CN104865307A - Analysis method for alcohol compound in biological sample - Google Patents
Analysis method for alcohol compound in biological sample Download PDFInfo
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- CN104865307A CN104865307A CN201510164576.6A CN201510164576A CN104865307A CN 104865307 A CN104865307 A CN 104865307A CN 201510164576 A CN201510164576 A CN 201510164576A CN 104865307 A CN104865307 A CN 104865307A
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Abstract
The invention relates to a target derivatization-matrix assisted laser desorption-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) qualitative analysis method for alcohol compounds in a biological sample. The method comprises the steps of: conducting a pretreatment on a target analyte sample; reacting a derivative reagent and matrix of quinoline carboxylic acid with alcohol compound on a mass spectrum target plate to form a derivatized product; meanwhile conducting matrix-assisted laser desorption-Fourier transform ion cyclotron resonance mass spectrometry qualitative analysis by using excess quinoline acid as a substrate. The method is rapid, efficient and sensitive, and lays foundation for alcohol compound detection.
Description
Technical field
Derivatization-Matrix Assisted Laser Desorption mass spectrum-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FT ICR MS) analytical approach on the target that the present invention relates to a kind of alcohol compound, it is characterized in that taking quinoline carboxylic acid as derivatization reagent and matrix, mass spectrographic target plate reacts with alcohol compound, generate derivatization product, excessive quinoline carboxylic acid is matrix simultaneously, thus improve MALDI Ionization Efficiency, through Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry alcohol compound.
Background technology
Alcohol compound such as cholesterol, fatty alcohol, vitamin A, steroid hormone, ergosterol etc. are metabolin important in human body and antifungal drug action target spot, relevant to the function of multiple metabolic enzyme.The content of these compounds can react the exception of disease and human body metabolism's enzyme, such as, cholesterol in human body content Ahl tribulus sea silent sickness, the various diseases such as coronary heart disease are correlated with; Fatty alcohol content and Sjogren-Larsson syndrome in human body, the disease associations such as DHAP synthase shortage; The sterol metabolism of fungal cell is one of antifungal drug action target spot and antifungal agent resistance mechanism.Therefore, qualitative and quantitative analysis is carried out to alcohol compound in biological specimen (as blood plasma, urine, hair, cell) and have very important effect to the many physiological change of research, medical diagnosis on disease and drug screening.
Biological specimen is (as urine, hair, cell) in analytical approach mainly High Performance Liquid Chromatography/Mass Spectrometry (LC-MS) and the gas chromatography-mass spectrum (GC-MS) of alcohol compound, but pre-treatment is more complicated, need separation and purification, the needs of high flux screening can not be met, substance assistant laser desorpted ionization-Fourier transform Ion cyclotron Resonance Mass Spectrometry (MALDI-FT ICR MS) is due to its high resolving power, high flux and high sensitivity and there is great advantage, simplify loaded down with trivial details sample handling procedure, the high resolving power of FTMS enables us to the very close compound of mass-to-charge ratio in detection of complex sample.MALDI-FT ICR MS has been widely used in the qualitative and quantitative analysis of endogenous material in various biological specimen.
Have been reported biological specimen in alcohol compound analytical approach in, alcohol compound is due to its more weak proton affinity and more weak acidity, not easily ionizable, and in MALDI and ESI, signal is more weak, sensitivity is lower, and is easily suppressed by the material of other easily ionizables.In order to improve the response of its mass spectrum, existing various different derivatization reagent is developed out, and betaine aldehyde is (see Wu, C.P.; Ifa, D.R.; Manicke, N.E.; Cooks, R.G.Anal.Chem.2009,81,7618 – 7624.), 2-pyridine carboxylic acid is (see Higashi, T.; Shibayama, Y.J.; Shimada, K.J.Chromatogr.B2007,846,195-201.), 2-Aminopyrazine (Analyst, 2013,138,6270-6276) and dansyl Cl are (see Tang, Z.M.; Peter Guengerich, F.Anal.Chem.2010,82,7706 – 7712.) be used to derivatization alcohol compound.But these methods all exist shortcoming, comprise pre-treatment more complicated, excessive derivatization reagent needs removing, and derivatization reagent itself has stronger Ionization Efficiency, can suppress the detection of determinand.
