CN113671082A - Gas chromatography-ultra-high resolution mass spectrometry-based method for screening migration hazards in gutta-percha - Google Patents

Gas chromatography-ultra-high resolution mass spectrometry-based method for screening migration hazards in gutta-percha Download PDF

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CN113671082A
CN113671082A CN202110958078.4A CN202110958078A CN113671082A CN 113671082 A CN113671082 A CN 113671082A CN 202110958078 A CN202110958078 A CN 202110958078A CN 113671082 A CN113671082 A CN 113671082A
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gutta
percha
migration
mass
hazards
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吕庆
刘雅慧
王志娟
王婉
张庆
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Chinese Academy of Inspection and Quarantine CAIQ
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention discloses a method for screening migration hazards in gutta-percha based on gas chromatography-ultrahigh resolution mass spectrometry, which comprises the following steps: sample processing, namely analyzing by adopting a gas chromatography-ultra-high resolution mass spectrometry method, performing deconvolution processing on acquired data, retrieving a mass spectrogram of an unknown substance in a commercial standard spectrogram library, and identifying by utilizing isotope information in an EI spectrogram; and measuring the molecular ions and molecular formulas of the unknown substances through data obtained in a PCI mode of mass spectrum. The invention provides a brand new research path, is different from the traditional target detection of target substances, can find more non-target potential unknown hazards, can realize accurate qualitative identification, and has positive effects on monitoring potential chemical migration hazards in toys such as gutta percha and the like and promoting the quality safety of products.

Description

Gas chromatography-ultra-high resolution mass spectrometry-based method for screening migration hazards in gutta-percha
Technical Field
The invention relates to a method for screening hazards, in particular to a method for screening the migration hazards in gutta percha based on gas chromatography-ultrahigh resolution mass spectrometry.
Background
Infants under 1 year old can exercise hand-eye coordination and initially explore the world by biting things. The gutta-percha is a special toy for infants and is mainly made of silica gel or TPU plastic. By chewing the gutta-percha, discomfort in the teething period can be effectively relieved, the growth of deciduous teeth is stimulated, and the chewing pleasure is increased. Potentially chemically hazardous substances in gutta percha can be introduced during manufacture (using chemical additives and processing aids, including impurities and unintentionally added substances), packaging, shipping, and storage, and can be transferred into the infant through saliva, causing cumulative chemical hazards. Currently, gutta-percha products are managed worldwide with reference to the regulations of toys and children's products. The primary regulations include European Community toy safety directives 2009/48/EC, European Community Committee toy safety coordination Standard EN71, the U.S. Consumer safety improvement Act, the list of highly interesting chemicals in the Washington State Children's safety protection Act (the CHCC list), the national Standard of toy safety GB6675-2014, and the like.
In recent years, the detection of chemical substances in toys such as gutta percha has been limited mainly to the list of substances prescribed by the above-mentioned regulations. Researchers have sequentially detected heavy metals, phthalates, nitrosamines, preservatives, bisphenol a, and other substances in gutta percha in order to determine whether the product meets regulatory requirements. Some researchers have further conducted risk assessment studies of these substances in order to explore the hazards these chemicals pose to infants. Although some target chemical indicators in gutta percha meet the regulatory requirements, the "qualified" product is not necessarily absolutely "safe" and the unknown chemical risks present in the product still pose a potential threat to the health of the infant.
Harmful chemical substances in the gutta-percha toy can enter the bodies of infants through contact with saliva in the oral cavity, and the health of the infants is harmed. In recent years, researchers have conducted some research (i.e., routine targeted testing) on known target chemicals in gutta-percha and other toys, but no unknown chemicals have been reported to be present in such products.
