CN109507335B - Method for high-throughput detection of various environmental pollutants in urine by using LC-MS-MS - Google Patents
Method for high-throughput detection of various environmental pollutants in urine by using LC-MS-MS Download PDFInfo
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Abstract
The invention discloses a method for detecting various environmental pollutants in urine at high flux by utilizing LC-MS/MS, which comprises the following steps: (1) sample pretreatment: treating the urine after enzymolysis with acetonitrile containing formic acid, fully whirling, centrifuging to obtain a supernatant, and performing vacuum centrifugal concentration on the supernatant to obtain a sample introduction sample for on-machine detection; (2) detecting the sample injection sample by adopting LC-MS/MS: the liquid phase conditions were: the chromatographic column adopts a reversed phase C18 chromatographic column; the mobile phase A is 4-6 mmol/L ammonium acetate aqueous solution; the mobile phase B is methanol; gradient elution is adopted; the detection mass spectrum conditions are as follows: and a dynamic multi-ion detection mode under an electrospray ion source is adopted. The method has the characteristics of quick detection, high efficiency, high flux, strong universality, high sensitivity, good stability and good reproducibility, and is suitable for the mass detection of biological samples in epidemiological research.
Description
Technical Field
The invention belongs to the field of environmental analysis, and particularly relates to development, verification and application of a method for detecting multiple different types of environmental pollutants in urine by utilizing LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) in a high-throughput manner.
Background
While the Chinese economy is rapidly developed, the environmental pollution caused by the Chinese economy, particularly the influence on health, is concerned. In recent years, people have been engaged in the detection work of environmental pollutants in human body samples, and found that different types of environmental pollutants can be detected in the urine of common people, but the detection methods are respectively based on different detection methods. Because people inevitably bear the accumulated exposure of various pollutants, but not the exposure of single pollutants, how to comprehensively evaluate the exposure level of various pollutants of a human body is the premise and the basis for researching the influence of environmental pollution on health. In the modern scarce biological sample, the development of a high-throughput analysis method for detecting various pollutants at one time is the key point for comprehensively evaluating the pollutants of the population.
China is a big agricultural and industrial country, and pesticides and chemical products are widely used for a long time, which brings various environmental pollutions. Among them, 9 kinds of pollutants such as four kinds of pesticides (organic phosphorus and herbicides, carbamates, pyrethroids and neonicotinoids), three kinds of antiseptics/bacteriostats (parabens, phthalates, triclosan, triclocarban and the like), phenols and other chemical products (benzophenone-3 types and the like) and the like widely exist in daily life, have the characteristics of low dose persistence, specificity, passability, tardiness and the like, are the most concerned important pollutants at home and abroad at present, and the specific representative compounds are shown in table 1. Persistent Organic Pollutants (POPs) in these environments tend to pose certain hazards to the human body under sustained exposure at low concentrations, thereby affecting human health development. Therefore, there is a need for a comprehensive and accurate assessment of the exposure of the above 9 categories of contaminants to the human population.
The detection method for various environmental pollutants disclosed at present has the advantages of less types of pollutants, low universality and low sensitivity. For example, a method for detecting single phenol environmental pollutants in urine by GC-MS/MS, a method for detecting two major environmental pollutants, namely benzoate and phenol, in urine by LC-MS/MS, and a method for detecting three environmental pollutants, namely organophosphorus pesticide, phthalate and bisphenol A (BPA), in urine by GC-MS/MS, and the like. The GC-MS method needs complex steps such as acidification, liquid-liquid extraction, solid-phase extraction and derivatization for sample pretreatment, is time-consuming and labor-consuming, is difficult to apply to compounds with fragile heated structures, and has certain limitations in application. The types of pollutants related to the LC-MS method are not wide enough, and when various types of pollutants are detected, the pollutants need to be detected for many times, so that the sample consumption is large, and the time consumption is long. Therefore, the prior art still faces the challenge of fully evaluating the exposure of the pollutants, and the development of a sensitive and stable detection method for detecting a plurality of different types of pollutants by using a small amount of samples at a time is urgently needed.