Summary of the invention
The problem to be solved in the present invention is: provide a kind of matrix derivatization method based on quinoline carboxylic acid in conjunction with the method for MALDI-FT ICR MS for alcohol compound in qualitative analysis biological specimen, a kind of by esterification on target furtherly, these alcohol compounds are converted into quinoline ester, excessive reagent plays the effect of matrix, greatly can improve the sensitivity for analysis of target compound in substance assistant laser desorpted ionization-Fourier transform mass spectrophotometry subsequently and specificity like this, sample pretreatment process that need not be complicated, for the alcohol compound in high flux qualitative analysis biosome sample, for one is quick, efficiently, sensitive analytical approach, for studying this compounds content and physiological function in vivo further, the relation of disease lays the foundation.
For achieving the above object, the invention provides the analytical approach of alcohol compound in a kind of biological sample, it comprises the steps:
By after the sample organic solvent extraction of pre-treatment, get after organic layer carries out drying and add the acetonitrile solution of quinoline carboxylic acid and the dichloromethane solution of phosgene, obtain reactant liquor;
By described reactant liquor point sample 0.5 ~ 1 μ L on the target of laser desorption-Fourier Transform Ion cyclotron Resonance mass spectrometer, react 30 ~ 60 minutes at 15 ~ 40 DEG C, carry out Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry using excessive quinoline carboxylic acid as matrix, the mol ratio of described quinoline carboxylic acid and alcohol compound to be measured is 20:1-50:1;
By contrast high-resolution data analysis collection of illustrative plates, obtain the qualitative results of alcohol compound;
Wherein, described sample is the biosome material containing alcohol compound, and described alcohol compound is the one in cholesterol, fungi ergosterol, androsterone, vitamine D3, cholestanol, dehydrocholesterol, stigmasterol, dehydrogenation oil recovery sterol, lanosterol.
Principle of the present invention is: the generation chemical reaction making quinoline carboxylic acid and alcohol compound, utilizes quinoline carboxylic acid's derivative products that reaction product is carried out through Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry alcohol compound.Reaction equation as shown in Figure 1.
Preferably, in the acetonitrile solution of described quinoline carboxylic acid, the concentration of quinoline carboxylic acid is 2 ~ 5mg/mL, volume 50-100 μ L.
Preferably, described quinoline carboxylic acid is quinoline-3-carboxylic acid, Cinchonic Acid, quinoline-5-carboxylic acid, QUINOLINE-6-CARBOXYLIC ACID, quinoline-7-carboxylic acid or quinoline-8-carboxylic acid.
Preferably, in the dichloromethane solution of described phosgene, the volume fraction of phosgene is 10 ~ 40%, volume 50-100 μ L.
Preferably, the pretreatment process of described sample is: potassium hydroxide/ethanolic solution sample being added 1 ~ 2mL volume ratio 1:1, saponification 1 ~ 2h at 60 ~ 70 DEG C.
Preferably, the condition of described Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry is: laser energy 20 ~ 30%, sweep limit: m/z 100 ~ 1500, frequency: 1000Hz, spot diameter 500 ~ 1000 μm, data acquisition is carried out all in the positive-ion mode; The daughter ion condition of scanning: impact energy 10-30eV.
Preferably, described sample is hair or yeast cells.
Compared with prior art, the present invention has following beneficial effect:
1, for specificity derivatization alcoholic extract hydroxyl group on MALDI target, more targeted in the process of mass spectrophotometry, improve the Ionization Efficiency of determinand, enhance sensitivity for analysis (detectability 0.2-0.5ng/mL);
2, on target after reaction without the need to the sample pretreatment process of complexity, Direct Analysis after reaction, realizes high throughput analysis, easy and simple to handle, and high resolving power enables us to the very close compound of mass-to-charge ratio in detection of complex sample;
3, this to invent derivatization reagent used simple, be easy to get, mark part relatively little (173Da), structure is also relatively simple, Nature comparison is stablized, can produce under impact energy effect in mass spectrum daughter ion scanning analysis slough a part quinoline carboxylic ester fragmention [M-172]+, improve selectivity, be conducive to strengthening the accuracy of qualitative analysis.
