CN107543891B - Screening method for androgen chemical risk substances in washing and caring products - Google Patents

Abstract

The invention discloses a screening method for androgen chemical risk substances in toiletries, which comprises the following steps: (1) establishing an accurate mass database and a mass spectrum library of a compound to be tested; (2) pre-processing and detection of actual samples: The toiletries samples are subjected to pretreatment steps such as extraction, centrifugation, purification, and filtration, and the obtained sample solution is subjected to ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection; (3) Step (2) The obtained test results are compared and analyzed with the accurate mass database and mass spectral library established in step (1) to screen androgen chemical risk substances. The screening method for androgen chemical risk substances in the toiletries established by the invention has high accuracy, strong specificity and high sensitivity, and can be suitable for daily detection and quality control of the toiletries.

Description

A screening method for androgen chemical risk substances in toiletries

technical field

The invention relates to a screening method for chemical substances, in particular to a screening method for androgen chemical risk substances in toiletries based on ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry combined technology Check method.

Background technique

1. The chemical risk substances in the toiletries have a potential harmful effect on the personal health of consumers.

From the beginning of its birth, toiletries have provided guarantee for people's pursuit of a clean, healthy and fashionable life. With the continuous development and progress of society, people's pursuit of a clean, healthy and fashionable lifestyle has also been continuously upgraded, thus driving the development of toiletries. The industry has entered the fast lane of development and upgrading. At present, the products in the toiletries market are dazzling, and functional, subdivided and specialized products are constantly emerging to meet the diversified needs of consumers.

Today, when toiletries are increasingly becoming necessities of people's lives, their safety has also received more and more attention. In 2014, Hanyi shampoo was detected by a third-party authoritative testing agency to contain the possible carcinogen acrylamide. Two of the preservatives, methylisothiazolinone and methylchloroisothiazolinone, were also found to exceed the standard by nearly two times. It is very easy to cause human skin allergies; as one of Johnson & Johnson's signature products, the biggest selling point of baby shampoo is that it does not irritate the eyes. In 2010, in the process of random inspection of domestic toiletries by the Beijing Center for Disease Control and Prevention, a considerable proportion of products were detected with phthalates, of which the detection rate of perfumes was as high as 92.3% (phthalates). Formate esters are environmental hormones that harm human reproduction); in addition, triclosan in toiletries and antibiotics and hormones in other toiletries are also potential chemical risk substances.

Long-term use of cleaning products containing the above toxic and harmful substances can lead to problems such as dark spots, atrophy and thinning of facial skin, osteoporosis, muscle atrophy, and metabolic disorders, which can seriously lead to the occurrence of cancer. In order to attract customers to buy it for the short-term effect, merchants still use the combination of suits, adding hormones and other similar active ingredients to one or several products, which is also easy to evade inspection. Therefore, its danger cannot be underestimated.

2. The supervision of chemical risk substances in toiletries is relatively lagging behind, and the prevention ability is insufficient.

According to the US National Electronic Injury Surveillance System (NEISS) statistics, from 2001 to 2009, there were a total of 7,405 safety incidents caused by various types of toiletries. The European Union's Rapid Alert System for Non-Food Consumer Products (RAPEX), the US Consumer Product Safety Commission (CPSC), and the US Food and Drug Administration (FDA) have also issued risk warnings for illegally adding chemical substances to toiletries.

In recent years, my country has invested more and more financial, human and material resources in the safety supervision of toiletries, but in the face of intensifying product safety incidents, it still seems powerless. The main problem is that my country's supervision mode is mainly based on post-event remediation, and efforts to prevent products in the early stage are still insufficient. The main problem is that developed countries have repeatedly introduced new regulations and systems, resulting in frequent recalls of products in my country, such as In 2013, Johnson & Johnson's baby toiletries were recalled 48 times; Procter & Gamble (China) Co., Ltd. recalled problems such as mouthwash in 2011.

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

At present, for the quantitative detection of target compounds, domestic and foreign researches mainly use triple quadrupole tandem mass spectrometry. However, the detection method of toiletries using high-resolution mass spectrometry has not been reported.