Disclosure of Invention
The identification of unknown substances usually requires a complicated analysis process, the analysis time is long, the final identification result must be confirmed at multiple confirmation points, and high reliability is required. The current method is gas chromatography-low resolution mass spectrometry (GC-MS) combined with commercial standard spectral libraries or self-established spectral libraries for retrieval. Qualitative analysis of unknown substances relies on matching of measured and standard spectra. However, if the spectra of different compounds are very similar, or the difference in fragment ions between isomers is very subtle, the low resolution mass spectrometry identification results are inaccurate. The resolution of the ultrahigh resolution mass spectrum and the electrostatic field Orbitrap mass spectrum (Orbitrap, with the highest resolution of 12-24 ten thousand) is far higher than that of the traditional low resolution mass spectrum (such as quadrupole and unit resolution) and common high resolution mass spectra such as time of flight mass spectrum (TOF, with the highest resolution of 1-2 ten thousand), sufficient and accurate chemical element mass information is provided, so that researchers can determine unknown element compositions and chemical structures of substances by utilizing natural isotope ratio and fragment information. The GC-Orbitrap MS has the advantages of high resolution, high sensitivity, high quality and precision, wide dynamic range, spectrum data deconvolution function and the like, and has great potential in the aspect of carrying out analysis on unknown chemical substances in the gutta percha.
The aim of the present invention is to non-target screen for unknown chemical risk substances that may migrate from gutta-percha to infant saliva based on GC-Orbitrap MS. To date, this is the first report of gas chromatography-ultra high resolution mass spectrometry to study unknown chemical risk substances in gutta percha toys.
The invention discloses a method for screening migration hazards in gutta-percha based on gas chromatography-ultrahigh resolution mass spectrometry, which comprises the following steps:
step one, sample treatment: cutting gutta-percha into small pieces, performing an experiment in simulated saliva to obtain a migration solution, adding an ethyl acetate-n-hexane mixed solution for liquid-liquid extraction, vibrating, standing and concentrating to obtain a liquid to be detected, and performing detection on a machine;
analyzing the liquid to be detected by adopting a gas chromatography-ultra-high resolution mass spectrometry method, wherein an EI mode and a PCI mode are respectively adopted for analysis during mass spectrometry detection;
thirdly, obtaining the accurate mass number of fragment ions of unknown substances according to the result of full-scan detection on the extracting solution of the gutta-percha sample in an EI mode of a mass spectrum, carrying out deconvolution processing on the collected data, removing interference ions to obtain a clean mass spectrogram, deducting impurity peaks existing in a solvent or a system through comparison with a blank sample, and identifying unique chromatographic peaks in the gutta-percha;
searching the mass spectrogram of the unknown substance in a commercial standard spectrogram library, and further filtering the matching result by using the accurate mass information of each ion peak;
step five, when a plurality of candidate results with similar retrieval scores exist in a certain unknown object, identifying by utilizing isotope information in an EI spectrogram;
and step six, measuring the molecular ions and molecular formulas of the unknown substances through data obtained in the PCI mode of the mass spectrum, and further selecting the best matching from the candidate results.
The invention relates to a method for screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry, wherein in the first step, the sample treatment specifically comprises the following steps:
cutting the smooth part of the surface of the gutta-percha sample into (10 +/-1) cm2Weighing, placing into a 40mL glass colorimetric tube with a plug, adding 20mL simulated saliva into the colorimetric tube, shaking the sample in a water bath at 37 ℃ and 100r/min for 240min to obtain a migration solution, transferring the migration solution to a separation funnel, adding 5mL of an ethyl acetate-n-hexane mixed solution with the volume ratio of 1:1, vibrating for 1min, standing for 30min, concentrating the organic phase to 0.5mL under the mild nitrogen flow purging, and measuring on a machine.
The invention relates to a method for screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry, wherein the chromatographic separation conditions in the second step are as follows:
HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrying high-purity helium (99.999%) with the flow rate of 1 mL/min; sample introduction volume: 1 μ L, the injection port temperature is 280 ℃, and the temperature rise program: the initial temperature was 40 deg.C, held for 1min, then ramped up to 310 deg.C at a rate of 8 deg.C/min, held for 5 min.