For liquid phase analysis, the difficulty in detecting many different types of compounds at once is that: complete separation of individual compounds of different types in the liquid phase, reduced sensitivity of individual compounds to mass spectrometry, etc. There is also a difficulty in developing a sensitive and stable assay for detecting a variety of different types of contaminants in a single sample. Due to the fact that the sensitivity of the compound is influenced, the function of a matrix in a sample is realized besides the performance of a detection instrument, a sample pretreatment method which can effectively remove the matrix of the sample and fully enrich the target compound is developed, and the combination of LC-MS/MS becomes the key for successfully establishing an analysis method.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting multiple different types of environmental pollutants in urine at one time in a high-throughput manner, and the method has the characteristics of quick detection, high efficiency, high throughput, strong universality, high sensitivity, and good stability and reproducibility.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for high-throughput detection of a plurality of different types of environmental pollutants in urine by LC-MS/MS comprises the following steps:
(1) sample pretreatment: adding an internal standard substance into a urine sample with a certain volume, adding ammonium acetate/acetic acid buffer salt with half of the volume of the urine sample to adjust the ph to about 5.0, adding beta-glucuronidase and incubating for a period of time; adding acetonitrile containing formic acid with the volume fraction of 0.1-2% after incubation is finished, centrifuging after full vortex, taking supernatant, and carrying out vacuum centrifugal concentration on the supernatant to obtain a sample for on-machine detection;
(2) detecting the sample injection sample by adopting LC-MS/MS:
the detection liquid phase conditions are as follows: the chromatographic column adopts a reversed phase C18 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 4-6 mmol/L ammonium acetate aqueous solution; the mobile phase B is methanol; gradient elution was used, the gradient elution procedure was performed as follows: mobile phase A + mobile phase B is 100%; the initial ratio of mobile phase was 95% mobile phase a and 5% mobile phase B; the volume percentage of the mobile phase B is increased from 5% to 10% in 0-4 min; the volume percentage of the mobile phase B is increased from 10% to 60% in 4-12 min; the volume percentage of the mobile phase B is increased from 60% to 85% in 12-14 min; the volume percentage of the mobile phase B is increased from 85% to 90% in 14-16 min; keeping the volume percentage of the mobile phase B at 90% for 16-18 min;
the detection mass spectrum conditions are as follows: dynamic multiple ion detection mode (DMRM) under electrospray ion source (ESI) was employed.
In one specific embodiment, in step (1), the pH is adjusted with an aqueous solution of ammonium acetate. Preferably, the concentration of the ammonium acetate aqueous solution is 1 mol/L.
In a specific embodiment, in the step (1), the incubation condition is incubation at 37 ℃ for 2-4 h. Preferably, incubation is for 3 h.
In a specific embodiment, in the step (1), the volume of 0.1-2% formic acid added in acetonitrile is 3-8 times of the volume of the urine sample. Preferably 4 to 7 times. More preferably 5 to 6 times.
In a specific embodiment, in the step (1), the volume fraction of formic acid in the acetonitrile is 0.5-1.5%. Preferably 0.8 to 1.2%. More preferably 0.9 to 1.1%.
In a specific embodiment, the mobile phase A may be an aqueous ammonium acetate solution having a concentration of 4mmol/L, 4.5mmol/L, 5mmol/L, 5.5mmol/L, or 6 mmol/L.
In a specific embodiment, the mobile phase A is 5mmol/L ammonium acetate water solution; the mobile phase B is methanol.
In a preferred embodiment, the column size is 3.0mm by 100 mm.
In a preferred embodiment, the sample injection amount is 15-25 μ l; further preferably 18 to 22. mu.l; more preferably 20. mu.l.
In a preferred embodiment, the flow rate is 0.3-0.7 ml/min; preferably, the flow rate is 0.4-0.6 ml/min; more preferably 0.5 ml/min.
In a preferred embodiment, the column temperature is controlled to be 39-41 ℃; more preferably 40 deg.c.
In one specific embodiment, the ion source parameters for mass spectrometry conditions are set as:
gas temperature (Gas Temp) 200 ℃; the Gas Flow rate (Gas Flow) was 14 l/min; spray air pressure (Nebulizer) 35 psi; the Sheath Gas temperature (Sheath Gas Heater) was 350 ℃; the Sheath Gas Flow rate (Sheath Gas Flow) was 11 l/min; capillary voltage (capliliary): 3000 v; nozzle voltage 1500 v.
In a particular embodiment, the method detects a target compound that is at least one or more of the following compounds:
chlorpyrifos (Chlorpyrifos), Chlorpyrifos-methyl, 3,5, 6-trichloro-2-hydroxypyridine (TCPY) metabolites of Chlorpyrifos-methyl, Chlorpyrifos (Chlorpyrifos) and Chlorpyrifos-methyl, p-Nitrophenol (NTP), pentachlorophenol (PCP), bisphenol A (BPA), benzophenone-3 (BP-3), Triclosan (TCS), methyl p-hydroxybenzoate (4-OH-methyl ester), ethyl p-hydroxybenzoate (4-OH-ethyl ester), propyl p-hydroxybenzoate (4-OH-propyl ester), diethyl phthalate (MEHP), Triclocarban (TCC), 3-phenoxybenzoic acid (3-phenoxy), 3- (2, 2-dichlorovinyl) -2, 2-dimethylcyclopropanecarboxylic acid (DCCA), Methomyl (Methomyl), Carbofuran (3-OH-Carbofuran), a specific metabolite of Carbofuran (Carbofuran), Acetamiprid (Acetamiprid), N-demethylacetamiprid (N-DeM-Acetaprid), a major metabolite of Acetamiprid, and (2-Cl-1,3-T-5-CA), Shirenshu (Imidacloprid). Specific information on these target compounds is shown in table 1.
