CN104991026A - Method for detecting methyl hippuric acid in urine - Google Patents
Method for detecting methyl hippuric acid in urine Download PDFInfo
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- CN104991026A CN104991026A CN201510365202.0A CN201510365202A CN104991026A CN 104991026 A CN104991026 A CN 104991026A CN 201510365202 A CN201510365202 A CN 201510365202A CN 104991026 A CN104991026 A CN 104991026A
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Abstract
The invention provides a method for detecting methyl hippuric acid in urine. The method comprises the following steps: taking a urine sample, adding parachlorobenzoic-acid, hydroquinone and 3,4-dihydroxyphenylanaline into the urine sample, and oscillating, mixing and centrifuging; then adding a hydrochlric solution, sodium chloride and ethyl acetate into a supernatant obtained through centrifugation, and oscillating, mixing and centrifuging; then conducting rotary evaporation on a supernatant obtained through centrifugation; finally, adding residues obtained in the step 3 into distilled water, concentrating by nanofiltration, and conducting efficient liquid phase analysis on the concentrated solution. The method provided by the invention is high in sensitivity, precision and recovery rate, and excellent in stability; all methodological indexes conform to related requirements of Guide for Establishing Occupational Health Standards Part 5-Method for Detecting Chemical Substances in Biomaterials; the method provided by the invention can be used for detecting 3 isomers of methyl hippuric acid in urine of both occupational workers exposed to dimethylbenzene and non-occupational people exposed to dimethylbenzene.
Description
Technical field
The invention belongs to bioanalytical method, be specifically related to the detection method of toluric acid in a kind of urine.
Background technology
Dimethylbenzene is water white transparency, has the liquid of aromatic hydrocarbon special odor, be on phenyl ring two hydrogen by methyl substituted product, exist adjacent, to three kinds of isomeride.Dimethylbenzene has volatility, special smell, inflammable, can mix arbitrarily with ethanol, chloroform or ether, insoluble in water.As a kind of very important industrial chemicals, industrial dimethylbenzene refer to by the m-xylene of 45% ~ 70%, the P-xylene of 15% ~ 25% and 10% ~ 15% the potpourri that forms of o-xylene 3 kinds of isomerss.
Industrial dimethylbenzene is mainly used in the industry such as paint and thinning agent, adhesive and pharmacy, and the adjuvant of various coating, the Effective Anti detonator of aviation fuel, fiber, plastics, rubber, the synthesis of dyestuff and the production of phthalic acid, in addition m-xylene is also for the production of different peptide acid, and P-xylene is used for the synthesis of terephthalic acids and the production of polyester.Non-industry exposes and mainly comes from auto exhaust, the use of petroleum products, storage, transport, refuse process.People also can be exposed to dimethylbenzene by smoking, or consumption is containing the food of dimethylbenzene, or by the commodity of skin contact containing dimethylbenzene, the dimethylbenzene of average suction every day of population is 0.3-8.6ug/kg, be 0.06ug/kg by taking in the amount exposed potable water every day, therefore dimethylbenzene has typical low-level background.
Research shows that a large amount of or Long Term Contact dimethylbenzene can produce serious toxic action to human body, dimethylbenzene is similar with split isomeride Drug Pharmacokinetics, and poisonous effect is also similar, sucks high concentration dimethylbenzene can cause nose, throat irritation through respiratory tract, expiratory dyspnea, pneumonia, pulmonary edema.Can stimulation be caused, erythema through direct skin contact, dry, degreasing, scorching hot, play blister, chap, finally cause dermatitis.Direct contact eye can cause Eye irritation, conjunctivitis, ambustio corneae.In urine, toluric acid is the characteristic metabolic products that dimethylbenzene enters human body, and urine in methylhippuric acid concentration with contact xylene concentration height correlation, can be used as the specific biological mark of Exposed dimethylbenzene.Because in urine, toluric acid has high specificity, highly sensitive feature; and sampling is convenient; many countries have all promulgated that the Biological exposure limit of dimethylbenzene is to protect the health of laborer, regularly carries out biological monitoring to laborer, are combined the degree of exposure to evaluate dimethylbenzene with environmental monitoring.At present, the research of China's P-xylene urine metabolic product is still in the starting stage, has formulated the air exposure limit of dimethylbenzene, has not yet formulated corresponding Biological Limit.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art and the detection method that toluric acid in a kind of urine is provided, this detection method is highly sensitive, precision good, the recovery is high, good stability, and every methodology index all meets the related request of chemical substance assay method in occupational health standard formulation guide Part V-biomaterial.