The invention relates to a method for screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry, wherein in the second step, when the mass spectrometry adopts an EI mode, a chromatograph performs split sample injection with a split ratio of 10:1, and when the mass spectrometry adopts a PCI mode, the chromatograph does not perform split sample injection;
the mass spectrometry conditions were as follows: the method comprises the steps of performing Full-scanning Full-Scan in a Full-Scan mode, scanning the mass range of ions within 50-600 m/z, analyzing each sample by adopting an EI mode and a PCI mode respectively, wherein EI ionization energy is 70eV, when the PCI mode is adopted, methane is used as a reaction gas, the flow rate of the methane is 1.5mL/min, the temperatures of a transmission line and an ion source are 250 ℃ and 280 ℃ respectively, C-Trap and HCD collision gases are high-purity nitrogen (99.999%), the solvent delay time is 3.5min, under the condition of m/z 200, the mass resolution is set to be 60000FWHM, and the TIC intensity threshold is 1e6The maximum injection time was set to 200ms and the mass tolerance window was set to 5 ppm.
The invention relates to a method for screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry, wherein data deconvolution parameters in the third step are as follows:
deconvoluting with Tracefinder resolution plug software, setting the mass number tolerance window range of the extracted ion current to + -5 ppm to reduce the chemical substance interference in the matrix, obtaining the chromatogram of the extracted ion current with high selectivity, setting the accurate mass deviation to + -5 ppm, the threshold of signal-to-noise ratio to be 3, and the threshold of TIC intensity to be 1e6When the matching value of the deconvolution fragment ions is 99% and the retention time correction window is 10s, the deconvolution separation effect is the best.
The invention relates to a method for screening migration hazards in gutta percha based on gas chromatography-ultra-high resolution mass spectrometry, wherein in the retrieval and matching process of the fourth step, a positive matching index SI represents a matching value of a comparison between a measured spectrogram and a spectrogram in a standard spectrogram library, an HRF value represents an accurate mass percentage of an actually measured value of fragment ions and a corresponding fragment ion element composition in the standard spectrogram library, a retention index is also an important index of qualitative analysis, the smaller the deviation Delta RI of the retention index is, the better the comprehensive score of the retrieval is larger than 90, the SI is larger than 700, the HRF is larger than 90, and the Delta RI is smaller than 100, a plurality of interference items can be filtered, the time of data analysis is effectively reduced, and the accuracy of an identification result is improved.
The invention relates to a method for screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry, which is different from the prior art in that:
the gas chromatography-electrostatic field orbit trap ultrahigh resolution mass spectrometry detection technology established by the invention can be used for carrying out high-throughput screening and identification on unknown chemical substances which migrate from gutta-percha to saliva, and the method can be called non-targeted screening because the detection is initially a blind screen without a target. The invention provides a method for screening and identifying unknown substances in a gutta-percha toy for the first time, which comprises sample processing conditions, instrument analysis conditions, a qualitative identification process of the unknown substances and the like. In the aspect of qualitative identification of unknown substances, the cases possibly encountered are given and corresponding solutions are given from easy to difficult, and the solutions comprise multi-index comprehensive score comparison, isotope-assisted qualitative analysis of substances, Positive Chemical Ionization (PCI) identification of substance molecular formulas and the like. The invention provides a brand-new research path in the aspect of safety research of gutta-percha toy products, is different from the traditional target detection of target substances, can find more non-target potential unknown hazards, can realize accurate qualitative identification, and has positive effects on monitoring potential chemical migration hazards in gutta-percha and other toys and promoting the quality safety of products.
The method for screening the migration hazards in the gutta percha based on the gas chromatography-ultra-high resolution mass spectrometry is further described below with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a process for identifying isophorone in an example of the present invention; wherein, (A) a list of unknown chromatographic peaks detected in a sample; (B) a candidate substance list ordered according to comprehensive scores after a certain unknown chromatographic peak is subjected to spectral library retrieval; (C) a fragment ion overlay for identifying a substance; (D) comparing the actual spectrogram with the standard spectrogram; (E) the abundance of each fragment ion and the deviation between the measured accurate mass number and the theoretical accurate mass number;
FIG. 3 is an illustration of an embodiment of the invention utilizing isotopic information for assisting identification of a substance; after the spectrum library is searched, a candidate substance list is sorted according to the comprehensive scores; (B) comparing the actual spectrum with the theoretical spectrum of the benzocaine;
FIG. 4 is an illustration of the identification of molecular formula of an unknown using chemical ionization PCI in an embodiment of the present invention; wherein, (A) the materials list is sorted according to the comprehensive score after the spectrum library is searched; (B) EI spectrogram of N-methylformanilide; (C) PCI spectrum of N-methyl formanilide; note: including the measured mass, elemental composition, theoretical and measured mass deviation (ppm) of fragment ions by [ M + H [ + ]]+、[M+C2H5]+The precise mass number, confirming the presence of N-methylformanilide;
FIG. 5 is a diagram illustrating identification of an unknown in a gutta percha toy according to an embodiment of the present invention.