TABLE 122 Classification of the Compounds of interest
In a particular embodiment, the internal standard is selected to be added according to the target compound to be detected. A general requirement is that the internal standard should be stable and not a sample endogenous substance, and the retention time should be close to that of the target compound to be detected, optimally with almost the same chemical properties as the target compound. If a plurality of target compounds are detected and the retention time of each target compound is greatly different, a plurality of internal standard substances can be added to correspond to different retention time periods respectively, and the internal standard substance with the retention time closer to the target compound is used as a reference for calculation when the content of a certain target compound is calculated. Preferably, the internal standard substance may be an isotopic label of 22 target compounds in table 1, and one internal standard substance may be used for calculation of the content of one or more detected target compounds with retention time closer thereto.
In a specific embodiment, the internal standard is one or more.
In a specific embodiment, the internal standard is an isotope label of the detected target compound, which is indicated by YES in "Inner stand" in table 3, i.e. the isotope label of the corresponding target compound is added as the internal standard.
In a specific embodiment, the amount of the target compound detected is calculated by plotting a standard curve according to the internal standard method.
In a specific embodiment, the mixed standard solution of 22 target compounds with different concentrations is added into a urine sample to be detected respectively to prepare the urine sample with different concentrations, 5 batches are continuously made in different days, the linear correlation of the 22 compounds in a certain range is considered, and the content of the detected target compound is calculated according to a linear equation curve.
One of the major technical difficulties faced in the development of the analytical method of the present invention is: the analysis method needs to be fast and accurate. The method is highly efficient due to the large number of biological samples to be tested in epidemiological studies, namely: the pretreatment method is simple and rapid, and the analysis part of the instrument is required to realize the separation and accurate quantification of a plurality of target compounds in at least table 1 in a short time by single-needle sample injection. In the existing method, the pretreatment of the urine sample is complex and tedious, most urine samples need to be subjected to enzymolysis treatment, and then are subjected to solid phase extraction for filtration and enrichment through a series of operations, the time of single needle analysis of the instrument is long, and some urine samples even are injected for 30min through a single needle.
The second major technical difficulty faced in developing the analytical method of the present invention is: the sensitivity of the method for detecting multiple pollutants by using a trace sample is high. Since all compounds in table 1 in biological samples in epidemiological investigation belong to trace levels, the sensitivity of the method is high to meet the trace sample detection requirement. Since sampling of biological samples involved in epidemiological investigations is very difficult, the amount of samples is small, and the resources of the samples are precious, a relatively large amount of contaminants can be detected with the smallest amount of samples possible. The existing method uses urine samples with different sizes of 2ml-4ml, the sample size is larger, and the forest sensitivity is lower when the sample size is small.
In developing the method of the present invention, it was found that matrix interference in the sample is one of the main factors affecting the sensitivity of the method, so that matrix removal during sample pretreatment is critical.
The existing method generally adopts Solid Phase Extraction (SPE) technology, namely, a chromatographic column made of a certain material of filler is selected, a urine sample is filtered, a target compound is adsorbed on the filler of the small column firstly, then a matrix adsorbed on the filler is washed by a specific solution, finally the target compound adsorbed on the filler is eluted by the specific solution, and the eluent is concentrated and then is subjected to on-machine detection. Because the 22 target chemical properties related by the method are different and different, it is difficult to find an SPE column which meets the properties of all compounds. Even if a suitable SPE column is found, this method is not only complicated and time consuming, but also the target compound is lost: firstly, the urine sample can not be completely adsorbed on the small column filler when passing through the SPE small column; secondly, elution of the matrix also carries away a portion of the target compound. Therefore, the method needs a larger sample size to achieve higher sensitivity.
In order not to lose the target compound, urine is firstly directly centrifugally sampled in the development process of the method, and the detection result shows that the responsiveness of the compound with larger polarity and early appearance and the compound with smaller polarity and late appearance is very low, as shown in figure 1, the peak type and the separation degree of each peak are very poor. Analysis shows that as the organic phase is increased, a large amount of matrix in urine is flushed out, and enters a mass spectrum along with a target compound so as to interfere with the responsiveness of the compound. From the ratio of the organic phase in the mobile phase when the matrix is flushed out we conclude that these matrices may be small proteins contained in urine.
