CN111983090A - Method for simultaneously detecting BTEX metabolites in urine - Google Patents
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Abstract
The invention provides a method for simultaneously detecting BTEX metabolites in urine, which comprises the following steps: (a) sample pretreatment: acidifying the urine sample and filtering; (b) preparing a standard solution; (c) high performance liquid chromatography determination: the chromatographic column is a C18 chromatographic column; the mobile phase is a mixed solution of methanol and 0.5 percent of (acetic acid + phosphoric acid), and the elution mode is gradient elution; (d) and calculating the concentration of each metabolite in the urine sample according to a linear regression equation of a standard curve obtained by the standard solution through a chromatographic system and the peak area of the urine sample. The method established by the invention has the advantages of simple sample pretreatment process, simple operation, good separation effect, good accuracy, precision and recovery rate, can complete one-time detection within 30 minutes by selecting and optimizing the chromatographic column and the elution condition, and can simultaneously detect and analyze various BTEX metabolites.
Description
Technical Field
The invention relates to the technical field of biological analysis and detection, in particular to a method for simultaneously detecting BTEX metabolites in urine.
Background
BTEX generally refers to benzene series, and is a generic term for benzene derivatives, mainly including benzene, toluene, ethylbenzene, xylene, styrene, etc., and widely exists in petroleum, gas stations, plastic products and industrial by-products. BTEX has a high carcinogenic risk and is toxic to organisms, and is a major concern by the International Agency for Research on Cancer (IARC). BTEX has three general routes of human exposure: respiration, ingestion, and skin contact, with respiratory exposure being the most dominant exposure route.
The main sources of BTEX contact in the living environment comprise automobile exhaust, gasoline volatilization, organic matter combustion and the like, and BTEX contact in the professional environment mainly comprises painters, shoe factories, petrochemical workers and the like. Health risks of BTEX include central nervous system anesthesia caused by short-term bolus exposure and hematopoietic toxicity and cancer caused by long-term exposure. European and American countries recommend 1ppm as the 8h occupational contact limit of BTEX, and China also revises the time weighted average allowable concentration of BTEX to 6mg/m in 20023. At present, the contact level of BTEX in part of professional population is mainly ppb level, and the BTEX contact of 1-20ppb is also present in the common population daily.
The current situation of exposure to BTEX at low doses makes the results less reliable when using traditional external exposure for risk assessment. The biological detection can reflect the real risk of human body exposure, and can effectively reflect the load degree of pollutants in individual samples through the detection of biomarkers in biological samples (such as blood, urine, alveoli and the like). The urine has the characteristics of no harm to a subject, easiness in acquisition and the like in the collection process, and is a main means of biological detection. Biomarkers are biochemical indicators for determining occupational exposure levels, and can be used to assess levels of exposure to exogenous chemicals and their resulting harmful biological effects. Research on human biomarkers has shown that trans, trans muconic acid (t, t-MA) can be used as a biomarker for benzene contact; hippuric acid can be used as a biomarker of toluene; the mandelic acid and the phenylglyoxylic acid can be used as contact biomarkers of ethylbenzene and styrene; methylhippuric acid can be used as a contact biomarker of xylene.
Common detection methods for BTEX urine biomarkers include spectrophotometry, gas chromatography, and High Performance Liquid Chromatography (HPLC). Spectrophotometry is a method for qualitative and quantitative analysis by measuring the absorbance of a substance to be measured at a specific wavelength or within a certain wavelength range. Wangjiaxing (1990) used spectrophotometry to determine hippuric acid concentration in urine samples, the lowest detection concentration of which was 6 mg/L; however, in the experimental process, sunlight has a destructive effect on hippuric acid reaction products, the color development effect is influenced by improper operation, and the repeatability is low. The gas chromatography uses gas as a mobile phase, and separates sample substances according to the distribution coefficient difference of the substances in the mobile phase and a fixed phase. The high performance liquid chromatography is not limited by the volatility and the thermal stability of a sample, and has the advantages of high efficiency, convenience and the like, and is a main detection means adopted in a laboratory at present. For the detection of the BTEX metabolites in urine, most of the BTEX metabolites can be independently detected by HPLC at present, BTEX often exists at the same time in the actual environment, and how to synchronously detect the BTEX metabolites in urine, the establishment of a rapid and efficient detection method is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting BTEX metabolites in urine, which can simultaneously and rapidly detect and analyze a plurality of BTEX metabolites, and has the advantages of simple operation, low cost and good separation effect.
