CN103837624B - Liquid chromatogram tandem mass spectrum measurement method for phenylglyoxylic acid and amygdalinic acid in urine - Google Patents
Liquid chromatogram tandem mass spectrum measurement method for phenylglyoxylic acid and amygdalinic acid in urine Download PDFInfo
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- CN103837624B CN103837624B CN201410094988.2A CN201410094988A CN103837624B CN 103837624 B CN103837624 B CN 103837624B CN 201410094988 A CN201410094988 A CN 201410094988A CN 103837624 B CN103837624 B CN 103837624B
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Abstract
The invention relates to a liquid chromatogram tandem mass spectrum measurement method for phenylglyoxylic acid and amygdalinic acid in urine. The liquid chromatogram tandem mass spectrum measurement method is characterized by comprising the step of directly introducing a liquid chromatogram-electrospray ionization-tandem mass spectrometer for measurement after a sample is diluted so as to detect the contents of phenylglyoxylic acid and amygdalinic acid in the urine rapidly and accurately. According to the brand-new measurement method for phenylglyoxylic acid and amygdalinic acid in the urine, an analyte is quantified by adopting an external standard method; the selectivity and the accuracy of the method are well improved by using a tandem mass spectrography. By selecting and optimizing a chromatographic column and an elution condition, the chromatographic separation process is well improved, and the chromatographic analysis time is shortened.
Description
technical field:
The invention belongs to the physical and chemical inspection technical field of urine specimen, is a kind of method adopting liquid chromatography-electrospray ionisation-tandem mass spectrometer to measure phenylacetaldehyde acid and mandelic acid content in urine specifically.
background technology:
Styrene is the important source material of plastic, rubber industry, in order to manufacture various plastic resin, synthetic rubber, is widely used in daily life and commercial production containing cinnamic plastic rubber product, as various wrappage, heat-barrier material, disposable lunch box, dixie cup, pipeline, container etc.Styrene is also present in cigarette smoke.Styrene mainly in gaseous form by respiratory tract, or enters human body by skin contact, and short-time contact high concentration styrene can cause the respiratory tract symptoms such as cough, pharyngalgia; Long contact time styrene can cause lung tissue structure to damage, thus people is easily suffered from an inflammation of the lungs, the disease such as pulmonary emphysema.International cancer research organization is classified as Group 2A, is namely probably carcinogenic substance to the mankind, to the carcinogenic substance of animal then for determining.The metabolism under the effect of CYP2B6 and CYP2E enzyme of styrene in suction body is styrene 7,8-oxide, styrene 7, the further metabolism under the effect of epoxide hydrolase of 8-oxide is phenylacetaldehyde acid and mandelic acid, phenylacetaldehyde acid and mandelic acid have been used as the biomarker that styrene exposes at present, and the structural formula of phenylacetaldehyde acid and mandelic acid is as follows:
At present, the method analyzing cinnamic metabolin in urine-phenylacetaldehyde acid and mandelic acid mainly contains: the method for liquid phase chromatography, vapor-phase chromatography, GC-MS and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).Due to urine matrix more complicated, the pre-treatment adopting the method for liquid chromatography to detect often to need through complexity to purify sample, and due to instrumental sensitivity inadequate, there is challenge in the detection for phenylacetaldehyde acid in non-professional exposed population group's urine; And gaschromatographic mass spectrometry method needs first to carry out derivatization; Liquid Chromatography-Tandem Mass Spectrometry, because it is highly sensitive, detectability is low, is therefore widely used in the research of biomarker in urine.
summary of the invention:
Object of the present invention is intended to overcome prior art defect, set up a kind of LC-MS/MS method of simple, phenylacetaldehyde acid fast, in the good mensuration urine of selectivity and mandelic acid, quick, the accurate content detecting phenylacetaldehyde acid and mandelic acid in urine of the method energy, measurement result is accurate, mensuration is disturbed few.
The object of the invention is to be achieved through the following technical solutions:
A Liquid Chromatography-Tandem Mass Spectrometry assay method for phenylacetaldehyde acid and mandelic acid in urine, comprises following processing step:
A, collection volunteer urine.
B, sample pre-treatments: thawed at room temperature urine sample, after mixing, through the membrane filtration of 0.22 μm, get the urine of 200 μ L, add 0.80 mL pure water, gets 2 μ L filtrates and introduce LC-MS/MS compartment analysis.
C, standard working solution are prepared: with phenylacetaldehyde acid and the mandelic acid mixed standard solution working solution of pure water preparation variable concentrations, concentration is respectively 1,2,5,10,50 and 100 ng/mL and 5,10,20,50 and 100 ng/mL.
D, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw phenylacetaldehyde acid and the mandelic acid standard working solution of the variable concentrations prepared, inject LC-MS/MS system, the equation of linear regression of phenylacetaldehyde acid and mandelic acid is respectively y=1.49 × 10
4x+3.81 × 10
3with y=1.35 × 10
4x-1.34 × 10
4, wherein the peak area of thing is analyzed in Y representative, and X represents the concentration of target analytes in urine.Related coefficient is greater than 0.999, measures sample liquid to be measured, records the peak area analyzing thing, substitutes into unary linear regression equation, tries to achieve the content analyzing thing in sample liquid to be measured.
Have chosen Agilent Zorbax Eclipse XDB-C18 chromatographic column (150 mm × 2.1 mm in the present invention, 3.5 μm), initial flow phase system chooses 50% solvent orange 2 A (aqueous solution) and 50% solvent B(methanol solution), isocratic elution, flow rate of mobile phase is 400 μ L/min, analysis time is 10 min, and sample size is 2 μ L.
The condition of tandem mass spectrum detecting device: electric spray ion source, multiple-reaction monitoring negative ion scan mode; Electron spray voltage: 4500V, atomization gas: 50 psi, gas curtain gas: 35 psi, auxiliary heating gas: 30 psi, injects voltage: 9 V, residence time: 100 ms.Choose m/z 149/77 as quota ion pair, m/z 149/105 is qualitative ion pair.
The range of linearity of the inventive method and detection limit:
With the mixing blank diaper preparation series standard curve processed, the point of the typical curve of phenylacetaldehyde acid and mandelic acid is respectively 1,2,5,10,50 and 100 ng/mL and 5,10,20,50 and 100 ng/mL, compound linear good, related coefficient is greater than 0.9990.Utilize mark-on to dilute and obtain quantitative limit and the detection limit of method, concentration corresponding to target peak 10 times of signal to noise ratio (S/N ratio)s is quantitative limit, and concentration corresponding to 3 times of signal to noise ratio (S/N ratio)s is detection limit.The quantitative limit of phenylacetaldehyde acid and mandelic acid is respectively 0.81 ng/mL and 2.24 ng/mL.
The inventive method recovery of standard addition and repeatability:
Urine matrix is adopted to add the recovery of the method assay method of standard items.Choose five urine specimens, after mixing, choose low, neutralize high three Pitch-based sphere and add standard items, each concentration is parallel does 6 repetitions, obtain the recovery and the precision (in relative standard deviation (RSD)) of method, in table 1, the recovery scope of method is 85.0-110.2%, RSD < 10%.
The recovery of table 1 method and precision (n=6)
| Compound | Background/(ng/mL) | Add scalar/(ng/mL) | Detect/(ng/mL) | Average recovery rate/% | Relative standard deviation/% |
| 10 | 41.7 | 104.7 | 8.36 | ||
| Phenylacetaldehyde acid | 31.2 | 50 | 82.5 | 102.6 | 2.02 |
| 80 | 114.6 | 104.3 | 1.64 | ||
| 20 | 48.6 | 85.0 | 4.30 | ||
| Mandelic acid | 31.6 | 50 | 86.7 | 110.2 | 2.36 |
| 80 | 118.3 | 108.4 | 1.84 |
Method of the present invention overcomes the deficiency of prior art sample treatment, optimizes pre-treating method for urine specimen, and is optimized the coherent detection condition of LC-MS/MS, mainly optimizes ion gun condition, chromatographic column and flow visualizing.Compared with prior art the inventive method has following excellent results:
1. compare with traditional HPLC-FLD, higher (the phenylacetaldehyde acid detection limit: 0.24 ng/mL of LC-MS/MS method sensitivity; Mandelic acid detection limit: 0.67 ng/mL, pre-treatment only needs simple filtration and dilution, and the sample size of needs is few (200 μ L).
2. this method has easy and simple to handle, quick, accurate, sensitivity and reproducible advantage.
Accompanying drawing explanation
Fig. 1. the chromatogram of phenylacetaldehyde acid, the chromatogram of mandelic acid in smoker's urine.
Embodiment
The present invention is described further below in conjunction with example, but is not restriction the present invention.
A Liquid Chromatography-Tandem Mass Spectrometry assay method for phenylacetaldehyde acid and mandelic acid in urine, its test process is that urine directly introduces LC-MS/MS systematic analysis after dilution.
Example 1:
1. instrument and reagent: Agilent 1200 liquid chromatography (Agilent company of the U.S.), is furnished with G1329A automatic sampler, G1311A quaternary mixing pump and G1316A column oven; The triple quadrupole rods tandem mass spectrometry instrument (Applied biosystems) of API 4000, is furnished with electric spray ion source (ESI) and analyst 1.5 software data disposal system.
Phenylacetaldehyde acid standard items (purity: 98%, Toronto Research Chemicals Inc.); Mandelic acid standard items (purity: 99.5%, U.S. ChemService Inc.); Methyl alcohol (TEDIA company Inc., Ohio, USA), all solvents are chromatographically pure, and in experiment, water used is all deionized water.