The detection method of toluric acid in urine, comprises the following steps:
Step 1, gets urine sample, adds parachlorobenzoic-acid, p-dihydroxy-benzene, 3,4-bis-para hydroxybenzene alanine, vibration mixing, centrifugal;
Step 2, adds hydrochloric acid solution, sodium chloride and ethyl acetate in the centrifugal gained supernatant of step 1, vibration mixing, centrifugal;
Step 3, gets the centrifugal gained supernatant of step 2, rotary evaporation;
Step 4, adds in distilled water by residue obtained for step 3, and nanofiltration concentrates, and concentrate enters high-efficient liquid phase analysis.
As the further improvement of foregoing invention, in step 1, urine consumption is 50 ~ 100mL, and parachlorobenzoic-acid consumption is 1 ~ 5g, p-dihydroxy-benzene consumption be 2 ~ 6g, 3,4-bis-para hydroxybenzene employed amount of alanine be 1 ~ 4g.
As the further improvement of foregoing invention, in step 1, centrifugal condition is 5000 ~ 7000rpm, and centrifugation time is 3 ~ 5min.
As the further improvement of foregoing invention, in step 2, supernatant consumption is 50 ~ 100mL, and hydrochloric acid solution is 5 ~ 10mL, sodium chloride 3 ~ 7g, ethyl acetate 1 ~ 6mL.
As the further improvement of foregoing invention, in step 2, centrifugal condition is 3000 ~ 5000rpm, and centrifugation time is 3 ~ 5min.
As the further improvement of foregoing invention, in step 3, rotating evaporation temperature is 50 ~ 65 DEG C, and vacuum tightness is 0.1MPa.
As the further improvement of foregoing invention, in step 4, the material of NF membrane is pottery, polyethersulfone or polyamide, and the molecular cut off of NF membrane is 100 ~ 500Da, and the pressure limit of nanofiltration is 1.0 ~ 4.0Mpa.
As the further improvement of foregoing invention, in step 4, the chromatographic condition of efficient liquid phase is chromatographic column C18 reverse-phase chromatographic column, column temperature 35 DEG C, mobile phase methanol: 0.1v/v% glacial acetic acid=30:70, flow velocity 0.8mL/min, measures wavelength 230nm, sample size 10 μ L.
Detection method provided by the invention is highly sensitive, precision good, the recovery is high, good stability, every methodology index all meets the related request of chemical substance assay method in occupational health standard formulation guide Part V-biomaterial, can be used for the mensuration of toluric acid 3 kinds of isomerss in Exposed dimethylbenzene workman and non-professional contact dimethylbenzene crowd urine.
Embodiment
Embodiment 1
The detection method of toluric acid in urine, comprises the following steps:
Step 1, gets urine sample, adds parachlorobenzoic-acid, p-dihydroxy-benzene, 3,4-bis-para hydroxybenzene alanine, vibration mixing, centrifugal;
Step 2, adds hydrochloric acid solution, sodium chloride and ethyl acetate in the centrifugal gained supernatant of step 1, vibration mixing, centrifugal;
Step 3, gets the centrifugal gained supernatant of step 2, rotary evaporation;
Step 4, adds in distilled water by residue obtained for step 3, and nanofiltration concentrates, and concentrate enters high-efficient liquid phase analysis.
In step 1, urine consumption is 50mL, and parachlorobenzoic-acid consumption is 1g, p-dihydroxy-benzene consumption be 2g, 3,4-bis-para hydroxybenzene employed amount of alanine be 1g.
In step 1, centrifugal condition is 5000rpm, and centrifugation time is 5min.
In step 2, supernatant consumption is 50mL, and hydrochloric acid solution is 5mL, sodium chloride 3g, ethyl acetate 1mL.
In step 2, centrifugal condition is 3000rpm, and centrifugation time is 3min.
In step 3, rotating evaporation temperature is 50 DEG C, and vacuum tightness is 0.1MPa.
In step 4, the material of NF membrane is pottery, polyethersulfone or polyamide, and the molecular cut off of NF membrane is 100Da, and the pressure limit of nanofiltration is 4.0Mpa.
In step 4, the chromatographic condition of efficient liquid phase is chromatographic column C18 reverse-phase chromatographic column, column temperature 35 DEG C, mobile phase methanol: 0.1v/v% glacial acetic acid=30:70, flow velocity 0.8mL/min, measures wavelength 230nm, sample size 10 μ L.