Detailed Description
1. Materials and methods
1.1 reagents and materials
And (3) standard substance: phenol was purchased from dr. N-hexane and ethyl acetate were chromatographically pure and purchased from j.t.baker, usa. C7-C40The normal alkane mixed solution was purchased from Sigma-Aldrich, USA.
Simulated saliva: 0.17g MgCl2·6H2O，0.15g CaCl2·6H2O，0.76g K2HPO4·2H2O，0.53g K2CO30.33g NaCI and 0.75g KCI, dissolving potassium and sodium salt in 900mL deionized water when preparing simulated saliva, adding calcium and magnesium salt, adjusting pH to 6.8 +/-0.1 by using 1% hydrochloric acid aqueous solution after completely dissolving, and then fixing the volume to 1L by using deionized water. The product is preserved in dark, and the pH value before use is ensured to be approximately equal to 6.8 +/-0.1.
Q active GC Orbitrap type gas chromatography-quadrupole-electrostatic field Orbitrap mass spectrometer equipped with Triplus RSH autosampler (Thermo Fisher, USA); an NTS-4000 constant temperature water bath shaker (Eyela, Japan); TurboVap model II automatic nitrogen-blown concentrator (Biotage, USA).
10 samples of gutta percha toys of different brands were randomly drawn from the supermarket and the marketplace in Beijing. The origin is China (Guangdong province, 5; Zhejiang province, 3; Shanghai, 1) and Korea (1). These samples were individually sealed prior to analysis to avoid cross-contamination.
1.2 sample treatment method
Cutting the smooth part of the surface of the gutta-percha sample into (10 +/-1) cm2. After weighing, the sample was placed in a 40mL glass cuvette with a stopper, and 20mL of simulated saliva was added to the cuvette. The sample was shaken in a water bath at 37 ℃ and 100r/min for 240min to obtain a migration solution. Transferring the migration solution to a separation funnel, adding 5mL (1:1, v/v) of ethyl acetate-n-hexane mixed solution, vibrating for 1min, and standing for 30 min. The organic phase was concentrated to 0.5mL under a gentle stream of nitrogen and measured on the machine.
1.3 conditions of analysis
Chromatographic separation conditions: HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrying high-purity helium (99.999%) with the flow rate of 1 mL/min; sample introduction volume: 1 μ L, injection port temperature 280 ℃. Temperature rising procedure: the initial temperature was 40 deg.C, held for 1min, then ramped up to 310 deg.C at a rate of 8 deg.C/min, held for 5 min. When the mass spectrum adopts an EI mode, the chromatograph performs split sample injection with a split ratio of 10: 1. When the mass spectrum adopts a PCI mode, the chromatograph does not split the sample injection so as to increase the sensitivity.
Mass spectrum detection conditions: the Full scanning Full-Scan mode is adopted, and the mass range of the scanned ions is 50-600 m/z. Each sample was analyzed using electron ionization (EI mode) and positive chemical ionization (PCI mode), respectively. The EI ionization energy was 70 eV. When the PCI mode is adopted, methane is used as reaction gas, and the flow rate of the methane is 1.5 mL/min. The transfer line and ion source temperatures were 250 ℃ and 280 ℃, respectively. The C-Trap and HCD collision gases were high purity nitrogen (99.999%). The solvent delay time was 3.5 min. Under the condition of m/z 200, the mass resolution is set to 60000FWHM, and the TIC intensity threshold is 1e6. The maximum injection time was set to 200ms and the mass tolerance window was set to 5 ppm.