SUMMARY OF THE INVENTION

With the increasingly stringent regulations, more and more chemical risk substances are required to be detected. The traditional method of detection through multiple reaction monitoring mode can no longer meet the requirements of such high-throughput screening. The determination of chemical risk substances should be carried out rapidly. and high-throughput development. The resolution and mass accuracy of high-resolution mass spectrometry are significantly better than triple quadrupole mass spectrometry, and it is a type of mass spectrometry that can be qualitative and quantitative at the same time; it can achieve high resolution in a wide mass range and obtain accurate molecular weights of substances; obtain true isotopic distribution; High-sensitivity tandem mass spectrometry enables accurate mass determination of precursor and product ions. Therefore, the technical problem to be solved by the present invention is to provide a method with high resolution and mass accuracy, which can obtain accurate molecular weight information of substances in a wide mass range, obtain true isotope distribution, and have the function of high-sensitivity tandem mass spectrometry, which can realize parent ions and daughter ions and daughters. Screening method for chemical risk substances in toiletries by accurate mass determination of ions.

A method for detecting androgen chemical risk substances in toiletries, comprising the following steps:

Pretreatment and detection of samples: The samples of toiletries are subjected to pretreatment steps such as extraction, centrifugation, purification, and filtration, and the obtained sample solution is subjected to ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection; In the above-mentioned ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the chromatographic separation conditions are as follows:

Chromatographic column: HSS T3 chromatographic column, length 100mm, inner diameter 2.1mm, particle size 1.8μm; column temperature: 25°C; flow rate: 0.35mL min ^-1 ; injection volume: 5μL;

The androgens include trenbolone, testosterone, nandrolone, boldenone, androstenedione, oxandrolone, dihydrotestosterone, clotestosterone acetate and testosterone propionate.

The method for detecting androgen chemical risk substances in toiletries according to the present invention, wherein, in the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the mobile phase and gradient elution procedures See Table 1:

Table 1 Chromatographic mobile phase and gradient elution procedure

In the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the quadrupole-electrostatic field orbitrap high-resolution mass spectrometry detection conditions are as follows:

Electrospray voltage: positive ion mode 3.2kV; sheath gas pressure: 36, arbitrary unit; auxiliary gas pressure: 6, arbitrary unit; ion source temperature: 330℃; transmission metal capillary temperature: 300℃; lens RF voltage: 45V; scan Range: mass-to-charge ratio 100-800; MS full scan resolution: 70000, full width at half maximum; Orbitrap maximum capacity: 1×10 ⁶ ; Orbitrap maximum injection time: 95ms;

Data-dependent secondary product ion full scan resolution: 17500, full width at half maximum; isolation window: mass-to-charge ratio ±2; normalized collision energy: 20, 40, 60 eV; maximum orbitrap capacity: 1×10 ⁵ ; Orbitrap maximum injection time: 95ms; dynamic exclusion time: 5s.

The method for detecting androgen chemical risk substances in the toiletries of the present invention, wherein, the pretreatment comprises the following steps:

Weigh 0.1 g of a sample of toiletries into a 10 mL plastic centrifuge tube, add 1 mL of saturated sodium chloride solution, then add 5 mL of methanol and 2 mL of tetrahydrofuran, fully vortex for 30 s, and ultrasonically extract for 15 min; the extract is centrifuged at 12,000 rpm for 15 min , suck the supernatant extract, filter through a 0.22 μm microporous membrane, and carry out ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection.

A screening method for androgen chemical risk substances in toiletries, comprising the following steps:

(1) Establish an accurate mass database and mass spectral library of the compound to be tested. The accurate mass database includes compound name, molecular formula, chromatographic retention time, and accurate mass information of one precursor ion and two characteristic fragment ions. The mass spectrometer The spectral library includes the secondary mass spectra generated by applying different collision energies to the compounds to be tested;

(2) sample pretreatment and detection method of the present invention;

(3) Compare and analyze the test results obtained by the sample pretreatment and detection methods with the established accurate mass database and mass spectral library of the compound to be tested. Only the accurate mass and chromatographic retention of the current precursor ion and two characteristic fragment ions are When the time, the isotopic peak distribution of precursor ions and the secondary mass spectrum all match with the accurate mass database and mass spectral library information, it can be determined that the compound to be tested is screened and detected in the actual sample.