2. sample preparation: urine sample, in thawed at room temperature, after mixing, through the membrane filtration of 0.22 μm, is got the urine of 200 μ L, added 0.80 mL pure water, gets 2 μ L filtrates and introduces LC-MS/MS compartment analysis.
3. assay method: the point drawing the typical curve of phenylacetaldehyde acid and mandelic acid is respectively 1,2,5,10,50 and 100 ng/mL and 5,10,20, the each 2 μ L of sample after the standard solution of 50 and 100 ng/mL and dilute filtration, inject LC-MS/MS system and carry out compartment analysis, record the content of phenylacetaldehyde acid and mandelic acid in sample.
Example 2: as described in Example 1, leaves and takes 10 urine specimens.The content recording phenylacetaldehyde acid and mandelic acid in urine is as shown in table 2.
The contents level (μ g/mL) of phenylacetaldehyde acid and mandelic acid in table 2 urine
| Sequence number | Phenylacetaldehyde acid | Mandelic acid | Sequence number | Phenylacetaldehyde acid | Mandelic acid |
| 1 | 0.20 | 0.12 | 6 | 0.10 | 0.18 |
| 2 | 0.36 | 0.11 | 7 | 0.16 | 0.17 |
| 3 | 0.52 | 0.2 | 8 | 0.42 | 0.21 |
| 4 | 0.15 | 0.24 | 9 | 0.44 | 0.24 |
| 5 | 0.61 | 0.14 | 10 | 0.17 | 0.35 |
Claims (2)
1. phenylacetaldehyde acid and the Liquid Chromatography-Tandem Mass Spectrometry assay method of mandelic acid in urine, is characterized in that: comprise following processing step:
A, collection volunteer urine;
B, sample pre-treatments: thawed at room temperature urine sample, after mixing, through the membrane filtration of 0.22 μm, get the urine of 200 μ L, add 0.80 mL pure water, gets 2 μ L filtrates and introduce Liquid Chromatography-Tandem Mass Spectrometry compartment analysis;
C, standard working solution are prepared: with phenylacetaldehyde acid and the mandelic acid hybrid standard working solution of pure water preparation variable concentrations;
D, Liquid Chromatography-Tandem Mass Spectrometry measure, draw phenylacetaldehyde acid and the mandelic acid standard working solution of the variable concentrations prepared, inject liquid chromatography tandem mass spectrometer system, choose Agilent Zorbax Eclipse XDB-C18 chromatographic column, specification 150 mm × 2.1 mm, 3.5 μm, initial flow phase system chooses 50% solvent orange 2 A: aqueous solution and 50% solvent B: methanol solution, isocratic elution, and flow rate of mobile phase is 400 μ L/min, analysis time is 10 min, and sample size is 2 μ L; The condition of tandem mass spectrum detecting device: electric spray ion source, multiple-reaction monitoring negative ion scan mode; Electron spray voltage: 4500V, atomization gas: 50 psi, gas curtain gas: 35 psi, auxiliary heating gas: 30 psi, injects voltage: 9 V, residence time: 100 ms; The equation of linear regression of phenylacetaldehyde acid and mandelic acid is respectively y=1.49 × 10
4x+3.81 × 10
3with y=1.35 × 10
4x-1.34 × 10
4, wherein the peak area of thing is analyzed in Y representative, and X represents the concentration of target analytes in urine; Related coefficient is greater than 0.999, measures sample liquid to be measured, records the peak area analyzing thing, substitutes into unary linear regression equation, tries to achieve the content analyzing thing in sample liquid to be measured.
2. phenylacetaldehyde acid and the Liquid Chromatography-Tandem Mass Spectrometry assay method of mandelic acid in urine according to claim 1, is characterized in that: the sour and mandelic acid hybrid standard working solution concentration of phenylacetaldehyde is respectively 1,2,5,10,50 and 100 ng/mL and 5,10,20,50 and 100 ng/mL.
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Non-Patent Citations (4)
| Title |
|---|
| Liquid chromatography/electrospray tandem mass spectrometry characterization of styrene metabolism in man and in rat;Paola Manini et al.;《Rapid Communications in Mass Spectrometry》;20021230;第16卷(第24期);2239-2248 * |
| Simultaneous analysis of 28 urinary VOC metabolites using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI/MSMS);K.Udeni Alwis et al.;《Analytica Chimica Acta》;20120421;第750卷;152– 160 * |
| Simultaneous determination of mandelic acid enantiomers and phenylglyoxylic acid in urine by high-performance liquid chromatography with precolumn derivatization;Jin-Zhao Wang et al.;《Journal of Chromatography B》;20060619;第840卷;50–55 * |
| UPLC法同时测定吸烟者尿液中苯乙醛酸和苯乙醇酸含量;石龙凯等;《烟草化学》;20140228(第2期);33-37 * |
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