Data deconvolution parameters: deconvolution was performed using tracefine Deconvolution plug software, and the mass number tolerance window range of the extracted ion flux was set to ± 5ppm in order to reduce chemical interference in the matrix, to obtain an extracted ion flux chromatogram with high selectivity. Setting the accurate mass deviation to be +/-5 ppm, the threshold value of the signal-to-noise ratio to be 3 and the threshold value of the TIC intensity to be 1e6When the matching value of the deconvolution fragment ions is 99% and the retention time correction window is 10s, the deconvolution separation effect is the best. 1.4 working process for screening and identifying unknown substances in gutta-percha toy
The working flow of the invention is shown in figure 1. The method comprises the following steps of firstly, carrying out full-scan detection on an extracting solution of a gutta-percha sample in an EI mode of a mass spectrum, and measuring the accurate mass number of an unknown substance by using an ultra-high resolution mass spectrum. And secondly, performing deconvolution processing on the acquired data to remove interfering ions and obtain a relatively clean mass spectrogram. The unique chromatographic peaks in the gutta percha were identified by subtracting the impurity peaks present in the solvent or system by comparison with the blank (i.e., the same procedure as the rest without the gutta percha sample in 1.2). And thirdly, retrieving the mass spectrogram of the unknown substance in a commercial standard spectrogram library, and further filtering the matching result by using the accurate mass information of each ion peak. The positive match index SI represents the match of the measured spectra against spectra in the standard library, and the HRF value represents the exact mass percentage of the measured values of fragment ions that match the elemental composition of the corresponding fragment ions in the standard library. The retention index is also an important indicator of qualitative analysis, and the smaller the retention index deviation Δ RI, the better. When the retrieved comprehensive score is larger than 90, the SI is larger than 700, the HRF is larger than 90, and the Delta RI is smaller than 100, a plurality of interference items can be filtered, the data analysis time is effectively reduced, and the accuracy of the identification result is improved. And fourthly, when a plurality of candidate results with similar retrieval scores exist in an unknown substance, identifying by utilizing isotope information and fragment ions in an EI spectrogram, measuring molecular ions and molecular formulas of the unknown substance by obtaining PCI data, and further selecting the best matching candidate result from the candidate results.
2. Results and discussion
2.1 optimization of chromatography columns and sample processing methods
In order to identify potential substances in gutta-percha, there are two necessary requirements for the choice of chromatographic column. First, the column should have good thermal stability so that more higher boiling substances are detected. Second, the column should have a certain polarity to separate substances of different polarity. The highest working temperature of the highly polar HP-INNOWAX and DB-WAX is only 250 ℃ and is not suitable for high boiling compounds. Therefore, polysiloxane columns with high thermal stability should be used, such as HP-5MS columns with weak polarity and DB-17MS or DB-35MS columns with medium polarity. In addition, retention index is an important reference index in the qualitative process of unknown substances, and is usually measured using non-polar, weakly polar and polar columns. Therefore, subsequent studies were performed using HP-5 MS.
Gutta-percha is a product that comes into contact with the oral cavity of infants. Potential chemicals in gutta-percha migrate into the body primarily through saliva contact. Therefore, the migration amount of chemical substances in artificial saliva is mainly studied. Solid Phase Extraction (SPE), Solid Phase Microextraction (SPME) and liquid-liquid extraction (LLE) are three commonly used methods for extracting substances from a migrating solution. SPE and SPME involve complex optimization processes including optimization of the extraction materials, the elution Solvent (SPE), and the adsorption and desorption conditions (SPME). Optimizing these parameters is difficult because there is no defined target analyte. The LLE method using ethyl acetate and n-hexane (1:1, v/v) as extraction solvents is simple to operate and is suitable for extracting unknown substances with different polarities in a solution. After the liquid-liquid extraction, quantitative concentration was performed using a gentle nitrogen flow purge to improve the sensitivity of the detection.