The screening method for androgen chemical risk substances in the toiletries of the present invention, wherein, the method for establishing an accurate mass database and a mass spectrometry library of the compound to be tested specifically includes the following steps:

Accurate mass database: respectively prepare standard solutions of the compounds to be tested with a concentration of 100 μg/L, and directly inject the samples with the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer. Perform analysis and detection in mode to determine the exact mass of the precursor ion of the corresponding compound to be tested;

Apply collision energy to each compound to be tested to obtain fragment ions of each compound, and select two fragment ions with higher response intensities as characteristic fragment ions;

In the above process, the key parameters of mass spectrometry such as electrospray voltage, ion source temperature, sheath gas pressure and resolution were optimized respectively;

Prepare a mixed standard solution of the compound to be tested with a concentration of 100 μg/L, optimize the separation conditions of ultra-high pressure liquid chromatography, and obtain the chromatographic retention time of each compound;

Establish an accurate mass database: Enter the name, molecular formula, CAS number, accurate mass number of precursor ions, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window for each compound separately, as well as the compound to be tested When the signal response of the compound to be tested exceeds this threshold, the corresponding precursor ion is further analyzed by secondary mass spectrometry; the exact mass information of the compound to be tested and its precursor ions and fragment ions is shown in Table 2;

Table 2 Accurate mass information of the compounds to be tested and their precursor ions and fragment ions

Establish a mass spectrometer library: prepare standard solutions of the compounds to be tested with a concentration of 100 μg/L respectively, and use the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer to directly inject samples for analysis, and set up a series of With different collision energies, crush the target compound to obtain the secondary mass spectrum of each compound; input and store all secondary mass spectra to obtain the mass spectrum library of all the compounds to be tested.

The mass spectrum of the androgen chemical risk substance in the present invention is shown in FIG. 1 , wherein, the relationship between each code is as follows: 1. Trenbolone; 2. Testosterone; 3. Nandrolone; 4. Boldenone; 5. Androgen 6. Oxandrolone; 7. Dihydrotestosterone; 8. Chlortestosterone acetate; 9. Testosterone propionate.

The screening method for chemical risk substances in toiletries according to the present invention, wherein the compounds to be tested are analyzed by mass spectrometry in a positive ion mode;

The preparation of the standard solution includes the following steps: prepare standard stock solutions of 500-1000 μg mL ^-1 respectively, and store them in the dark at 4°C; prepare a mixed standard solution of 10 μg mL ^-1 , which is then diluted with methanol to prepare a series of different concentrations The matrix matched standard solution;

Androgens were dissolved in methanol solvent. The difference between the screening method for androgen chemical risk substances in the toiletries of the present invention and the prior art is that:

The invention establishes a screening method for androgen chemical risk substances in toiletries based on ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry technology, which will effectively solve the problem of androgen in toiletries. Screening and confirming technical problems can be used to carry out risk assessment of toxic and harmful substances in toiletries and formulation of limit standards, thereby reducing the probability of injury to consumers caused by chemical risk substances and creating a healthy and good consumption environment; research; The results can also guide enterprises to avoid the use of chemical risk substances that lead to potential safety hazards in the production process of products, so that consumers can more clearly understand the potential hazards of chemical risk substances in toiletries, thereby improving the overall safety consumption concept of the society, and producing good results. social benefit.

The relevant laws, regulations and standards of toiletries are the main means and technical basis for measuring and controlling the quality characteristics of products. Only by understanding these laws, regulations and standards, can enterprises effectively promote the healthy and standardized development of toiletries, so that products can obtain Good economic benefits. The screening method established by the invention can provide effective technical support for relevant testing institutions, further improve and improve various management regulations and standards of the toiletries industry, standardize the market economic rules and business order of the toiletries industry, and improve the cleaning and care products industry. The ability of technological innovation and shortening the technological gap with the international toiletries industry will have important economic significance for accelerating the sustainable and healthy development of related Chinese enterprises.

The method of the invention has high resolution and mass accuracy, can obtain the accurate molecular weight of the substance in a wide mass range, obtains the real isotope distribution, has the function of high-sensitivity tandem mass spectrometry, and realizes the accurate mass determination of parent ions and product ions. Toiletries Screening methods for androgen chemical risk substances in China.

