at half maximum; isolating the window: mass-to-charge ratio of +/-2; normalized collision energy: 20,40,60 eV; maximum capacity of orbit trap 1X 10⁵(ii) a Maximum injection time of the orbitrap: 80 ms; dynamic exclusion time: and 6 s.

Sixthly, experimental data comparison analysis

And comparing and analyzing the sample detection experimental data result with the established accurate mass database and the mass spectrum library of the compound to be detected, wherein the result side shows that the sample is a positive sample only when the accurate mass number, the chromatographic retention time, the isotope peak distribution and the secondary mass spectrum of the precursor ion are all matched with the accurate mass database and the mass spectrum library of the precursor ion and the two characteristic fragment ions.

The method specifically comprises the following steps:

setting a mass number extraction window, a retention time window and isotope distribution threshold parameters, and comparing and analyzing the collected sample data according to the set parameters:

first, according to the set parameters, such as mass number extraction window + -5 ppm, chromatographic peak area not less than 1 × 10⁶Extracting the accurate mass number of the precursor ions in the database; if the mass number of the precursor ions of the target compound in the database appears in the data acquired by the full scanning of the sample, and the chromatographic peak area is not less than 1 x 10⁶Then, the comparison of the accurate mass number of the precursor ions is regarded as successful;

secondly, setting the standard deviation of a retention time window to be +/-3, and if the chromatographic retention time of the precursor ions falls within the standard deviation of +/-3 of the corresponding retention time in the database, judging that the retention time matching is successful;

thirdly, performing isotope distribution calculation according to the molecular formula of the compound to be detected, wherein the set threshold value is 90%, and when the isotope distribution of the precursor ions in the sample data is compared with the database by the matching degree of 90%, the matching is regarded as successful;

fourthly, according to the comparison condition of the two characteristic fragment ions in the database and the two fragment ions acquired by the experimental result, if the deviation between the experimental value and the accurate mass number of the fragment ions in the database is not more than +/-5 ppm, the matching is regarded as successful;

fifthly, comparing the secondary mass spectrogram acquired by the experiment with a mass spectrogram library; at this stage, the precursor ions, all fragment ions and the relative ion abundance ratios are compared and if all match, the match is deemed successful.

Seven, result in

55 samples of the washware were tested for glucocorticoid chemical risk substances using the method of example 2. The results show that 2 samples containing dexamethasone (with the content of 127.561mg/kg and 53.463mg/kg respectively) were screened, 1 sample containing betamethasone valerate (with the content of 17.896mg/kg) and 1 sample containing clobetasol propionate (with the content of 53.972 mg/kg).

Eight, discussion

1. Establishment of screening method

In order to fully exert the advantages of the electrostatic field orbit trap high-resolution mass spectrometry in screening analysis, a full-scanning-data-dependent secondary ion scanning mode is adopted in the research. In this collection mode, the mass spectrum is first scanned by the total mass number (m/z 100- & lt800- & gt), and when some target compound ions in the database are detected and the intensity is not lower than a certain set threshold, the ions are sent into the high-energy collision cell to be fragmented, and the daughter ion fragments are generated.