In order to achieve the above objects, the present invention provides a method for simultaneously detecting metabolites of BTEX in urine, the method comprising the steps of:
(a) sample pretreatment: acidifying the urine sample and filtering;
(b) preparing a standard solution of trans-muconic acid, hippuric acid, mandelic acid, phenylglyoxylic acid and methylhippuric acid;
(c) high performance liquid chromatography determination: the chromatographic column is a C18 chromatographic column; the mobile phase is a mixed solution of methanol and 0.5 percent (acetic acid and phosphoric acid), and the elution mode is gradient elution;
(d) and calculating the concentration of each metabolite in the urine sample according to a linear regression equation of a standard curve obtained by the standard solution through a chromatographic system and the peak area of the urine sample.
In one embodiment, in step (a), the urine sample is a sample having a urine specific gravity of 1.010 to 1.030.
In one embodiment of the present invention, the specific gravity of the urine sample is measured in a laboratory in time after the urine sample is collected, and the sample with the specific gravity of 1.010-1.030 is selected as the effective urine sample.
In one embodiment of the present invention, in step (a), the acidification is performed by using a hydrochloric acid buffer solution, preferably, the hydrochloric acid buffer solution is a buffer solution of concentrated hydrochloric acid to water in a volume ratio of 1: 1; more preferably, the acidification time is 10min or more; the volume of the urine sample is 50-150 times, more preferably 100 times of the volume of the buffer solution; the acidified sample is filtered through a filter, preferably a 0.22 μm filter.
In a specific embodiment of the invention, an effective sample to be tested is placed in a centrifuge tube, a hydrochloric acid buffer solution is added and mixed uniformly, and the acidified sample is filtered through a 0.22 mu m filter membrane and placed in a brown sample bottle for later use.
In one embodiment of the present invention, in step (C), the C18 chromatography column employs a TC-C18 reverse phase chromatography column; the column temperature is from 32 to 35 ℃ and preferably 32 ℃. The filler of the chromatographic column is activated carbon.
In one embodiment of the present invention, in step (c), the elution mode in the high performance liquid chromatography is gradient elution, and the mobile phase is a mixture of methanol and 0.5% (acetic acid + phosphoric acid); the sample amount is 1-10 μ L, preferably 10 μ L; the flow rate of the mobile phase is 0.5-1mL/min, preferably 1 mL/min.
In one embodiment of the invention, the procedure of the gradient elution is:
0-3min, 20% of methanol and 80% of mixed solution of acetic acid and phosphoric acid;
3-12min, 20% of methanol and 80% of acetic acid and phosphoric acid mixed solution;
12-22min, 40% of methanol and 60% of acetic acid and phosphoric acid mixed solution;
22-25min, 80% of methanol and 20% of mixed solution of acetic acid and phosphoric acid;
25-26min, 80% of methanol and 20% of mixed solution of acetic acid and phosphoric acid;
26min, 20 percent of methanol and 80 percent of mixed solution of acetic acid and phosphoric acid.
In one embodiment of the present invention, in step (c), the high performance liquid chromatography uses a general-purpose ultraviolet detector, and the ultraviolet lamp detection wavelength is 237 nm.
In one embodiment of the present invention, the standard solution is prepared by the following method: dissolving trans-muconic acid and hippuric acid with methanol, dissolving hippuric acid, mandelic acid, phenylglyoxylic acid and methylhippuric acid standard substances with deionized water, and diluting to obtain standard solutions of 1, 5, 10, 20, 40 and 80 mg/mL.
In one embodiment of the present invention, the trans, trans muconic acid, hippuric acid, mandelic acid, phenylglyoxylic acid and methylhippuric acid standard samples are respectively weighed and dissolved in deionized water (wherein the trans, trans muconic acid is dissolved in methanol) to prepare standard stock solutions, and the stock solutions are diluted to prepare standard solutions with the concentrations of 1, 5, 10, 20, 40 and 80 mg/mL.
In one embodiment of the present invention, the methylhippuric acid comprises 2-methylhippuric acid, 3-methylhippuric acid and 4-methylhippuric acid.