The toluric acid assay method of the present embodiment is good in the sensing range internal linear of 0.015 ~ 0.2mg/mL, 2, the related coefficient of 3,4-toluric acid is respectively 0.9997,0.9996,0.9998, and detection limit reaches 0.39mg/L, 0.37mg/L, 0.41mg/L.The recovery of standard addition scope of basic, normal, high concentration is 93.5 ~ 102.3%, and precision scope is 1.9 ~ 8.7%.
Embodiment 2
The detection method of toluric acid in urine, comprises the following steps:
Step 1, gets urine sample, adds parachlorobenzoic-acid, p-dihydroxy-benzene, 3,4-bis-para hydroxybenzene alanine, vibration mixing, centrifugal;
Step 2, adds hydrochloric acid solution, sodium chloride and ethyl acetate in the centrifugal gained supernatant of step 1, vibration mixing, centrifugal;
Step 3, gets the centrifugal gained supernatant of step 2, rotary evaporation;
Step 4, adds in distilled water by residue obtained for step 3, and nanofiltration concentrates, and concentrate enters high-efficient liquid phase analysis.
In step 1, urine consumption is 75mL, and parachlorobenzoic-acid consumption is 3g, p-dihydroxy-benzene consumption be 5g, 3,4-bis-para hydroxybenzene employed amount of alanine be 2g.
In step 1, centrifugal condition is 6000rpm, and centrifugation time is 4min.
In step 2, supernatant consumption is 80mL, and hydrochloric acid solution is 7mL, sodium chloride 5g, ethyl acetate 3mL.
In step 2, centrifugal condition is 4000rpm, and centrifugation time is 4min.
In step 3, rotating evaporation temperature is 60 DEG C, and vacuum tightness is 0.1MPa.
In step 4, the material of NF membrane is pottery, polyethersulfone or polyamide, and the molecular cut off of NF membrane is 300Da, and the pressure limit of nanofiltration is 2.0Mpa.
In step 4, the chromatographic condition of efficient liquid phase is chromatographic column C18 reverse-phase chromatographic column, column temperature 35 DEG C, mobile phase methanol: 0.1v/v% glacial acetic acid=30:70, flow velocity 0.8mL/min, measures wavelength 230nm, sample size 10 μ L.
The toluric acid assay method of the present embodiment is good in the sensing range internal linear of 0.015 ~ 0.2mg/mL, 2, the related coefficient of 3,4-toluric acid is respectively 0.9997,0.9996,0.9998, and detection limit reaches 0.39mg/L, 0.37mg/L, 0.41mg/L.The recovery of standard addition scope of basic, normal, high concentration is 93.5 ~ 102.3%, and precision scope is 1.9 ~ 8.7%.
Embodiment 3
The detection method of toluric acid in urine, comprises the following steps:
Step 1, gets urine sample, adds parachlorobenzoic-acid, p-dihydroxy-benzene, 3,4-bis-para hydroxybenzene alanine, vibration mixing, centrifugal;
Step 2, adds hydrochloric acid solution, sodium chloride and ethyl acetate in the centrifugal gained supernatant of step 1, vibration mixing, centrifugal;
Step 3, gets the centrifugal gained supernatant of step 2, rotary evaporation;
Step 4, adds in distilled water by residue obtained for step 3, and nanofiltration concentrates, and concentrate enters high-efficient liquid phase analysis.
In step 1, urine consumption is 100mL, and parachlorobenzoic-acid consumption is 5g, p-dihydroxy-benzene consumption be 6g, 3,4-bis-para hydroxybenzene employed amount of alanine be 4g.
In step 1, centrifugal condition is 7000rpm, and centrifugation time is 3min.
In step 2, supernatant consumption is 100mL, and hydrochloric acid solution is 10mL, sodium chloride 7g, ethyl acetate 6mL.
In step 2, centrifugal condition is 5000rpm, and centrifugation time is 5min.
In step 3, rotating evaporation temperature is 65 DEG C, and vacuum tightness is 0.1MPa.
In step 4, the material of NF membrane is pottery, polyethersulfone or polyamide, and the molecular cut off of NF membrane is 500Da, and the pressure limit of nanofiltration is 1.0Mpa.
In step 4, the chromatographic condition of efficient liquid phase is chromatographic column C18 reverse-phase chromatographic column, column temperature 35 DEG C, mobile phase methanol: 0.1v/v% glacial acetic acid=30:70, flow velocity 0.8mL/min, measures wavelength 230nm, sample size 10 μ L.