In the compound screening stage, the detection of the target substance is mainly based on three key parameters: the exact mass number of the precursor ion, the retention time, and the isotopic peak distribution. The setting of the exact mass number extraction window has a crucial impact on the detection sensitivity and selectivity of the method. In the present invention, the concentrations of 100. mu. g L were used respectively^-1And 500. mu.g kg of the mixed standard solution^-1The marked sample is optimized for the parameters. The experimental results show that the deviation of the mass number in the two solutions is better than 2ppm and 3.5ppm respectively. Considering the follow-up confirmation of characteristic fragment ions and a secondary spectrogram, and simultaneously avoiding the occurrence of false negative results as much as possible, the research sets the extraction window of mass number to be 5ppm, so that the method can ensure no false negative results and has no obvious influence on the selectivity and sensitivity of the method detection. For the retention time parameter, the reasonable setting of the retention time window plays an important role in the accuracy of the screening result. The retention time may drift slightly due to the application of different high resolution mass spectrometry systems and the operation of different personnel, and therefore the study set the retention time window to mean retention time ± 3 × retention time standard deviation. In the full-scanning stage of the mass spectrum, due to the fact that the setting of different resolutions has certain influence on the selectivity and sensitivity of the method, the research also carries out detailed optimization on mass spectrum resolution parameters. Three different resolutions were set for the experiment (30000,50000)70000 full width at half maximum) are respectively carried out, and the result shows that the mass precision of all target compounds is highest under the resolution of 70000 full width at half maximum, the sensitivity is not obviously different from the former two, and 10-15 scanning points can still be provided to obtain excellent chromatographic peak shapes.

In the validation stage, the study collected secondary mass spectra of each target compound and recorded the exact mass numbers of the two characteristic fragment ions for each compound. Resolution of 70000 full width at half maximum already provides sufficiently high selectivity in the primary mass spectrometry scan stage, where sensitivity should be a major consideration, so we set mass spectrometry resolution to the lower and commonly used 17500 full width at half maximum in the validation stage. To obtain a more complete secondary mass spectrum, the study used normalized collision energies of 20,40 and 60eV and recorded the exact mass numbers of two of the fragment ions with higher response intensities into the database. In high resolution mass spectrometry, some key parameters, such as spray voltage, capillary temperature, air curtain gas, purge gas, and lens rf voltage, are also optimized in detail.

2. Optimization of sample extraction and purification methods

The invention adopts a sample extraction mode of ultrasonic-assisted extraction. First, the recovery efficiency was examined for methanol and acetonitrile, which are common organic solvents, and a mixed solvent of methanol + acetonitrile (1:1, v/v) which is a mixture of both solvents at an equal volume ratio. Considering the high efficiency and the equilibrium of the solvent for all the extraction of the target compounds, the present study first selects methanol + acetonitrile (1:1, v/v) as extraction solvent.

When only solvent is used for extraction, and the extract is put into a machine for analysis after passing through a microporous filter membrane, the matrix in the washing and protecting product still has large matrix interference on the analysis of the target compound, so that the invention adopts a simpler and more efficient sample purification mode, namely dispersed solid phase extraction, for further purifying the sample extract. The result shows that 100 mgN-propyl ethylenediamine has good purification effect on the sample. Therefore, the present invention selects 100mg of N-propylethylenediamine as the scavenger for the sample.

3. Linear relationship, detection limit and quantification limit

The mixed standard stock solution is sequentially diluted into a mixed working solution with a series of concentration gradients by the processed sample solution, the mixed working solution is measured under the optimized chromatographic mass spectrum condition, the concentration (x) is linearly investigated by the molecular ion peak area (y) of each target substance, and the results of the linear range, the detection limit and the quantification limit of the target compound are shown in a table 5. The linear regression coefficients of the target compound are all larger than 0.99, which shows that the method has good linear relation and can carry out accurate quantification. The detection limit and the quantification limit of the target compound were calculated at 3-fold and 10-fold signal-to-noise ratios, respectively, and the results are shown in table 5.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (6)

1. A method for detecting chemical risk substances of glucocorticoids in washing products is characterized by comprising the following steps: the method comprises the following steps:

sample pretreatment and detection methods: extracting, centrifuging, purifying and filtering the washing and protecting product sample to obtain a sample solution, and performing ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection on the sample solution; in the ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometry detection, the ultrahigh pressure liquid chromatography separation conditions are as follows:

a chromatographic column: an ACQUITY UPLC BEH Shield RP18 column with a length of 150mm, an inner diameter of 2.1mm and a particle size of 1.7 μm; column temperature: 40 ℃; flow rate: 0.4mL min^-1(ii) a Sample introduction amount: 10 mu L of the solution;

the mobile phase and gradient elution procedure are shown in table 1:

TABLE 1 chromatographic mobile phase and gradient elution procedure

The glucocorticoid comprises triamcinolone, prednisolone, hydrocortisone, prednisone, cortisone, methylprednisolone, betamethasone, dexamethasone, flumethasone, beclomethasone, triamcinolone acetonide, flurandrenolide, triamcinolone diacetate, prednisolone acetate, fluoromethalone, hydrocortisone acetate, deflazacort, fludrocortisone acetate, prednisolone acetate, cortisone acetate, methylprednisolone acetate, betamethasone acetate, budesonide, hydrocortisone butyrate, dexamethasone acetate, fluoromethalone acetate, hydrocortisone valerate, triamcinolone acetonide acetate, fluocinolone acetate, diflorasone diacetate, betamethasone valerate, prednisolone kate, halcinonide, alclomethasone dipropionate, amcinonide, clobetasol propionate, fluticasone propionate, betamethasone dipropionate, Beclomethasone dipropionate and clobetasone butyrate;

in the ultrahigh-pressure liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection, the quadrupole-electrostatic field orbit trap high-resolution mass spectrometry detection conditions are as follows:

electrospray voltage: positive ion mode 3.4 kV; sheath gas pressure: 55, arbitrary units; auxiliary gas pressure: 6, arbitrary units; ion source temperature: 370 ℃; temperature of transmission metal capillary: 320 ℃; lens and lens assemblyRadio frequency voltage: 55V; scanning range: mass to charge ratio 100-; first-order mass spectrum full-scan resolution: 70000 full width at half maximum; maximum capacity of the orbitrap: 1X 10⁶(ii) a Maximum injection time of the orbitrap: 80 ms;

data dependent secondary ion full scan resolution: 17500 full width at half maximum; isolating the window: mass-to-charge ratio of +/-2; normalized collision energy: 20,40,60 eV; maximum capacity of orbit trap 1X 10⁵(ii) a Maximum injection time of the orbitrap: 80 ms; dynamic exclusion time: and 6 s.

2. The method for detecting chemical risk substances of glucocorticoids in washing products according to claim 1, wherein the method comprises the following steps: the pretreatment comprises the following steps:

weighing 0.3g of a washing and protecting article sample into a 10mL plastic centrifuge tube, adding 2mL of saturated sodium sulfate solution, performing mixed demulsification by swirling for 30s, then adding 4mL of methanol and 4mL of acetonitrile, performing sufficient swirling for 30s, and performing ultrasonic extraction for 30 min; centrifuging the extractive solution at 12000rpm for 8min, transferring the centrifuged supernatant into another 10mL plastic centrifuge tube, adding 100mg N-propyl ethylenediamine solid phase extraction powder, vortexing for 1min, sucking the supernatant extractive solution, filtering with 0.22 μm microporous membrane, and performing ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry.

3. A screening method for chemical risk substances of glucocorticoid in washing products is characterized in that: the method comprises the following steps:

(1) establishing an accurate mass database and a mass spectrum library of a compound to be detected, wherein the accurate mass database comprises the name, molecular formula and chromatographic retention time of the compound, and accurate mass number information of a precursor ion and two characteristic fragment ions, and the mass spectrum library comprises secondary mass spectrograms generated after different collision energies are respectively applied to the compound to be detected;

(2) a method for detecting a chemical risk substance for glucocorticoids in a cleaning composition according to claim 1;

(3) and (3) comparing and analyzing the test result obtained in the step (2) with the established accurate mass database and mass spectrum library of the compound to be tested, and determining that the compound to be tested is detected in the actual sample only when the accurate mass number, the chromatographic retention time, the isotope peak distribution and the secondary mass spectrum of the precursor ions and the accurate mass database and mass spectrum library information of the precursor ions and the two characteristic fragment ions are all matched.