In one embodiment of the present invention, in step (a), the volume of the effective sample is 5-15mL, preferably 10 mL.
For the protocol of simultaneously measuring a plurality of different metabolites using the liquid chromatography method, the pretreatment process of the sample and the optimization of the chromatographic separation conditions are the most critical factors. In many chromatographic separation methods, it is difficult to separate different components efficiently, and the degree of separation is not required to a large extent depending on the handling of the sample and the setting of conditions during chromatography. In the embodiments of the present application, the applicant found in the research that if a complicated extraction process is performed by using an organic extraction solvent during the pretreatment process, the final recovery rate of the BTEX metabolite is poor. The scheme of the application directly adopts a pretreatment method of acidification and filtration, is simple and time-saving, and has good subsequent separation effect. In the present embodiment, the chromatographic conditions including, in particular, the mobile phase ratio, the gradient elution procedure and the column temperature are obtained from a large number of experiments, and under the conditions of the present invention, the solubility of the metabolite is good and the retention time is optimized. After gradient elution, the retention capacity of the target substance on the chromatographic column is good, the chromatographic peak shape symmetry is good, the separation effect is excellent, and the optimal separation result can be obtained within the shortest analysis time.
Has the advantages that:
the invention provides a method for detecting and analyzing corresponding biological indicators in urine of a human body in an environment of benzene, toluene, xylene and styrene exposure, which can simultaneously detect BTEX metabolites such as trans-muconic acid, mandelic acid, phenylglyoxylic acid, hippuric acid and methylhippuric acid in the urine.
The method established by the invention has the advantages of simple and efficient sample pretreatment process, low cost of reagents used in the pretreatment process and contribution to popularization and application.
The method can complete one-time detection within 30 minutes by selecting and optimizing the optimal experimental conditions such as chromatographic columns, elution conditions and the like in the liquid chromatogram, can simultaneously detect and analyze various BTEX metabolites, and has simple operation and good separation effect. The linear correlation coefficient of the working curve of each component is greater than 0.998, the average standard addition recovery rate of the sample is between 80 and 120 percent, the daily precision and the daytime precision are both less than 7 percent, and each sample component has good recovery rate and precision.
The method disclosed by the invention is accurate in determination and strong in specificity, and can be used for well separating trans-muconic acid, mandelic acid, phenylglyoxylic acid, hippuric acid, 2-methylhippuric acid, 3-methylhippuric acid and 4-methylhippuric acid and respectively detecting the content of each substance to be detected. The method has important application value for analyzing the benzene metabolites in the urine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a chromatographic apparatus;
FIG. 2 is a chromatogram of a standard (a: phenylglyoxylic acid, b: trans, trans muconic acid, c: mandelic acid, d: hippuric acid, e: 2-methylhippuric acid, f: 4-methylhippuric acid, g: 3-methylhippuric acid);
FIG. 3 is a BTEX non-contact urine chromatogram;
FIG. 4 is a chromatogram of BTEX-contactor urine;
FIG. 5 is a phenylglyoxylic acid chromatogram (retention time: 9.948 min);
FIG. 6 is a trans, trans muconic acid chromatogram (retention time 10.374 min);
FIG. 7 is a mandelic acid chromatogram (retention time 11.611 min);
FIG. 8 is a hippuric acid chromatogram (retention time: 12.299 min);
FIG. 9 is a chromatogram of 2-methylhippuric acid (retention time: 14.271 min);
FIG. 10 is a chromatogram of 4-methylhippuric acid (retention time: 17.370 min);
FIG. 11 is a chromatogram of 3-methylhippuric acid (retention time: 17.552 min);
FIG. 12 is a chromatogram of the urine of a BTEX contactor of comparative example 1.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
1. chromatographic conditions are as follows:
HPLC system: a high performance liquid chromatograph (including a quaternary pump, an ultraviolet detector, an automatic sample injector, a column oven and a chromatographic workstation); a chromatographic column: agilent TC-C18 (250X 4.6mm, 5 μm); mobile phase: the gradient elution method is adopted, the elution procedure is shown in table 1, and the volume ratio of methanol to 0.5% (acetic acid + phosphoric acid) mixed solution is 80: 20; flow rate: 1 mL/min; the column temperature was 32 ℃; the sample injection amount is 10 mu L; wavelength of ultraviolet lamp: 237 nm. FIG. 1 is a schematic diagram of a chromatographic apparatus.