The present invention is applied to the alcohol compound in qualitative analysis different kind organism sample, cholesterol, cholestanol, fungi ergosterol, androsterone, vitamine D3, dehydrocholesterol, stigmasterol, sitosterol, campesterol, dehydrogenation campesterol, lanosterol etc.Quick, efficient, sensitive detection alcohol compound can be realized by the method.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the chemical equation of middle alcohol compound of the present invention and quinoline carboxylic acid;
Fig. 2 is the cholesterol standards Matrix Assisted Laser Desorption after esterification on quinoline-3-carboxylic acid/phosgene target-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FT ICR MS) collection of illustrative plates, parent ion m/z 542.4, daughter ion m/z 369.4;
Fig. 3 be alcohol compound in biological specimen target on derivatization-Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FT ICR MS) qualitative analysis process schematic;
Fig. 4 is the MALDI-FT ICR MS collection of illustrative plates of sample of hair without derivatization;
Fig. 5 is the MALDI-FT ICR MS collection of illustrative plates of sample of hair after derivatization on quinoline-3-carboxylic acid/phosgene target, m/z 542.4.
Fig. 6 is the MALDI-FT ICR MS collection of illustrative plates of yeast cells without derivatization;
Fig. 7 is the MALDI-FT ICR MS collection of illustrative plates of yeast cells after derivatization on quinoline-3-carboxylic acid/phosgene target, m/z 542.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
As indicated with 1, technological process of the present invention as shown in Figure 3 for the structural formula of quinoline carboxylic acid of the present invention and alcohol compound to be measured.
embodiment 1the qualitative analysis of cholesterol in hair sample
Operation steps is as follows:
Hair distilled water, methylene chloride and washed with isopropyl alcohol, 50 ~ 60 DEG C of dryings, hair is cut into 1 ~ 2mm, take 0.3 ~ 0.4mg, add 1 ~ 2mL potassium hydroxide: ethanol (volume ratio 1:1), 60 ~ 70 DEG C of saponification 1 ~ 2h, add the extracted by ether twice of 2.5 ~ 5mL respectively, after merging organic layer, KOH is dry, nitrogen dries up, and adds the quinoline-3-carboxylic acid/acetonitrile solution of 50 ~ 100 μ L 2 ~ 5mg/mL, then mixes with the phosgene/dichloromethane solution of 50 ~ 100 μ L 10 ~ 40v/v%; Reactant liquor point sample 0.5 ~ 1 μ L is on target, 15 ~ 40 DEG C are reacted 30 ~ 60 minutes, Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FT ICR MS) is analyzed, as shown in Figure 2, the MALDI-FT ICR MS collection of illustrative plates before and after sample of hair derivatization is as shown in accompanying drawing 4,5 for spectrogram.Can find out that hair sample is after reaction, can obtain the derivatization product mass spectra peak m/z 542.4 of cholesterol, and without in the hair sample of reacting, not detect the peak of cholesterol.
embodiment 2the qualitative analysis of ergosterol in yeast cells
Yeast cells takes 200-500mg, add 1 ~ 2mL potassium hydroxide: ethanol (volume ratio 1:1), 60 ~ 70 DEG C of saponification 1 ~ 2h, add the extracted by ether twice of 2.5 ~ 5mL respectively, after merging organic layer, KOH is dry, nitrogen dries up, and adds the quinoline-3-carboxylic acid/acetonitrile solution of 50 ~ 100 μ L 2-5mg/mL, then mixes with the phosgene/dichloromethane solution of 50 ~ 100 μ L10 ~ 40v/v%; Reactant liquor point sample 0.5 ~ 1 μ L is on target, and 15 ~ 40 DEG C are reacted 30 ~ 60 minutes, and Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrum (MALDI-FT ICR MS) is analyzed.
MALDI-FT ICR MS collection of illustrative plates before and after yeast cells derivatization as illustrated in 6,7.Can find out that yeast cells sample is after reaction, can obtain the derivatization product mass spectra peak m/z 536.4 of ergosterol, and without in the yeast cells sample reacted, not detect the peak of ergosterol.
The present invention adopts the exact mass determination techniques of high-resolution FTMS, 5ppm is less than for standard with error, the accurate mass-to-charge ratio of the mass-to-charge ratio in Comparative map and theoretical target compound derivatization product, and the element composition extrapolating the derivatization product of target compound further.In experiment, exact mass mensuration is carried out to cholesterol derivative products and ergosterol derivative products, by comparing the high resolution mass spectrum data after sample of hair or yeast cells sample derivatization, thus obtain corresponding element composition, qualitative analysis is carried out to the cholesterol in hair and the ergosterol in yeast cells, as seen from Table 1, the theoretical mass-to-charge ratio of derivative products and experiment value error are within 5ppm.