4. The method for screening chemical risk substances of glucocorticoids in toiletries according to claim 3, wherein: the method for establishing the accurate mass database and the mass spectrum library specifically comprises the following steps:

accurate mass database: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mug/L, directly injecting samples by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, respectively carrying out analysis and detection in a positive ion mode and a negative ion mode, and determining the accurate mass number of precursor ions of the corresponding compounds to be detected;

applying collision energy to each compound to be detected to obtain fragment ions of each compound, and selecting two fragment ions with higher response intensity as characteristic fragment ions;

in the analysis and detection process, optimizing mass spectrum key parameters such as electrospray voltage, ion source temperature, sheath gas pressure, resolution ratio and the like;

preparing a mixed standard solution of the compounds to be detected with the concentration of 100 mu g/L, and optimizing the separation condition of the ultrahigh pressure liquid chromatography to obtain the chromatographic retention time of each compound;

establishing an accurate quality database: respectively inputting the name, molecular formula, chemical abstract number, precursor ion accurate mass number, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window of each compound, and additionally inputting a response threshold of the compound to be detected, and when the signal response of the compound to be detected exceeds the threshold, further performing secondary mass spectrometry on the corresponding precursor ions; the accurate mass number information of the compound to be tested, the precursor ions and the fragment ions thereof is shown in Table 2;

TABLE 2 accurate mass number information for the test compounds and their precursor and fragment ions

Establishing a mass spectrum library: respectively preparing standard solutions of compounds to be detected with the concentration of 100 mu g/L, directly injecting and analyzing by using a peristaltic pump equipped with an ultrahigh pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, setting a series of different collision energies, and crushing target compounds to obtain a secondary mass spectrogram of each compound; and inputting and storing all secondary mass spectrograms to obtain a mass spectrographic library of all compounds to be detected.

5. The method for screening chemical risk substances of glucocorticoids in toiletries according to claim 4, wherein: the specific steps for performing the alignment analysis are as follows:

setting a mass number extraction window, a retention time window and isotope distribution threshold parameters, and comparing and analyzing the collected sample data according to the set parameters:

first, according to the set parameters, such as mass number extraction window + -5 ppm, chromatographic peak area not less than 1 × 10⁶Extracting the accurate mass number of the precursor ions in the database; if the mass number of the precursor ions of the target compound in the database appears in the data acquired by the full scanning of the sample, and the chromatographic peak area is not less than 1 x 10⁶Then, the comparison of the accurate mass number of the precursor ions is regarded as successful;

secondly, setting the standard deviation of a retention time window to be +/-3, and if the chromatographic retention time of the precursor ions falls within the standard deviation of +/-3 of the corresponding retention time in the database, judging that the retention time matching is successful;

thirdly, performing isotope distribution calculation according to the molecular formula of the compound to be detected, wherein the set threshold value is 90%, and when the isotope distribution of the precursor ions in the sample data is compared with the database by the matching degree of 90%, the matching is regarded as successful;

fourthly, according to the comparison condition of the two characteristic fragment ions in the database and the two fragment ions acquired by the experimental result, if the deviation between the experimental value and the accurate mass number of the fragment ions in the database is not more than +/-5 ppm, the matching is regarded as successful;

fifthly, comparing the secondary mass spectrogram acquired by the experiment with a mass spectrogram library; at this stage, the precursor ions, all fragment ions and the relative ion abundance ratios are compared and if all match, the match is deemed successful.

6. The method for screening chemical risk substances of glucocorticoids in toiletries according to claim 5, wherein:

carrying out mass spectrometry on the compound to be detected in a positive ion mode;

the preparation of the standard solution comprises the following steps: 500-1000. mu.g mL of the mixture was prepared^-1The standard stock solution is stored in the dark at the temperature of 4 ℃; 10. mu.g mL of the composition^-1The mixed standard solution is diluted by methanol to prepare a series of matrix matching standard solutions with different concentrations;

the standard substance of glucocorticoid is dissolved by methanol solvent.

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