The invention establishes a rapid screening and quantitative analysis strategy for androgens in toiletries based on ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry combined with an accurate mass database and a spectral library. Through the investigation of the detection limit, quantification limit, linear relationship, stability and matrix effect of the method of the present invention and the detection of actual samples, the rapid analysis method established by the present invention has high accuracy, strong specificity and high sensitivity, and can be applied to Daily inspection and production quality control of toiletries.

The screening method for androgen chemical risk substances in the toiletries of the present invention will be further described below with reference to the accompanying drawings.

Description of drawings

Fig. 1 is the mass spectrum of androgen chemical risk substance in the present invention, wherein, the reference relationship of each code is as follows: 1. Trenbolone; 2. Testosterone; 3. Nandrolone; 4. Boldenone; 5. Androstendi 6. Oxandrolone; 7. Dihydrotestosterone; 8. Chlortestosterone acetate; 9. Testosterone propionate.

Detailed ways

Example 1

A method for detecting androgen chemical risk substances in toiletries, comprising the following steps:

The samples of toiletries are extracted, centrifuged, purified, filtered and other pretreatment steps, and the obtained sample solution is subjected to ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection; ultra-high pressure liquid chromatography-quadrupole In the rod-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the chromatographic separation conditions are as follows:

Chromatographic column: HSS T3 chromatographic column, length 100mm, inner diameter 2.1mm, particle size 1.8μm; column temperature: 25°C; flow rate: 0.35mL min ⁻¹ ; injection volume: 5μL, mobile phase and gradient elution procedure are shown in Table 1 :

Table 1 Chromatographic mobile phase and gradient elution procedure

In the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the quadrupole-electrostatic field orbitrap high-resolution mass spectrometry detection conditions are as follows:

Electrospray voltage: positive ion mode 3.2kV; sheath gas pressure: 36, arbitrary unit; auxiliary gas pressure: 6, arbitrary unit; ion source temperature: 330℃; transmission metal capillary temperature: 300℃; lens RF voltage: 45V; scan Range: mass-to-charge ratio 100-800; MS full scan resolution: 70000, full width at half maximum; Orbitrap maximum capacity: 1×10 ⁶ ; Orbitrap maximum injection time: 95ms;

Data-dependent secondary product ion full scan resolution: 17500, full width at half maximum; isolation window: mass-to-charge ratio ±2; normalized collision energy: 20, 40, 60 eV; maximum orbitrap capacity: 1×10 ⁵ ; Orbitrap maximum injection time: 95ms; dynamic exclusion time: 5s.

Preprocessing includes the following steps:

Weigh 0.1 g of a sample of toiletries into a 10 mL plastic centrifuge tube, add 1 mL of saturated sodium chloride solution, then add 5 mL of methanol and 2 mL of tetrahydrofuran, fully vortex for 30 s, and ultrasonically extract for 15 min; the extract is centrifuged at 12,000 rpm for 15 min , suck the supernatant extract, filter through a 0.22 μm microporous membrane, and carry out ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection.

The androgens include trenbolone, testosterone, nandrolone, boldenone, androstenedione, oxandrolone, dihydrotestosterone, clotestosterone acetate and testosterone propionate.

Using the method of Example 1, 30 kinds of toiletries samples were tested for androgen chemical risk substances. The results showed that no samples were screened to contain banned androgens.

Example 2

1. Instruments and Reagents

Dionex UltiMate 3000 fast high-performance liquid chromatography system (Thermo Fisher, USA); QExactive Focus quadrupole-electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher, USA); Milli-Q ultrapure water instrument (Millipore, USA); Vortex-Genie 2 vortex shaker (Scientific Industries, USA).

Methanol, acetonitrile (chromatographically pure, Thermo Fisher Company, USA); ammonium hydroxide, formic acid (chromatographically pure, DimaTechnology Company, USA); laboratory water is deionized water. Carbon eighteen, N-propylethylenediamine and graphitized carbon black (Macherey-Nagel, Germany); multi-walled carbon nanotubes (Miltenyi Biotec GmbH, Germany); anhydrous magnesium sulfate, sodium chloride (Guangzhou Xilong Chemical Co., Ltd.) Limited Liability Company); 0.22 μm microporous membrane (Pall Corporation, USA).