TABLE 1 elution procedure
2. Pretreatment of urine samples
10mL of sample to be detected is placed in a 15mL centrifuge tube, then 0.1mL of hydrochloric acid buffer solution (the volume ratio of concentrated hydrochloric acid to deionized water is 1: 1) is added into the centrifuge tube and mixed uniformly, the acidified sample is filtered through a 0.22 mu m filter membrane and placed in a brown sample bottle to be detected.
3. Specificity
Volunteers were non-smoking, non-occupational BTEX-exposed office workers who did not take any medication 3 days prior to sampling.
4. Linear test
Polyglycolic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid and hippuric acid standards are dissolved by deionized water, trans-and trans-muconic acid is dissolved by methanol, and each standard is diluted into standard solutions of 1, 5, 10, 20, 40 and 80 mg/ml. The operation is carried out according to a urine sample pretreatment method, the peak area is taken as the ordinate, the standard concentration of each component is taken as the abscissa, and a working curve is drawn. The linear regression equation, the linear correlation coefficient and the linear range of each working curve were calculated, and the results are shown in table 2.
TABLE 2 metabolite Linear equation and Linear Range
The experimental result shows that the linear correlation coefficient of the working curve of each substance to be detected is high (the linear correlation coefficient is greater than 0.998), the linear range is wide, and the requirement of quantitative detection of the urine sample can be met.
5. Accuracy and precision experiment
2 parts of fresh urine (the specific gravity of the urine is 1.010-1.030) of volunteers are taken to prepare a uniformly mixed sample. Then divided into 3 groups, each group of 7 samples, respectively adding high, middle and low 3 concentrations of standard solution, namely 80mg/mL, 20mg/mL and 5mg/mL of trans, trans muconic acid, mandelic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid, hippuric acid. Each measurement is carried out for 3 times, an average value is taken, and blank is subtracted to calculate the precision in the day and the average standard addition recovery rate. And measured once on each of day 2 and day three according to the experimental method to calculate the daytime precision. The results are shown in Table 3. Except for the reaction, the recovery rate of the trans-muconic acid is 73.82 percent, and the average recovery rate of the metabolites in the rest 6 is between 80 and 120 percent. The precision in the day and the precision in the day are both less than 7 percent, which shows that the method has good repeatability experiment effect.
TABLE 3 concentration levels in urine of volunteers
Example 2:
urine samples of 4 professional contacts (working in BTEX exposure factories for a long time) and 4 non-professional contacts were selected, and the concentration contents of trans-muconic acid, mandelic acid, phenylglyoxylic acid, methylhippuric acid and hippuric acid in the urine samples of the contacts and the non-contacts were measured by the sample pretreatment method, and the results are shown in tables 4 and 5. FIG. 2 is a chromatogram of a standard (a: phenylglyoxylic acid, b: trans, trans muconic acid, c: mandelic acid, d: hippuric acid, e: 2-methylhippuric acid, f: 4-methylhippuric acid, g: 3-methylhippuric acid); FIG. 3 is a BTEX non-contact urine chromatogram; FIG. 4 is a chromatogram of BTEX-contactor urine; FIGS. 5 to 11 are respectively a phenylglyoxylic acid chromatogram, a trans-muconic acid chromatogram, a phenylglycolic acid chromatogram, a hippuric acid chromatogram, a 2-methylhippuric acid chromatogram, a 4-methylhippuric acid chromatogram and a 3-methylhippuric acid chromatogram.
The average concentration of trans, trans muconic acid, mandelic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid and hippuric acid in the urine of all non-contact persons is respectively 2.03mg/g Cre, 5.28mg/g Cre, 1.48mg/g Cre, 83.33mg/g Cre, 2.97mg/g Cre, 2.47mg/g Cre and 0.44mg/g Cre; the average concentration of trans, trans muconic acid, mandelic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid and hippuric acid in urine of a contact person is respectively 4.22mg/g Cre, 7.41mg/g Cre, 2.32mg/g Cre, 125.62mg/g Cre, 2.38mg/g Cre, 3.46mg/g Cre and 1.96mg/g Cre, which is obviously higher than the corresponding concentration of trans, trans muconic acid, mandelic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid and hippuric acid of a non-contact person.