2.2 screening and identification of unknown substances in gutta-percha toys
2.2.1 preliminary identification based on spectral library retrieval composite score and Retention index
And obtaining a spectrogram with relatively clean unknown substances in the sample through data deconvolution, performing compound retrieval and high-resolution filtration of candidate compounds in an NIST standard spectrum library, and performing comprehensive sequencing on all retrieval results by combining the positive correlation score SI and the HRF value of the retrieval. Retention index is also an important indicator of qualitative analysis. In analyzing the sampleBefore the preparation, study C was carried out by the same analytical method7-C40Mixed solution of normal alkane. The retention index of the compound was calculated by retention time and compared to the retention index of the substance in the standard library. Finally, the deviation Δ RI of the retention index is calculated. The smaller Δ RI, the more reliable the results. However, there are individual compounds that do not retain index data in the NIST library, so their Δ RI cannot be calculated.
In 10 gutta percha, a total screening identified 60 chromatographic peaks, each corresponding to numerous search results. Generally, the reliability of identifying a compound is higher when the composite score is greater than 90, the SI value is greater than 700, the HRF value is greater than 90, and the Δ RI is less than 100. For example, in a candidate for a peak of an unknown substance (fig. 2B), the overall score of isophorone is 96.2, SI is 807, HRF is 100, and Δ RI is 1. The other 7 candidates were excluded because the evaluation index did not meet the requirements. Therefore, the possibility that the unknown substance is isophorone is very high.
2.2.2 assisted identification Using isotopic information of substances
When multiple candidate compounds are closely scored, isotopic information in the EI spectrum can be used for identification. As seen in FIG. 3A, the best result of the unknown substance search was ethyl 4-acetamidobenzoate, with a composite score of 98.8, SI value of 945, and HRF value of 99.32. And the abundance ratio of the molecular ion peak m/z 165.07843 to the isotope 166.08177 of the unknown substance is 100: 9.6. According to the rule of isotopic abundance ratios, the compound has a maximum of 9C atoms, thus excluding the highest scoring results. The search result with the atomic number of C less than or equal to 9 is C9H11NO2And C7H7NO2. Considering that SI is greater than or equal to 700, HRF is greater than or equal to 90, and Delta RI is less than or equal to 100, benzocaine is selected from 4 candidate results (benzocaine, ethyl 3-aminobenzoate, ethyl 6-methylpyridine-3-carboxylate, 4-aminobenzoic acid). Benzocaine has a composite score of 98.1, an SI value of 914, an HRF value of 99.32, and a Δ RI of 20. The fragment ions observed in fig. 3B correlate well with the mechanism of benzocaine cleavage. Molecular ion cleavage at position 2, loss of one CH3CH2O-is the base peak m/z 12 in FIG. 30.04437, the exact mass deviation from theoretical fragmentation was-0.17 ppm. In addition, other characteristic ions can be observed at 137.04718, 92.04951, and 65.03862, all with mass deviations of less than 1 ppm. Thus, the most likely result of this unknown is benzocaine.
2.2.3 identification of molecular formula of unknown Agents Using Positive chemical ionization PCI
For several candidate compounds with similar composite scores, obtaining chemical ionization data using methane as the reactant gas is a reliable identification method. In positive chemical ionization PCI mode, [ M + H ] can be detected]+Or [ M + C2H5]+The molecular ions of (a) add up to a peak, thereby inferring the molecular formula of the compound. As shown in FIG. 4A, C11H18NO3P-composite scores were highest at 97.3, but SI values were only 562. Other candidate composite scores are relatively close, and Δ RI does not provide a reference. However, these candidate compounds have different molecular formulae, so that the compounds can be identified by PCI. As shown in fig. 4B, it is difficult to determine 135.06794 in the EI spectrum whether it is the molecular ion peak of the compound there. In FIG. 4C, [ M + H ] in the PCI spectrum]+The addition peak of (a) was 136.07564, which differs from the theoretical exact mass 136.07569 by-0.37 ppm. In addition, adduct ions such as [ M + C ] are common in PCI spectra2H5]+(-0.01ppm) can be used to verify elemental composition. Thus, the most likely formula of the unknown is C8H9NO, the most likely result of the substance is N-methylformanilide (composite score 97, SI 867, HRF 99.02).
