CN106383191B - The Liquid Chromatography-Tandem Mass Spectrometry measuring method of D-VB5 content in a kind of urine - Google Patents
The Liquid Chromatography-Tandem Mass Spectrometry measuring method of D-VB5 content in a kind of urine Download PDFInfo
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- CN106383191B CN106383191B CN201610702547.5A CN201610702547A CN106383191B CN 106383191 B CN106383191 B CN 106383191B CN 201610702547 A CN201610702547 A CN 201610702547A CN 106383191 B CN106383191 B CN 106383191B
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Abstract
The Liquid Chromatography-Tandem Mass Spectrometry measuring method of D-VB5 content in a kind of urine, it is characterized by: being that urine sample is introduced directly into liquid chromatogram-electrospray ionisation-tandem mass spectrograph to be measured after diluting, using quantified by external standard method analyte, the contents level of D-VB5 in urine can quickly, be accurately detected.The present invention is the measuring method of D-VB5 content in completely new urine, tandem mass spectrometry preferably improves the selectivity and accuracy of method, the quantitative limit of D-VB5 is respectively 1.54 ng/mL, and the rate of recovery range of method is 87.9-95.3%, RSD < 13.0%.By chromatographic column and the selection and optimization of elution requirement, chromatographic separation process is preferably improved, shortens the time of chromatography, is 8 min.
Description
Technical field:
The invention belongs to the physical and chemical inspection technical fields of urine, specifically a kind of to use liquid chromatogram-electrospray ionisation-
The method that tandem mass spectrometer measures D-VB5 content in urine.
Background technique:
D-VB5 is the precursor of coacetylase (CoA), and CoA is the biologically active form that it is functioned, and is produced in substance containing carbon skeleton
Pantothenic acid participation is both needed in raw energy and much cyto-architectural formation, insufficient or physiological status change of ingesting can cause pantothenic acid to lack
It is weary.Pantothenic acid shortage is suppressed the reaction for needing CoA, mainly influences fatty acid metabolism, is suppressed fatty acid beta oxidation,
Fatty acid oxidation especially in peroxisome is significantly suppressed, and can lead to hyperlipidemia etc., and serious person can produce brain damage
Evil.In addition, pantothenic acid have lipoid peroxidization resistant, to by lipid peroxidation injury cell and rat have well protect
Shield effect, it may be possible to free radical be removed by CoA form or CoA promotes phosphatide synthesis to promote to repair.It detects general in urine
Acid content can be used for evaluating individual for the utilization power of nutriment, have for evaluation human body trophic level and health status
There is certain meaning.
Currently, the test method of D-VB5 content mainly has High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FL) in urine
The method of post column derivatization is built using isotope reverse ion with liquid chromatography-mass spectrometry (LC-MS), such as Fukuwatari
The measuring method of D-VB5 content in urine is found, method is quantitatively limited to 32.5 μ g/mL；Heudi etc. establishes D- in urine
The LC-MS method of pantothenic acid content, urine are tested and analyzed after simple centrifugation, dilution, and the detection of method is limited to 0.1 μ
G/mL, LC-MS/MS have highly sensitive, highly selective, and that D-VB5 content in urine is measured using LC-MS/MS method
It has not been reported.
Summary of the invention:
The purpose of the present invention is intended to overcome prior art defect, establishes a kind of simple, quick, selectivity is good measurement urine
The LC-MS/MS method of middle D-VB5 content, this method can quickly, accurately detect the content of D-VB5 in urine, and measurement result is quasi-
Really, measurement interference is few.
The purpose of the present invention is achieved through the following technical solutions:
The Liquid Chromatography-Tandem Mass Spectrometry measuring method of D-VB5 content in a kind of urine, specifically includes following processing step:
A, urine is thawed at room temperature, after centrifugation, then pipettes 100 μ L urines in the volumetric flask of 10 mL；
B, it with the aqueous formic acid constant volume of volume ratio 0.1%, is uniformly mixed, then pipettes the color that about 1 mL is put into 1.8 mL
It composes in bottle, is analyzed to Liquid Chromatography-Tandem Mass Spectrometry；
C, standard working solution is prepared: preparing the D-VB5 standard working solution of various concentration with 0.1% aqueous formic acid；
D, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measures, and draws the standard work of above-mentioned prepared various concentration
Solution, injects LC-MS/MS system, and the linear equation of D-VB5 is y=3.84e3x+7.71 e3, wherein Y represents the peak of analyte
Area, X indicate the concentration of target analytes in sample, and related coefficient is greater than 0.999, is measured, measures to sample prepare liquid
The peak area of analyte substitutes into unary linear regression equation, acquires the content of analyte in sample prepare liquid.