Therefore, we followed the procedure of protein precipitation on urine, which first used 2 volumes of trichloroacetic acid and perchloric acid, without effect, but rather diluted urine further reduced the sensitivity of the target compound. In order to achieve the purpose that a sediment matrix does not dilute a urine sample, a volatile organic solvent acetonitrile is adopted for protein precipitation, 3 times of volume of acetonitrile is added into the urine sample and fully vortexed, the lower layer is centrifugated to find that the sediment exists in the lower layer, the supernatant is taken and put into a vacuum centrifugal concentrator to volatilize the acetonitrile, and the rest urine sample is detected on a computer, so that the responsiveness of a compound which generates a peak at a higher organic phase ratio is greatly improved, which shows that the matrixes such as small molecular proteins corresponding to interference target compounds in the urine are removed, and the response of the compound which has a polarity larger than the peak is not improved, as shown in figure 2.
The reason for the analysis again may be that some highly polar matrixes in urine or the pH value of urine influence the responses of the compounds, the pH value of a sample system is randomly adjusted, and after a plurality of tests, it is finally determined that the matrix with interference effect in the urine sample is precipitated by acetonitrile containing 1% formic acid, so that the problem of matrix interference can be perfectly solved, and the response of each compound can meet the detection requirement, as shown in fig. 3.
In developing the method of the present invention, proper setting of instrument parameters is also one of the factors affecting the sensitivity of the method. In order to complete the separation of each target compound in a relatively short time as much as possible, a C18 reverse phase chromatographic column with higher column efficiency is selected; and the mobile phase part is tested for many times, and the response of each compound is stronger when methanol is used as an organic phase than when acetonitrile is used as an organic phase. And the effect is best when 5mmol/L ammonium acetate is added into the water phase from the point of combining the peak type and the responsiveness of each target compound. Because the parent ions of the detected target compounds have positive ions and negative ions, the mass spectrum part adopts a dynamic multi-ion detection mode (DMRM) under an electrospray ionization source (ESI), and when the positive mode and the negative mode are used together and a plurality of target compounds are monitored simultaneously, the response of each compound in the DMRM mode is relatively high and stable.
Compared with other methods, the method provided by the invention has the advantages of small sample consumption, simple and rapid pretreatment, short single-needle sample analysis time, capability of completing all peaks in 18min, high sensitivity (low LOD value), capability of saving manpower and material cost and suitability for large-scale detection of biological samples in epidemiological research. The method is successfully applied to the detection of pollutants in pregnancy maternal urine samples in the eugenic queue project of 192 children in Shanghai city after the establishment and the examination are completed, and the result is shown in a table 10.
The method has the characteristics of strong universality, high sensitivity, high stability and good reproducibility, and is particularly suitable for the analysis and detection of high-throughput samples.
Drawings
FIG. 1 is a plot of Total Ion Current (TIC) of urine spiked and centrifuged direct sample application, showing that the peaks are of poor peak shape and resolution.
FIG. 2 is a Total Ion Current (TIC) spectrum of a urine sample treated with acetonitrile, from which it can be seen that the peak type and resolution of a compound having a late peak in the middle and the later peak of the spectrum are improved, while a compound having a large polarity and an early peak is not improved.
FIG. 3 is a Total Ion Current (TIC) spectrum of a urine sample after being treated by acidified acetonitrile, and it can be seen from the graph that the peak types and the separation degrees of various compounds are effectively improved.
Fig. 4A, 4B, and 4C are mass spectrum MRM maps obtained by detecting a mixed standard solution of 22 target compounds according to the method of the present invention in the first embodiment.
FIG. 5 is a diagram showing the overlapping of the MRM mass spectrum measured after the method of the present invention is used to detect the urine sample and 22 target compound mixed standard solutions (40ng/ml) in example two.
FIG. 6 is a mass spectrum obtained by detecting a blank aqueous solution according to the method of the present invention in example two, wherein each curve is an overlay of MRM (total mass spectrum) of each target compound.
Detailed Description
The inventor of the application finds out and obtains a method for detecting various environmental pollutants in urine in high flux by using LC-MS (liquid chromatography-mass spectrometry) through extensive research and a large number of experimentsThe method of (1), through the present experimental verification, the method can be used for the detection and quantitative analysis of at least 22 environmental pollutants and/or human body metabolites thereof (as shown in table 1), the applicable scope is wide, the universality is high; each environmental pollutant and/or the human metabolic product thereof has higher sensitivity, and can meet the detection requirement of trace samples; the method for producing various environmental pollutants and/or human body metabolism has good linearity, R2The quantitative detection results are all more than or equal to 0.99, and the quantitative detection results are accurate; the method has high precision and good stability, is suitable for long-time sample injection detection and analysis of mass samples, and is suitable for high-throughput sample injection analysis.