TABLE 4 metabolite concentration in urine (mg/g Cre) of contacters
TABLE 5 metabolite concentration in urine (mg/g Cre) of non-contacters
Comparative example
1. Chromatographic conditions
HPLC system: a high performance liquid chromatograph (including a quaternary pump, an ultraviolet detector, an automatic sample injector, a column oven and a chromatographic workstation); a chromatographic column: agilent TC-C18 (250X 4.6mm, 5 μm); mobile phase: the gradient elution method is adopted, the elution procedure is shown in table 1, methanol and 0.5% of (acetic acid + phosphoric acid) mixed solution are 20: 80; flow rate: 1 mL/min; the column temperature was 32 ℃; the sample injection amount is 10 mu L; wavelength of ultraviolet lamp: 237 nm.
TABLE 1 elution procedure
2. The result of the detection
The results are shown in FIG. 12.
As can be seen from fig. 12, no peak pattern of the target substance (trans, trans muconic acid, mandelic acid, phenylglyoxylic acid, 2-methylhippuric acid, 3-methylhippuric acid, 4-methylhippuric acid, hippuric acid) was observed, and qualitative and quantitative analysis of the target substance could not be carried out.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for simultaneously detecting BTEX metabolites in urine, the method comprising the steps of:
(a) sample pretreatment: acidifying the urine sample and filtering;
(b) preparing a standard solution of trans-muconic acid, hippuric acid, mandelic acid, phenylglyoxylic acid and methylhippuric acid;
(c) high performance liquid chromatography determination: the chromatographic column is a C18 chromatographic column; the mobile phase is a mixed solution of methanol and 0.5 percent of (acetic acid + phosphoric acid), and the elution mode is gradient elution;
(d) and calculating the concentration of each metabolite in the urine sample according to a linear regression equation of a standard curve obtained by the standard solution through a chromatographic system and the peak area of the urine sample.
2. The method of claim 1, wherein in step (a), the urine sample is a sample having a urine specific gravity of 1.010-1.030.
3. The method according to claim 1, wherein in step (a), the acidification is performed by using a hydrochloric acid buffer solution, preferably, the hydrochloric acid buffer solution is a buffer solution with a concentrated hydrochloric acid-water volume ratio of 1: 1; more preferably, the acidification time is 10min or more; the volume of the urine sample is 50-150 times, more preferably 100 times of the volume of the buffer solution; the acidified sample is filtered through a filter, preferably a 0.22 μm filter.
4. The method of claim 1, wherein in step (C), the C18 chromatography column is a TC-C18 reverse phase chromatography column; the column temperature is from 32 to 35 ℃ and preferably 32 ℃.
5. The method according to claim 1, wherein in step (c) the sample is taken in an amount of 1-10 μ L, preferably 10 μ L; the flow rate of the mobile phase is 0.5-1mL/min, preferably 1 mL/min.
6. The method of claim 1, wherein the gradient elution is performed by:
0-3min, 20% of methanol and 80% of mixed solution of acetic acid and phosphoric acid;
3-12min, 20% of methanol and 80% of acetic acid and phosphoric acid mixed solution;
12-22min, 40% of methanol and 60% of acetic acid and phosphoric acid mixed solution;
22-25min, 80% of methanol and 20% of mixed solution of acetic acid and phosphoric acid;
25-26min, 80% of methanol and 20% of mixed solution of acetic acid and phosphoric acid;
26min, 20 percent of methanol and 80 percent of mixed solution of acetic acid and phosphoric acid.
7. The method of claim 1, wherein in step (c), the high performance liquid chromatography uses an ultraviolet detector with a detection wavelength of 237 nm.
8. The method of claim 1, wherein the standard solution is prepared by: dissolving trans-muconic acid and hippuric acid with methanol, dissolving hippuric acid, mandelic acid, phenylglyoxylic acid and methylhippuric acid standard substances with deionized water, and diluting to obtain standard solutions of 1, 5, 10, 20, 40 and 80 mg/ml.
9. The method of claim 1, wherein the methylhippuric acid comprises 2-methylhippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid.
10. The method according to any one of claims 1 to 9, wherein in step (a) the volume of the urine sample is 5-15mL, preferably 10 mL.
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