Table 1. hair and yeast cells be gained Analysis of Sterol result after derivatization reaction on target and Mass Spectrometer Method
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (7)
1. the analytical approach of alcohol compound in biological sample, is characterized in that, comprise the steps:
By after the sample organic solvent extraction of pre-treatment, get after organic layer carries out drying and add the acetonitrile solution of quinoline carboxylic acid and the dichloromethane solution of phosgene, obtain reactant liquor;
By described reactant liquor point sample 0.5 ~ 1 μ L on the target of laser desorption-Fourier Transform Ion cyclotron Resonance mass spectrometer, react 30 ~ 60 minutes at 15 ~ 40 DEG C, to be 20:1-50:1 with alcohol compound mol ratio to be measured, quinoline carboxylic acid carries out Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry as matrix;
By contrast high-resolution data analysis collection of illustrative plates, obtain the qualitative analysis of alcohol compound;
Wherein, described sample is the biosome material containing alcohol compound, and described alcohol compound is the one in cholesterol, fungi ergosterol, androsterone, vitamine D3, cholestanol, dehydrocholesterol, stigmasterol, dehydrogenation oil recovery sterol, lanosterol.
2. analytical approach as claimed in claim 1, it is characterized in that, in the acetonitrile solution of described quinoline carboxylic acid, the concentration of quinoline carboxylic acid is 2 ~ 5mg/mL, and volume is 50-100 μ L.
3. analytical approach as claimed in claim 1 or 2, it is characterized in that, described quinoline carboxylic acid is quinoline-3-carboxylic acid, Cinchonic Acid, quinoline-5-carboxylic acid, QUINOLINE-6-CARBOXYLIC ACID, quinoline-7-carboxylic acid or quinoline-8-carboxylic acid.
4. analytical approach as claimed in claim 1, it is characterized in that, in the dichloromethane solution of described phosgene, the volume fraction of phosgene is 10 ~ 40%, and volume is 50-100 μ L.
5. analytical approach as claimed in claim 1, it is characterized in that, the pretreatment process of described sample is: potassium hydroxide/ethanolic solution sample being added 1 ~ 2mL volume ratio 1:1, saponification 1 ~ 2h at 60 ~ 70 DEG C.
6. analytical approach as claimed in claim 1, it is characterized in that, the condition of described Matrix Assisted Laser Desorption-Fourier Transform Ion cyclotron Resonance mass spectrophotometry is: laser energy 20 ~ 30%, sweep limit: m/z100 ~ 1500, frequency: 1000Hz, spot diameter 500 ~ 1000 μm, data acquisition is carried out all in the positive-ion mode; The daughter ion condition of scanning: impact energy 10 ~ 30eV.
7. analytical approach as claimed in claim 1, it is characterized in that, described sample is hair or yeast cells.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108051526A (en) * | 2018-01-11 | 2018-05-18 | 哈尔滨工业大学 | A kind of method of PREPOD pollutants in detection environment |
| CN113640368A (en) * | 2020-04-27 | 2021-11-12 | 株式会社岛津制作所 | Method for analyzing structure of organic compound |
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| CN102967647A (en) * | 2012-11-15 | 2013-03-13 | 中国科学院上海有机化学研究所 | Electrospray ionization-quadrupole-time-of-flight mass spectrometry analyzing method of sterol contained in edible oil |
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| CN102175750A (en) * | 2011-02-25 | 2011-09-07 | 中国科学院上海有机化学研究所 | Method for analyzing biological samples by using matrix assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectra |
| CN102967647A (en) * | 2012-11-15 | 2013-03-13 | 中国科学院上海有机化学研究所 | Electrospray ionization-quadrupole-time-of-flight mass spectrometry analyzing method of sterol contained in edible oil |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051526A (en) * | 2018-01-11 | 2018-05-18 | 哈尔滨工业大学 | A kind of method of PREPOD pollutants in detection environment |
| CN113640368A (en) * | 2020-04-27 | 2021-11-12 | 株式会社岛津制作所 | Method for analyzing structure of organic compound |
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