Prepare standard stock solutions of 500-1000 μg mL ^-1 respectively, and store them at 4°C in the dark. A mixed standard solution of 10 μg mL ^-1 was prepared and then diluted with methanol to prepare a series of matrix-matched standard solutions of different concentrations.

The molecular formula, molecular weight, CAS number, oil-water partition coefficient and preparation solvent of the target compound are shown in Table 3.

Table 3 Name, molecular formula, molecular weight, CAS number, oil-water partition coefficient and preparation solvent of the target compound

2. Establishment of accurate mass database and mass spectral library

The accurate mass database includes compound names, molecular formulas (used to calculate isotopic peak distributions), retention times, and accurate mass numbers of precursor ions and two characteristic fragment ions (Table 4). The spectral library includes MS spectra generated by analysis at different collision energies for each compound. The spectral library can be regarded as an auxiliary confirmation method after the sampling data has been screened by the accurate mass database.

The method for establishing an accurate mass database and mass spectral library of a compound to be tested specifically includes the following steps:

Accurate mass database: respectively prepare standard solutions of the compounds to be tested with a concentration of 100 μg/L, and directly inject the samples with the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer. Perform analysis and detection in mode to determine the exact mass of the precursor ion of the corresponding compound to be tested;

Apply collision energy to each compound to be tested to obtain fragment ions of each compound, and select two fragment ions with higher response intensities as characteristic fragment ions;

In the above process, the key parameters of mass spectrometry such as electrospray voltage, ion source temperature, sheath gas pressure and resolution were optimized respectively;

Prepare a mixed standard solution of the compound to be tested with a concentration of 100 μg/L, optimize the separation conditions of ultra-high pressure liquid chromatography, and obtain the chromatographic retention time of each compound;

Establish an accurate mass database: Enter the name, molecular formula, CAS number, accurate mass number of precursor ions, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window for each compound separately, as well as the compound to be tested When the signal response of the compound to be tested exceeds the threshold, the corresponding precursor ion is further subjected to secondary mass spectrometry analysis;

Establish a mass spectrometer library: prepare standard solutions of the compounds to be tested with a concentration of 100 μg/L respectively, and use the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer to directly inject samples for analysis, and set up a series of With different collision energies, crush the target compound to obtain the secondary mass spectrum of each compound; input and store all secondary mass spectra to obtain the mass spectrum library of all the compounds to be tested.

The mass spectrum of the androgen chemical risk substance in the present invention is shown in FIG. 1 , wherein, the relationship between each code is as follows: 1. Trenbolone; 2. Testosterone; 3. Nandrolone; 4. Boldenone; 5. Androgen 6. Oxandrolone; 7. Dihydrotestosterone; 8. Chlortestosterone acetate; 9. Testosterone propionate.

3. Sample pretreatment

Weigh 0.1 g of a sample of toiletries into a 10 mL plastic centrifuge tube, add 1 mL of saturated sodium chloride solution, then add 5 mL of methanol and 2 mL of tetrahydrofuran, fully vortex for 30 s, and ultrasonically extract for 15 min; the extract is centrifuged at 12,000 rpm for 15 min , suck the supernatant extract, filter through a 0.22 μm microporous membrane, and carry out ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection.

4. Chromatographic separation conditions

Chromatographic column: HSS T3 chromatographic column, length 100mm, inner diameter 2.1mm, particle size 1.8μm; column temperature: 25°C; flow rate: 0.35mL min ⁻¹ ; injection volume: 5μL, mobile phase and gradient elution procedure are shown in Table 1 :

Table 1 Chromatographic mobile phase and gradient elution procedure

Five, mass spectrometry detection conditions

Electrospray voltage: positive ion mode 3.2kV; sheath gas pressure: 36, arbitrary unit; auxiliary gas pressure: 6, arbitrary unit; ion source temperature: 330℃; transmission metal capillary temperature: 300℃; lens RF voltage: 45V; scan Range: mass-to-charge ratio 100-800; MS full scan resolution: 70000, full width at half maximum; Orbitrap maximum capacity: 1×10 ⁶ ; Orbitrap maximum injection time: 95ms;

Data-dependent secondary product ion full scan resolution: 17500, full width at half maximum; isolation window: mass-to-charge ratio ±2; normalized collision energy: 20, 40, 60 eV; maximum orbital capacity: 1×10 ⁵ ; Orbitrap maximum injection time: 95ms; dynamic exclusion time: 5s.