The D-VB5 standard working solution concentration is respectively 3.6,6,30,60,120,240 and 360 ng/mL.
In Step d, liquid phase chromatogram condition are as follows: Waters ACQUITY UPLC HSS T3 chromatographic column, specification 100
The mm of mm × 2. 1,1.8 μm, mobile phase: solvent A: 0.1% aqueous formic acid and solvent B:0.1% formic acid acetonitrile solution flow
Phase flow velocity be 200 μ L/min, condition of gradient elution: 0-1 min, 5%B-5%B；1-5 min, 5%B-100%B；5-6 min,
100%B；6-6.3 min, 100%B-5%B；6.3-8 min, 5%B, analysis time is 8 min, and sample volume is 5 μ L.
In Step d, the condition of tandem mass spectrum detector: electric spray ion source, multiple-reaction monitoring cation scanning mode；
Electron spray voltage: 5500V, atomization gas: 30 psi, gas curtain gas: 40 psi, auxiliary heating gas: 30 psi, injection voltage: 10
V, residence time: 100 ms；The selected quota ion pair of D-VB5 is respectively 220.3 → 90.1 and 220.3 → 184.3.
The range of linearity and detection limit of the method for the present invention:
Prepare series standard curve with the urine that blank mixes, D-VB5 standard working solution concentration is respectively 3.6,6,
30,60,120,240 and 360 ng/mL.Compound it is linear good, related coefficient is all larger than 0.999.Utilize the side of signal-to-noise ratio
Method calculates quantitative limit and detection limit, and the corresponding concentration of 10 times of signal-to-noise ratio of target peak is quantitative limit, and the corresponding concentration of 3 times of signal-to-noise ratio is
Detection limit.The quantitative limit of D-VB5 is respectively 1.54 ng/mL.
The method of the present invention recovery of standard addition and repeatability:
Using matrix plus the rate of recovery of the method measuring method of standard items.Five urine specimens are chosen, are surveyed after mixing
Its fixed content is 72.35 ng/mL, chooses low, medium and high three pitch-based spheres and standard items are added, each concentration does 6 weights in parallel
It is multiple, the rate of recovery and precision (in terms of relative standard deviation (RSD)) of method are obtained, is shown in Table 1, the rate of recovery range of method is
87.9-95.3%, RSD < 13.0%.
The rate of recovery and precision (n=6) of 1 method of table
Method of the invention overcomes the low deficiency of prior art sensitivity, for urine specimen to the correlation of LC-MS/MS
Testing conditions are optimized, and mainly optimize ion source condition, chromatographic column and flow visualizing.Compared with prior art originally
Inventive method has following excellent results:
1. LC-MS/MS sensitivity is higher compared with HPLC and LC-MS, the detection of D-VB5 is limited to 0.46 ng/mL, LC-
MS method is 100 ng/mL, is greatly improved compared with the Sensitivity disclosed by the documents method.
2. this method pre-treatment is easy, complicated solid phase extraction procedure is needed not move through, analysis throughput can be improved, have
Quickly, accurately, high sensitivity and reproducible advantage.
To sum up, the present invention is the measuring method of D-VB5 content in completely new urine, and tandem mass spectrometry preferably improves
The selectivity and accuracy of method, the quantitative limit of D-VB5 are respectively 1.54 ng/mL, and the rate of recovery range of method is 87.9-
95.3%, RSD < 13.0%.By chromatographic column and the selection and optimization of elution requirement, chromatographic separation process is preferably improved,
The time of chromatography is shortened, is 8 min.
Detailed description of the invention
The chromatogram of Fig. 1 D-VB5.
Specific embodiment
The present invention is described further below in conjunction with example, but is not the limitation present invention.
Example 1:
1. instrument and reagent: 1200 liquid chromatogram of Agilent (Agilent company of the U.S.) is furnished with G1329A automatic sampling
Device, G1311A quaternary mixing pump and G1316A, column oven；The triple quadrupole tandem mass spectrometers of API 4000 (U.S.'s application biology
System house), it is furnished with electric spray ion source (ESI) and 1.5 software data processing system of analyst.