2.3 detection of unknown substances in gutta-percha samples
Using the methodology of this study, 28 agents were identified among 10 gums. Table 1 lists the names, CAS numbers, base peak ions, molecular formulas, composite scores, SI values, HRF values,. DELTA.RI, and EI patterns of all the substances [ M]+*Accurate mass deviation of [ M + H ] in PCI mode]+The exact mass deviation of the base peak ion. The comprehensive score of each substance is greater than 93.8, the SI value is greater than 734, the HRF value is greater than 92.23, and the accurate mass deviation of ions is within 1 ppm. With the exception of 3, 4-dimethylbenzyl alcoholThe delta RI of the product is less than 21, and the delta RI of the 3, 4-dimethylbenzyl alcohol is 150, but the result is proved to be accurate through standard product verification. The retention indices for the 8 substances are not included in the NIST library, so the Δ RI for these substances is not listed.
FIG. 5 shows the results of detection of 28 substances in a sample. The range of the base peak area is divided into 8 intervals represented by the size of a circle. The detection rate of phenol is 90%, the content of the sample is from low to high, and a plurality of intervals are covered. Taking phenol as an example, the migration amount of 10 gutta-percha samples is quantitatively analyzed, and the migration amount is 3.40-386.60 mu g/L. For reference, the transport of other substances can be roughly estimated, and then further quantitative studies can be carried out if necessary. Phenol is listed as a suspected carcinogen by the international agency for research on cancer (category 3). According to the European Union toy safety directive 2009/48/EC, the phenol migration amount should not exceed 5 mg/L. All 10 samples meet the limit requirements of the regulations. In addition, some of the identified substances are also subject to childhood product regulations. For example, cyclohexanone is listed as a category 3 carcinogen. Isophorone has a stimulating effect on the eyes and skin. According to European Union toy Standard EN71, the migration limits for cyclohexanone and isophorone are 46 and 3.0mg/L, respectively.
Other identified substances are also potentially hazardous, but have not been addressed by corresponding regulations to date. For example, dimethyl phthalate, N-methylaniline and N-methyl toluidine are environmentally preferred contaminants. The benzothiazole compounds have mutagenic effect on microorganisms and carcinogenic effect on human beings. Photoinitiators 1173 and 4-methylbenzophenone are UV initiators with carcinogenic, skin contact toxicity and reproductive toxicity. Isobutyl benzoate has endocrine disrupting effects.
TABLE 1 list of identification of potentially harmful substances in gutta-percha toys
Figure BDA0003221079050000091
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 screening migration hazards in gutta-percha based on gas chromatography-ultra-high resolution mass spectrometry is characterized by comprising the following steps: the method comprises the following steps:
step one, sample treatment: cutting gutta-percha into small pieces, performing an experiment in simulated saliva to obtain a migration solution, adding an ethyl acetate-n-hexane mixed solution for liquid-liquid extraction, vibrating, standing and concentrating to obtain a liquid to be detected, and performing detection on a machine;
analyzing the liquid to be detected by adopting a gas chromatography-ultra-high resolution mass spectrometry method, wherein an EI mode and a PCI mode are respectively adopted for analysis during mass spectrometry detection;
thirdly, obtaining the accurate mass number of fragment ions of unknown substances according to the result of full-scan detection on the extracting solution of the gutta-percha sample in an EI mode of a mass spectrum, carrying out deconvolution processing on the collected data, removing interference ions to obtain a clean mass spectrogram, deducting impurity peaks existing in a solvent or a system through comparison with a blank sample, and identifying unique chromatographic peaks in the gutta-percha;
searching the mass spectrogram of the unknown substance in a commercial standard spectrogram library, and further filtering the matching result by using the accurate mass information of each ion peak;
step five, when a plurality of candidate results with similar retrieval scores exist in a certain unknown object, identifying by utilizing isotope information in an EI spectrogram;
and step six, measuring the molecular ions and molecular formulas of the unknown substances through data obtained in the PCI mode of the mass spectrum, and further selecting the best matching from the candidate results.