6. Comparative analysis of experimental data

Compare and analyze the results of the sample detection experiment data with the established accurate mass database and mass spectral library of the compounds to be tested. Only the accurate mass numbers of the current precursor ion and two characteristic fragment ions, the chromatographic retention time, and the isotopic peaks of the precursor ions are analyzed. A positive sample is only shown when the distribution and MS spectra match all of the accurate mass database and mass spectral library.

Specifically include the following steps:

Set the mass extraction window, retention time window and isotope distribution threshold parameters, and compare and analyze the collected sample data according to the set parameters:

First, according to the set parameters, such as mass number extraction window ±5ppm, chromatographic peak area not less than 1×10 ⁶ , extract the accurate mass number of precursor ions in the database; if the target in the database appears in the sample full scan acquisition data The precursor ion mass number of the compound, and the chromatographic peak area is not less than 1×10 ⁶ , it is considered that the accurate mass number comparison of the precursor ion is successful;

Second, the chromatographic retention time, the standard deviation of the set retention time window is ±3, if the chromatographic retention time of the precursor ion falls within the range of ±3 standard deviation of the corresponding retention time in the database, it is considered that the retention time matching is successful;

Third, according to the molecular formula of the compound to be tested, the isotope distribution is calculated, and the set threshold is 90%. When the isotope distribution of the precursor ions in the sample data is 90% matched with the database comparison, it is regarded as a successful match;

Fourth, according to the comparison between the two characteristic fragment ions in the database and the two fragment ions collected from the experimental results, if the deviation between the experimental value and the accurate mass of the fragment ions in the database does not exceed ±5ppm, the matching is considered successful;

Fifth, compare the secondary mass spectrum acquired in the experiment with the mass spectrum library; at this stage, the precursor ions, all fragment ions and relative ion abundance ratios are compared, and if they all match, it is regarded as a match success.

7. Results

Using the method of this Example 2, 30 kinds of toiletries samples were tested for androgen chemical risk substances. The results showed that no samples were screened to contain banned androgens.

8. Discussion

1. Establishment of screening methods

In order to give full play to the advantages of electrostatic field orbitrap high-resolution mass spectrometry in screening analysis, this study adopted a full-scan-data-dependent secondary product ion scanning method. In this acquisition method, the mass spectrum is first scanned at the full mass (m/z 100-800). When the ions of some target compounds in the database are detected and their intensity is not lower than a certain set threshold, the The ions are sent to a high-energy collision cell for fragmentation, which produces fragmented product ions.

In the compound screening stage, the detection of target substances is mainly based on three key parameters: accurate mass of precursor ions, retention time and isotopic peak distribution. The setting of the accurate mass extraction window has a crucial impact on the detection sensitivity and selectivity of the method. In the present invention, a mixed standard solution with a concentration of 100 μg L ^-1 and a spiked sample with a concentration of 500 μg kg ^-1 were used to optimize this parameter. The experimental results show that in the two solutions, the mass deviation is better than 2ppm and 3.5ppm, respectively. Considering the subsequent confirmation of characteristic fragment ions and secondary spectra, and trying to avoid the occurrence of false negative results, this study set the extraction window of the mass number to 5 ppm, which can not only ensure no false negative results, but also prevent the occurrence of false negative results. The selectivity and sensitivity of the method detection were not significantly affected. For the retention time parameters, the reasonable setting of the retention time window plays an important role in the accuracy of the screening results. Due to the application of different high-resolution mass spectrometry systems and the operation of different personnel, there may be slight drift in the retention time. Therefore, in this study, the retention time window was set as the average retention time ±3×the standard deviation of the retention time. In the full-scan stage of mass spectrometry, since different resolution settings also have certain influence on the selectivity and sensitivity of the method, this study also carried out detailed optimization of the mass spectrometry resolution parameters. In the experiment, three different resolutions (30000, 50000, 70000 full width at half maximum) were set for data collection respectively. The results showed that at the resolution of 70000 full width at half maximum, the mass accuracy of all target compounds was the highest, and the sensitivity was higher than before. There is no significant difference between the two, and it still provides 10-15 scan points for excellent chromatographic peak shape.