The chromatographic conditions of the detection method adopted by the invention are as follows:
(1) sample pretreatment: adding an internal standard substance into a urine sample with a certain volume, adding ammonium acetate/acetic acid buffer salt with half of the volume of the urine sample to adjust the ph to about 5.0, adding beta-glucuronidase and incubating for a period of time; adding acetonitrile containing formic acid with the volume fraction of 0.1-2% after incubation is finished, centrifuging after full vortex, taking supernatant, and carrying out vacuum centrifugal concentration on the supernatant to obtain a sample for on-machine detection;
(2) detecting the sample injection sample by adopting LC-MS:
the detection liquid phase conditions are as follows: the chromatographic column adopts a reversed phase C18 chromatographic column; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 4-6 mmol/L ammonium formate aqueous solution; the mobile phase B is methanol; gradient elution was used, the gradient elution procedure was performed as follows: mobile phase A + mobile phase B is 100%; the initial ratio of mobile phase was 95% mobile phase a and 5% mobile phase B; the volume percentage of the mobile phase B is increased from 5% to 10% in 0-4 min; the volume percentage of the mobile phase B is increased from 10% to 60% in 4-12 min; the volume percentage of the mobile phase B is increased from 60% to 85% in 12-14 min; the volume percentage of the mobile phase B is increased from 85% to 90% in 14-16 min; keeping the volume percentage of the mobile phase B at 90% for 16-18 min;
the detection mass spectrum conditions are as follows: dynamic multiple ion detection mode (DMRM) under electrospray ion source (ESI) was employed.
In one specific embodiment, in step (1), the pH is adjusted with an aqueous solution of ammonium acetate. Preferably, the concentration of the ammonium acetate aqueous solution is 1 mol/L.
In a specific embodiment, in the step (1), the incubation condition is incubation at 37 ℃ for 2-4 h. Preferably, incubation is for 3 h.
In a specific embodiment, in the step (1), the volume of 0.1-2% formic acid added in acetonitrile is 3-8 times of the volume of the urine sample. Preferably 4 to 7 times. More preferably 5 to 6 times.
In a specific embodiment, in the step (1), the volume fraction of formic acid in the acetonitrile is 0.5-1.5%. Preferably 0.8 to 1.2%. More preferably 0.9 to 1.1%.
In a specific embodiment, the mobile phase A may be an aqueous ammonium acetate solution having a concentration of 4mmol/L, 4.5mmol/L, 5mmol/L, 5.5mmol/L, or 6 mmol/L.
In a specific embodiment, the mobile phase A is 5mmol/L ammonium acetate water solution; the mobile phase B is methanol.
In a preferred embodiment, the column size is 3.0mm by 100 mm.
In a preferred embodiment, the sample injection amount is 15-25 μ l; further preferably 18 to 22. mu.l; more preferably 20. mu.l.
In a preferred embodiment, the flow rate is 0.3-0.7 ml/min; preferably, the flow rate is 0.4-0.6 ml/min; more preferably 0.5 ml/min.
In a preferred embodiment, the column temperature is controlled to be 39-41 ℃; more preferably 40 deg.c.
In one specific embodiment, the ion source parameters for mass spectrometry conditions are set as: gas temperature (Gas Temp) 200 ℃; the Gas Flow rate (Gas Flow) was 14 l/min; spray air pressure (Nebulizer) 35 psi; the Sheath Gas temperature (Sheath Gas Heater) was 350 ℃; the Sheath Gas Flow rate (Sheath Gas Flow) was 11 l/min; capillary voltage (capliliary): 3000 v; nozzle voltage 1500 v.
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example one
The sample pretreatment step is as follows: after 100ul of urine sample is added with the internal standard solution, 50ul of 1mol/L ammonium acetate aqueous solution (PH is adjusted to 5.0) is added, then the beta-glucuronidase is added, mixed evenly and incubated for 3h at 37 ℃. And after incubation, taking out and adding 500ul acetonitrile containing 1% formic acid, fully whirling for 15s, centrifuging to obtain a supernatant, carrying out vacuum centrifugal concentration on the supernatant to 100ul, and carrying out detection on a machine, wherein the sample amount is 20 ul. The method is simple to operate, the sample consumption is small, and the characteristics that pretreatment is complicated, complex, time-consuming and sample consumption in other methods are overcome.
Liquid phase conditions: the specification of the chromatographic column is C18 reverse chromatographic column, 3.0mm x 100 mm; the water phase is 5mmol/L ammonium acetate water solution, the organic phase is pure methanol, the initial proportion of the mobile phase is 95% water phase and 5% organic phase, the ratio is 10% organic phase in 4min, 60% organic phase in 12min, 85% organic phase in 14min, the ratio is maintained for 2min after 90% organic phase in 16min, the running time is 4min later, the mobile phase is returned to the initial proportion and reaches the equilibrium. The amount of the sample was 20. mu.L.