The toluric acid assay method of the present embodiment is good in the sensing range internal linear of 0.015 ~ 0.2mg/mL, 2, the related coefficient of 3,4-toluric acid is respectively 0.9997,0.9996,0.9998, and detection limit reaches 0.39mg/L, 0.37mg/L, 0.41mg/L.The recovery of standard addition scope of basic, normal, high concentration is 93.5 ~ 102.3%, and precision scope is 1.9 ~ 8.7%.
The detection method of Application Example 2, the result of occupational health rig-site utilization show workman Ban Mo urinate in the content of toluric acid and workplace air xylene concentration (TWA) linear, coefficient R
2=0.7643, each sample standard deviation detects toluric acid.In on-the-spot 500 the sample Zhong Liangge chemical pollution district residents urine of environmental health, toluric acid recall rate is respectively 25.9%, 15.4%, and check plot recall rate is 3%.
Claims (8)
1. the detection method of toluric acid in urine, is characterized in that: comprise the following steps:
Step 1, gets urine sample, adds parachlorobenzoic-acid, p-dihydroxy-benzene, 3,4-bis-para hydroxybenzene alanine, vibration mixing, centrifugal;
Step 2, adds hydrochloric acid solution, sodium chloride and ethyl acetate in the centrifugal gained supernatant of step 1, vibration mixing, centrifugal;
Step 3, gets the centrifugal gained supernatant of step 2, rotary evaporation;
Step 4, adds in distilled water by residue obtained for step 3, and nanofiltration concentrates, and concentrate enters high-efficient liquid phase analysis.
2. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 1, urine consumption is 50 ~ 100mL, parachlorobenzoic-acid consumption is 1 ~ 5g, and p-dihydroxy-benzene consumption is 2 ~ 6g, 3,4-bis-para hydroxybenzene employed amount of alanine is 1 ~ 4g.
3. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 1, centrifugal condition is 5000 ~ 7000rpm, centrifugation time is 3 ~ 5min.
4. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 2, supernatant consumption is 50 ~ 100mL, hydrochloric acid solution is 5 ~ 10mL, sodium chloride 3 ~ 7g, ethyl acetate 1 ~ 6mL.
5. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 2, centrifugal condition is 3000 ~ 5000rpm, centrifugation time is 3 ~ 5min.
6. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 3, rotating evaporation temperature is 50 ~ 65 DEG C, vacuum tightness is 0.1MPa.
7. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 4, the material of NF membrane is pottery, polyethersulfone or polyamide, the molecular cut off of NF membrane is 100 ~ 500Da, and the pressure limit of nanofiltration is 1.0 ~ 4.0Mpa.
8. the detection method of toluric acid in urine according to claim 1, it is characterized in that: in step 4, the chromatographic condition of efficient liquid phase is chromatographic column C18 reverse-phase chromatographic column, column temperature 35 DEG C, mobile phase methanol: 0.1v/v% glacial acetic acid=30:70, flow velocity 0.8mL/min, measure wavelength 230nm, sample size 10 μ L.
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| CN201510365202.0A CN104991026A (en) | 2015-06-29 | 2015-06-29 | Method for detecting methyl hippuric acid in urine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442784A (en) * | 2016-09-23 | 2017-02-22 | 瀚盟测试科技（天津）有限公司 | UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometry) detection method for concentrations of hippuric acid, methyl hippuric acid and asaronic acid in human urine |
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Patent Citations (4)
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| CN1690219A (en) * | 2004-02-12 | 2005-11-02 | 株式会社山田养蜂场 | Reduction pressure peptide derived from bee milk |
| CN1831529A (en) * | 2006-01-23 | 2006-09-13 | 湖南师范大学 | Method for quick screening angiotemsin invertase inhibitor combined using high effieient liquid chromatograph and mass spectrum |
| CN102985822A (en) * | 2010-06-28 | 2013-03-20 | 塔林科技大学 | A method and device for determining content of the middle and protein bound uremic toxins in a biological fluid |
| CN102621262A (en) * | 2012-02-28 | 2012-08-01 | 杭州博圣生物技术有限公司 | Method for detecting organic acid in urine by means of gas chromatography and mass spectrography |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442784A (en) * | 2016-09-23 | 2017-02-22 | 瀚盟测试科技（天津）有限公司 | UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometry) detection method for concentrations of hippuric acid, methyl hippuric acid and asaronic acid in human urine |
| CN106442784B (en) * | 2016-09-23 | 2019-01-04 | 瀚盟测试科技（天津）有限公司 | The UPLC-MS/MS detection method of hippuric acid, toluric acid and almond acid concentration in human urine |
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