In the confirmation phase, this study acquired MS MS spectra for each target compound and recorded two characteristic fragment ion accurate masses for each compound. In the first stage of mass spectrometry scanning, the resolution of 70000 full width at half maximum has provided high enough selectivity. At this time, sensitivity should be the main aspect to consider. Therefore, in the confirmation stage, we set the mass spectrometry resolution to be lower And the commonly used 17500 full width at half maximum. In order to obtain a more complete secondary mass spectrum, the normalized collision energies of 20, 40 and 60 eV were used in this study, and the accurate mass numbers of two fragment ions with higher response intensity were recorded into the database. In the high-resolution mass spectrometry analysis, some key parameters such as spray voltage, capillary temperature, curtain gas, purge gas and lens RF voltage were also optimized in detail.

2. Linear relationship, detection limit and quantification limit

The mixed standard stock solution was sequentially diluted into a mixed working solution with a series of concentration gradients with the treated sample solution, and the measurement was carried out under the optimized chromatographic mass spectrometry conditions. The molecular ion peak area (y) of each target substance was used to measure the concentration (x). Linearity investigation, the linear range, detection limit and quantification limit of the target compound are shown in Table 5. The linear regression coefficients of the target compounds were all greater than 0.99, indicating that the method had a good linear relationship and could be accurately quantified. The detection limit and quantification limit of the target compound were calculated with 3-fold and 10-fold signal-to-noise ratio, respectively, and the results are shown in Table 5.

The above-mentioned embodiments merely describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.

Claims (5)

1. the detection method of androgen chemical risk substance in a kind of toiletries, is characterized in that: comprise the steps: Sample pretreatment and detection method: Weigh 0.1g of the toiletries sample into a 10mL plastic centrifuge tube, add 1mL saturated sodium chloride solution, then add 5mL methanol and 2mL tetrahydrofuran, fully vortex for 30s, and extract ultrasonically for 15min; The solution was centrifuged at 12,000 rpm for 15 min, and the supernatant extract was drawn, filtered through a 0.22 μm microporous membrane, and then subjected to ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection; Androgens include trenbolone, testosterone, nandrolone, boldenone, androstenedione, oxandrolone, dihydrotestosterone, clotestosterone acetate, and testosterone propionate; In the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the ultra-high pressure liquid chromatography separation conditions are as follows: Chromatographic column: HSS T3 chromatographic column, length 100mm, inner diameter 2.1mm, particle size 1.8μm; column temperature: 25°C; flow rate: 0.35mLmin ^-1 ; injection volume: 5μL; The mobile phase and gradient elution procedure are shown in Table 1: Table 1 Chromatographic mobile phase and gradient elution procedure

In the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry analysis and detection, the quadrupole-electrostatic field orbitrap high-resolution mass spectrometry detection conditions are as follows: Electrospray voltage: positive ion mode 3.2kV; sheath gas pressure: 36, arbitrary unit; auxiliary gas pressure: 6, arbitrary unit; ion source temperature: 330℃; transmission metal capillary temperature: 300℃; lens RF voltage: 45V; scan Range: mass-to-charge ratio 100-800; MS full scan resolution: 70000, full width at half maximum; Orbitrap maximum capacity: 1×10 ⁶ ; Orbitrap maximum injection time: 95ms; Data-dependent secondary product ion full scan resolution: 17500, full width at half maximum; isolation window: mass-to-charge ratio ±2; normalized collision energy: 20, 40, 60 eV; maximum orbitrap capacity: 1×10 ⁵ ; Orbitrap maximum injection time: 95ms; dynamic exclusion time: 5s. 2. a screening method for androgen chemical risk substances in the toiletries, is characterized in that: comprise the steps: (1) establishing the accurate mass database and mass spectral library of the compound to be tested, and the method for establishing the accurate mass database specifically comprises the following steps: Accurate mass database: respectively prepare standard solutions of the compounds to be tested with a concentration of 100 μg/L, and directly inject the samples with the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer. Perform analysis and detection in mode to determine the exact mass of the precursor ion of the corresponding compound to be tested; Apply collision energy to each compound to be tested to obtain fragment ions of each compound, and select two fragment ions with higher response intensities as characteristic fragment ions; In the above process, the key parameters of mass spectrometry such as electrospray voltage, ion source temperature, sheath gas pressure and resolution were optimized respectively; Prepare a mixed standard solution of the compound to be tested with a concentration of 100 μg/L, optimize the separation conditions of ultra-high pressure liquid chromatography, and obtain the chromatographic retention time of each compound; Establish an accurate mass database: Enter the name, molecular formula, CAS number, accurate mass number of precursor ions, accurate mass numbers of two characteristic fragment ions, chromatographic retention time and retention time window for each compound separately, as well as the compound to be tested When the signal response of the compound to be tested exceeds this threshold, the corresponding precursor ion is further analyzed by secondary mass spectrometry; the exact mass information of the compound to be tested and its precursor ions and fragment ions is shown in Table 2; Table 2 Accurate mass information of the compounds to be tested and their precursor and fragment ions