D-VB5 standard items are to be greater than 98% purchased from Shanghai branch company, Wuhan Yi Tai Science and Technology Ltd. purity；Formic acid and
Acetonitrile is chromatographically pure, and water used is deionized water in experiment.
2. sample treatment: urine is thawed at room temperature, then pipettes 100 μ L urines in the volumetric flask of 10 mL, is used
0.1% aqueous formic acid constant volume is uniformly mixed, then pipettes about 1 mL and be put into the chromatogram bottle of 1.8 mL, to LC-MS/MS
It is analyzed.
3. measuring method: drawing standard solution, (standard working solution concentration is respectively 3.6,6,30,60,120,240 and
360 ng/mL) and dilute filtration after each 5 μ L of sample, injection LC-MS/MS system carry out separation analysis, measure D- in sample
The content of pantothenic acid.
Example 2: measuring method described in embodiment 1 is pressed, 8 urine specimens is chosen, measures the content such as table 2 of its D-VB5
It is shown.
The contents level (μ g/mL) of D-VB5 in 2 sample of table
| Serial number | D-VB5 | Serial number | D-VB5 |
| 1 | 15.6 | 5 | 7.99 |
| 2 | 10.7 | 6 | 11.5 |
| 3 | 8.94 | 7 | 12.0 |
| 4 | 6.88 | 8 | 20.1 |
Claims (1)
1. the Liquid Chromatography-Tandem Mass Spectrometry measuring method of D-VB5 content in a kind of urine, it is characterised in that: the following steps are included:
A, urine is thawed at room temperature, after centrifugation, then pipettes 100 μ L urines in the volumetric flask of 10 mL；
B, with 0.1% aqueous formic acid constant volume, be uniformly mixed, then pipette about 1 mL and be put into the chromatogram bottle of 1.8 mL, to
Liquid Chromatography-Tandem Mass Spectrometry is analyzed；
C, standard working solution is prepared: preparing the D-VB5 standard working solution of various concentration, concentration with 0.1% aqueous formic acid
The ng/mL of respectively 3.6,6,30,60,120,240 and 360；
D, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measures, and draws the standard working solution of above-mentioned prepared various concentration,
LC-MS/MS system is injected, the linear equation of D-VB5 is y=3.84e3x+7.71 e3, wherein y represents the peak area of analyte, x
Indicate the concentration of target analytes in sample, related coefficientrGreater than 0.9993, sample prepare liquid is measured, measures analysis
The peak area of object substitutes into unary linear regression equation, acquires the content of analyte in sample prepare liquid；
Liquid phase chromatogram condition: using ACQUITY UPLC HSS T3 chromatographic column, specification 100 mm × 2. 1 mm, and 1.8 μm,
Mobile phase: solvent A: 0.1% aqueous formic acid, solvent B:0.1% formic acid acetonitrile solution, flow rate of mobile phase are 200 μ L/min, ladder
Spend elution requirement: 0-1 min, 5%B-5%B；1-5 min, 5%B-100%B；5-6 min, 100%B；6-6.3 min, 100%B-5%
B；6.3-8 min, 5%B；Analysis time is 8 min, and sample volume is 5 μ L；
Tandem mass spectrum detector condition: electric spray ion source, multiple-reaction monitoring cation scanning mode；Electron spray voltage:
5500V, atomization gas: 30 psi, gas curtain gas: 40 psi, auxiliary heating gas: 30 psi inject voltage: 10 V, when being resident
Between: 100 ms；The selected quota ion pair of D-VB5 is respectively 220.3 → 90.1 and 220.3 → 184.3.
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Inventor after: Hu Qingyuan Inventor after: Fu Yaning Inventor after: Hou Hongwei Inventor after: Wang Hongjuan Inventor after: Zhang Xiaotao Inventor after: Chen Huan Inventor after: Liu Yong Inventor after: Wang An Inventor after: Liu Tong Inventor after: Han Shulei Inventor before: Hu Qingyuan Inventor before: Hou Hongwei Inventor before: Zhang Xiaotao Inventor before: Chen Huan Inventor before: Liu Yong Inventor before: Wang An Inventor before: Liu Tong Inventor before: Han Shulei Inventor before: Fu Yaning |
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