Mass spectrum parameters: the ESI source DMRM + -mode was used, and the specific parameters are shown in Table 2.
TABLE 2 ion Source parameters
| Parameter | Value(+) | Value(-) |
| Gas Temp(℃) | 200 | 200 |
| Gas Flow(l/min) | 14 | 14 |
| Nebulizer(psi) | 35 | 35 |
| SheathGasHeater | 350 | 350 |
| SheathGasFlow | 11 | 11 |
| Capillary(V) | 3000 | 3000 |
| Nozzle voltage(v) | 1500 | 1500 |
The method can be used for detecting 22 target compounds in the urine sample for quantitative detection and analysis, and can be used for comprehensively and accurately evaluating the pollutants of the population. The ion pair information for the 22 target compounds in the mass spectra is shown in table 3.
Table 322 target compound detected ion pairs and other parameter information
The results of mass spectrometry for the detection of 22 target compounds using the method of the present invention are shown in FIGS. 4A-4C.
Method specificity of embodiment two
The method has the characteristics that: the method is adopted to detect the blank double distilled water and the urine spiking sample, and whether the peak position of the target compound has an interference peak is judged to judge the specificity of the method to each target compound. The results of the experiment are shown in fig. 5 and 6.
Comparing fig. 5 and fig. 6, it can be seen that the method of the present invention has good specificity and good peak shape of each target compound, and the blank water sample measured by the method has no interference peak at the peak position of the target compound, and the method has high specificity.
Example three matrix Effect
And (3) detecting matrix effect: urine sample is added with mark to be directly tested on a computer, the urine sample is added with mark to be tested on the computer after matrix is removed through the pretreatment, the sample with certain concentration is prepared by double distilled water without matrix to be tested on the computer, response values of all target compounds are respectively tested, the influence of the matrix on the response of all the target compounds is seen, and the removal effect of the method on the matrix is also seen. The results are shown in Table 4.
Matrix Effect of 422 target Compounds
The experimental results show that: when the urine sample is directly put on a machine for detection, the matrix inhibition rate of the target compound which is most affected by the matrix is as high as 92.5 percent, and the response values are obviously increased after the sample is treated by a sample pretreatment mode in the method. Wherein, the response value of most target compounds is improved to more than 1.5 times of the original response value, and the response value of part of target compounds is improved to more than 3-4 times of the original response value. This indicates that the matrix effect is greatly improved, matrix interference is reduced, and the sensitivity of compound detection is higher.
Example four LOD value review
LOD value investigation: the LOD values of 22 target compounds were investigated using the method of the invention. The smaller the LOD value, the higher the detection sensitivity. The test results are shown in table 5.
TABLE 522 LOD values for the target Compounds
The experimental results show that: when the method of the invention is used for detecting the 22 compounds, the LOD value is between 0.003ng/ml and 0.2ng/ml, wherein the LOD value of 21 compounds is between 0.003ng/ml and 0.1ng/ml, and the detection sensitivity of each compound is very high.
Example five-line examination
Linear investigation: adding 22 kinds of mixed standard solutions of target compounds with different concentrations into human urine samples respectively to prepare urine samples with different concentrations. On different days, 5 batches were made in succession, and the linear correlation of 22 compounds was examined within a certain range. The test results are shown in table 6.