2. The method for screening the migration hazards in gutta percha based on gas chromatography-ultra high resolution mass spectrometry as claimed in claim 1, wherein: the sample treatment in the first step specifically comprises the following steps:
cutting the smooth part of the surface of the gutta-percha sample into (10 +/-1) cm2Weighing, placing into a 40mL glass colorimetric tube with a plug, adding 20mL simulated saliva into the colorimetric tube, shaking the sample in a water bath at 37 ℃ and 100r/min for 240min to obtain a migration solution, transferring the migration solution to a separation funnel, adding 5mL of an ethyl acetate-n-hexane mixed solution with the volume ratio of 1:1, vibrating for 1min, standing for 30min, concentrating the organic phase to 0.5mL under the mild nitrogen flow purging, and measuring on a machine.
3. The method for screening the migration hazards in gutta percha based on gas chromatography-ultra high resolution mass spectrometry as claimed in claim 1, wherein: the chromatographic separation conditions in the second step are as follows:
HP-5MS column (30 m.times.0.25 mm.times.0.25 μm); carrying high-purity helium (99.999%) with the flow rate of 1 mL/min; sample introduction volume: 1 μ L, the injection port temperature is 280 ℃, and the temperature rise program: the initial temperature was 40 deg.C, held for 1min, then ramped up to 310 deg.C at a rate of 8 deg.C/min, held for 5 min.
4. The method for screening the migration hazards in gutta percha based on gas chromatography-ultra high resolution mass spectrometry as claimed in claim 3, wherein: in the second step, when the mass spectrum adopts an EI mode, the chromatograph performs split sample injection with a split ratio of 10:1, and when the mass spectrum adopts a PCI mode, the chromatograph does not perform split sample injection;
the mass spectrometry conditions were as follows: the method comprises the steps of performing Full-scanning Full-Scan in a Full-Scan mode, scanning the mass range of ions within 50-600 m/z, analyzing each sample by adopting an EI mode and a PCI mode respectively, wherein EI ionization energy is 70eV, when the PCI mode is adopted, methane is used as a reaction gas, the flow rate of the methane is 1.5mL/min, the temperatures of a transmission line and an ion source are 250 ℃ and 280 ℃ respectively, C-Trap and HCD collision gases are high-purity nitrogen (99.999%), the solvent delay time is 3.5min, under the condition of m/z 200, the mass resolution is set to be 60000FWHM, and the TIC intensity threshold is 1e6The maximum injection time was set to 200ms and the mass tolerance window was set to 5 ppm.
5. The method for screening the migration hazards in gutta percha based on gas chromatography-ultra high resolution mass spectrometry as claimed in claim 4, wherein: the data deconvolution parameters in step three are as follows:
deconvoluting with Tracefinder resolution plug software, setting the mass number tolerance window range of the extracted ion current to + -5 ppm to reduce the chemical substance interference in the matrix, obtaining the chromatogram of the extracted ion current with high selectivity, setting the accurate mass deviation to + -5 ppm, the threshold of signal-to-noise ratio to be 3, and the threshold of TIC intensity to be 1e6When the matching value of the deconvolution fragment ions is 99% and the retention time correction window is 10s, the deconvolution separation effect is the best.
6. The method for screening the migration hazards in gutta percha based on gas chromatography-ultra high resolution mass spectrometry as claimed in claim 5, wherein: in the retrieval and matching process of the fourth step, the positive matching index SI represents a matching value of the comparison between the measured spectrogram and the spectrogram in the standard spectrogram library, the HRF value represents the accurate mass percentage of the measured value of the fragment ions in accordance with the corresponding fragment ion element composition in the standard spectrogram library, the retention index is also an important index of qualitative analysis, the smaller the retention index deviation Delta RI is, the better the retention index deviation Delta RI is, when the comprehensive score of the retrieval is larger than 90, the SI is larger than 700, the HRF is larger than 90, and the Delta RI is smaller than 100, a plurality of interference items can be filtered, the time of data analysis is effectively reduced, and the accuracy of the identification result is improved.
CN202110958078.4A 2021-08-20 2021-08-20 Gas chromatography-ultra-high resolution mass spectrometry-based method for screening migration hazards in gutta-percha Withdrawn CN113671082A (en)

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