The mass spectrum library includes secondary mass spectra generated by applying different collision energies to the compounds to be tested; (2) sample pretreatment and detection method as claimed in claim 1; (3) Compare and analyze the test results obtained by the sample pretreatment and detection methods with the established accurate mass database and mass spectral library of the compound to be tested. Only the accurate mass and chromatographic retention of the current precursor ion and two characteristic fragment ions are When the time, the isotopic peak distribution of precursor ions and the secondary mass spectrum all match with the accurate mass database and mass spectral library information, it is determined that the compound to be tested is screened and detected in the actual sample. 3. the screening method of androgen chemical risk substance in the toiletries according to claim 2, is characterized in that: described mass spectrometry library establishment method specifically comprises the steps: The standard solutions of the compounds to be tested with a concentration of 100 μg/L were prepared respectively, and the samples were directly injected and analyzed by the peristaltic pump equipped with the ultra-high pressure liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometer, and a series of different collision energies were set. The target compound is smashed to obtain the secondary mass spectrum of each compound; all secondary mass spectra are input and stored, that is, the mass spectrum library of all the compounds to be tested is obtained. 4. the screening method of chemical risk substance in the toiletries according to claim 3, is characterized in that: the concrete steps of carrying out comparative analysis are as follows: Set the mass extraction window, retention time window and isotope distribution threshold parameters, and compare and analyze the collected sample data according to the set parameters: First, according to the set parameters, such as mass number extraction window ±5ppm, chromatographic peak area not less than 1×10 ⁶ , extract the accurate mass number of precursor ions in the database; if the target in the database appears in the sample full scan acquisition data The precursor ion mass number of the compound, and the chromatographic peak area is not less than 1×10 ⁶ , it is considered that the accurate mass number comparison of the precursor ion is successful; Second, the chromatographic retention time, the standard deviation of the set retention time window is ±3, if the chromatographic retention time of the precursor ion falls within the range of ±3 standard deviation of the corresponding retention time in the database, it is considered that the retention time matching is successful; Third, according to the molecular formula of the compound to be tested, the isotope distribution is calculated, and the set threshold is 90%. When the isotope distribution of the precursor ions in the sample data is 90% matched with the database comparison, it is regarded as a successful match; Fourth, according to the comparison between the two characteristic fragment ions in the database and the two fragment ions collected from the experimental results, if the deviation between the experimental value and the accurate mass of the fragment ions in the database does not exceed ±5ppm, the matching is considered successful; Fifth, compare the secondary mass spectrum acquired in the experiment with the mass spectrum library; at this stage, the precursor ions, all fragment ions and relative ion abundance ratios are compared, and if they all match, it is regarded as a match success. 5. the screening method of chemical risk substance in the toiletries according to claim 4, is characterized in that: The compounds to be tested were analyzed by mass spectrometry in positive ion mode; The preparation of the standard solution includes the following steps: prepare standard stock solutions of 500-1000 μg mL ^-1 respectively, and store them in the dark at 4°C; prepare a mixed standard solution of 10 μg mL ^-1 , which is then diluted with methanol to prepare a series of different concentrations The matrix matched standard solution; Androgens were dissolved in methanol solvent.