Linear examination of the 622 target Compounds
| Compound (I) | Standard curve range(ng/ml) | Regression equation (n ═ 5) | R2(n＝5) | Weight of |
| 2-Cl-1,3-T-5-CA | 0.1-100 | Y＝1.07X+4.28*10-3 | 0.997 | 1/x2 |
| 6-CN | 0.1-100 | Y＝1.23X+7.06*10-3 | 0.998 | 1/x2 |
| Methomyl | 0.5-100 | Y＝0.60X+2.81*10-3 | 0.995 | 1/x2 |
| Imidacloprid | 0.01-100 | Y＝0.13X-2.67*10-4 | 0.997 | 1/x2 |
| NTP | 0.01-100 | Y＝0.26X-7.34*10-4 | 0.998 | 1/x2 |
| N-DeM-Acetamiprid | 0.1-100 | Y＝0.29X+8.75*10-4 | 0.999 | 1/x2 |
| 3-OH-Carbofuran | 0.01-100 | Y＝0.20X-5.19*10-4 | 0.995 | 1/x2 |
| Acetamiprid | 0.01-100 | Y＝0.63X-1.27*10-4 | 0.996 | 1/x2 |
| TCPY | 0.1-100 | Y＝2.33X-1.27*10-3 | 0.993 | 1/x2 |
| 3-PBA | 0.1-100 | Y＝0.94X-1.59*10-3 | 0.997 | 1/x2 |
| DCCA | 0.1-100 | Y＝0.59X+1.53*10-3 | 0.997 | 1/x2 |
| BPA | 0.1-100 | Y＝5.10X+1.35*10-2 | 0.997 | 1/x2 |
| PCP | 0.1-100 | Y＝0.46X+1.49*10-3 | 0.995 | 1/x2 |
| MEHP | 0.1-100 | Y＝0.94X+4.94*10-2 | 0.997 | 1/x2 |
| BP-3 | 0.1-100 | Y＝2.08X-1.03*10-2 | 0.995 | 1/x2 |
| Chlorpyrifos-methyl | 0.5-100 | Y＝2.31X-2.98*10-2 | 0.995 | 1/x2 |
| TCC | 0.1-100 | Y＝1.11X-2.50*10-3 | 0.999 | 1/x2 |
| TCS | 0.1-100 | Y＝0.10X+7.07*10-4 | 0.996 | 1/x2 |
| Chlorpyrifos | 0.1-100 | Y＝1.05X-8.43*10-3 | 0.999 | 1/x2 |
| 4-OH-methyl ester | 0.5-500 | Y＝0.58X-4.00*10-3 | 0.999 | 1/x2 |
| 4-OH-ethyl ester | 0.01-100 | Y＝0.73X-3.88*10-3 | 0.999 | 1/x2 |
| 4-OH-propyl ester | 0.1-100 | Y＝0.85X-3.16*10-3 | 0.997 | 1/x2 |
The experimental results show that: each compound has good linearity in the range of 0.01-500ng/ml and linear correlation coefficient R2And the accuracy of the method is high when the method is used for quantitative detection of the 22 target compounds.
Example six day precision and in-day precision review
Day time precision and day time precision: the precision in the daytime is that 5 batches of quality control samples are continuously made, 3 groups of low and high (0.4ng/ml and 40ng/ml) samples are made in each batch, and the standard recovery rate (REC%) and RSD% are counted; the precision in the same day is that 5 groups of low and high are made in the same day, the interval between each group is 2 hours, and the REC percent and the RSD percent of each quality control result are calculated. The results of the measurements are shown in tables 7A and 7B.
TABLE 7 daytime precision of 22 target compounds
TABLE 7 in-day precision of the 22 target compounds
The experimental results show that: the REC% of the daytime precision and the intraday precision are both 85-115%, and the RSD% is less than 15%, thus the method has good daytime precision and intraday precision, and good stability and repeatability.
Example seven Freeze thaw stability Studies
And (3) investigating freeze-thaw stability: in the testing of large batches of samples, storage and sampling of the samples may involve freeze-thawing, which may adversely affect the detection of poorly stable compounds. The method of the invention considers the freeze-thaw stability of 22 target compounds for 1 time (3 groups of low and high quality control) and the freeze-thaw stability for 2 times (3 groups of low and high quality control), and calculates the REC% and RSD% of each result. The test results are shown in tables 8A and 8B. The concentrations of the lower and the upper parts are respectively 0.4ng/ml and 40 ng/ml.
Table 8a freeze thaw stability of 22 target compounds 1 time
Table 8B freeze thaw 2 stability of 22 target compounds
And (3) displaying a detection result: the RSD of the quality control of the low concentration and the high concentration (0.4ng/ml and 40ng/ml) is less than 15 percent, and the REC percent is between 85 percent and 115 percent, which indicates that the stability is good after 1 time and 2 times of freeze thawing. The method of the invention is little influenced by sample freeze thawing, and has good stability and reproducibility.
Example eight Placement stability Studies
And (3) investigating freeze-thaw stability: in the detection of large numbers of samples, the sample to be detected may be left for a long time, and may adversely affect the detection of a compound having poor stability. The method of the invention is characterized by standing for 4h at 37 ℃ stability (3 groups each with low and high quality control) and treating the urine sample, standing for 48h stability (3 groups each with low and high quality control) in the sample tray, and calculating REC% and RSD% of each result. The test results are shown in tables 9A and 9B. The concentrations of the lower and the upper parts are respectively 0.4ng/ml and 40 ng/ml.
TABLE 9A stability of 22 target compounds at 37 ℃ for 4h
And (3) displaying a detection result: the RSD of the quality control of low and high concentrations (0.4ng/ml and 40ng/ml) is less than 15 percent, the REC percent is between 85 and 115 percent, and the stability of the sample placed for 4 hours at 37 ℃ and the stability of the sample placed for 48 hours in a sample feeding disc which is well processed are both good. The method of the invention is little influenced by the sample placement time, has good stability and reproducibility and is suitable for continuous detection of a large number of samples.
Example nine
After the method is established and examined, the method is successfully applied to the detection of pollutants in pregnancy maternal urine samples in 192 cases of senior citizen market child eugenic queue projects, and the results are shown in a table 10.
TABLE 10 levels (ng/ml) of 22 contaminants in maternal urine during pregnancy for the child eugenic cohort project in Shanghai
The method is used for detecting the items, and has the characteristics of high detection speed, high efficiency, high flux, strong universality, high sensitivity, high accuracy, and good stability and reproducibility.
In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (13)
1. A method for detecting a plurality of different types of environmental pollutants in urine by utilizing LC-MS-MS (liquid chromatography-mass spectrometry) at high flux, which is characterized by comprising the following steps:
(1) sample pretreatment: adding an internal standard substance into a urine sample with a certain volume, adding ammonium acetate/acetate buffer salt to adjust the pH value to about 5.0, adding beta-glucuronidase and incubating at 37 ℃ for 2-4 hours; after incubation, adding acetonitrile containing formic acid with the volume fraction of 0.1-2%, wherein the volume of the acetonitrile is 3-8 times of that of the urine sample; centrifuging after fully swirling to obtain a supernatant, and carrying out vacuum centrifugal concentration on the supernatant to obtain an on-machine detection sample;
(2) detecting the sample injection sample by adopting LC-MS-MS sample injection:
the detection liquid phase conditions are as follows: the chromatographic column adopts a reversed phase C18 chromatographic column, and the size of the chromatographic column is 3.0mm multiplied by 100 mm; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 4-6 mmol/L ammonium acetate aqueous solution; the mobile phase B is methanol; gradient elution was used, the gradient elution procedure was performed as follows: mobile phase A + mobile phase B is 100%; the initial ratio of mobile phase was 95% mobile phase a and 5% mobile phase B; the volume percentage of the mobile phase B is increased from 5% to 10% in 0-4 min; the volume percentage of the mobile phase B is increased from 10% to 60% in 4-12 min; the volume percentage of the mobile phase B is increased from 60% to 85% in 12-14 min; the volume percentage of the mobile phase B is increased from 85% to 90% in 14-16 min; keeping the volume percentage of the mobile phase B at 90% for 16-18 min; the flow rate is 0.3-0.7 ml/min; controlling the column temperature to be 39-41 ℃;
the detection mass spectrum conditions are as follows: a dynamic multi-ion detection mode under an electrospray ion source is adopted;
the method detects the following 22 target compounds: chlorpyrifos, chlorpyrifos-methyl, 3,5, 6-trichloro-2-hydroxypyridine, p-nitrophenol, pentachlorophenol, bisphenol A, benzophenone-3, triclosan, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, diethyl phthalate, triclocarban, 3-phenoxybenzoic acid, 3- (2, 2-dichlorovinyl) -2, 2-dimethylcyclopropanecarboxylic acid, methomyl, 3-hydroxyfuradane, acetamiprid, N-demethylacetamiprid, phillyrin, 6-chloronicotinic acid, 2-chloro-1, 3-thiazoline-5-carboxylic acid.
2. The method of claim 1, wherein in step (1), the pH is adjusted with an ammonium acetate/acetic acid buffer solution; the concentration of the ammonium acetate/acetic acid buffer solution is 1 mol/L.
3. The method of claim 1, wherein in step (1), the incubation is for 3 h.
4. The method of claim 1, wherein in step (1), 0.1-2% formic acid in acetonitrile is added in a volume of 4-7 times the volume of the urine sample.
5. The method of claim 4, wherein the 0.1-2% formic acid in acetonitrile is added in a volume of 5-6 times the volume of the urine sample.
6. The method of claim 1, wherein in step (1), the volume fraction of formic acid in the acetonitrile is 0.5 to 1.5%.
7. The method of claim 6, wherein in step (1), the volume fraction of formic acid in the acetonitrile is 0.8 to 1.2%.
8. The method of claim 7, wherein in step (1), the volume fraction of formic acid in the acetonitrile is 0.9 to 1.1%.
9. The method of claim 1, wherein the mobile phase a is a 5mmol/L aqueous ammonium acetate solution; the mobile phase B is methanol.
10. The method of claim 1, wherein the flow rate is 0.4 to 0.6ml/min under liquid phase conditions.
11. The method of claim 10, wherein the flow rate is 0.5ml/min under liquid phase conditions.
12. The method of claim 1, wherein the column temperature is controlled to 40 ℃ in the liquid phase condition.
13. The method of claim 1, wherein the ion source parameters for mass spectrometry conditions are set to:
the gas temperature is 200 ℃; the gas flow rate is 14 l/min; the spray gas pressure was 35 psi; the temperature of the sheath gas is 350 ℃; the flow rate of the sheath gas is 11 l/min; capillary voltage: 3000 v; nozzle voltage 1500 v.
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