CN104865307A - Analysis method for alcohol compound in biological sample - Google Patents

Abstract

The invention relates to a qualitative analysis method of on-target derivatization-matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) for alcohol compounds in biological samples. The method comprises the following steps: the target analyte is passed through the sample Pretreatment: use quinoline carboxylic acid as derivatization reagent and matrix, react with alcohol compounds on the target plate of mass spectrometry to generate derivatized products, and at the same time, excess quinoline carboxylic acid as matrix, and then undergo matrix-assisted laser analysis-Fourier transform Qualitative analysis by ion cyclotron resonance mass spectrometry. The method is fast, efficient and sensitive, and lays the foundation for the detection of alcohols.

Description

A method for analyzing alcohol compounds in biological samples

technical field

The present invention relates to a kind of on-target derivatization-matrix-assisted laser analysis mass spectrometry-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) analysis method of alcohol compounds, which is characterized in that quinoline carboxylic acid is used as derivatization reagent and matrix , react with alcohol compounds on the target plate of the mass spectrometer to generate derivatized products, and at the same time, excess quinoline carboxylic acid is used as the matrix to improve the ionization efficiency of MALDI, and the alcohol compounds are analyzed by matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry .

Background technique

Alcohol compounds such as cholesterol, fatty alcohols, vitamin A, steroid hormones, and ergosterol are important metabolites and targets of antifungal drugs in the human body, and are related to the functions of various metabolic enzymes. Changes in the content of these compounds can reflect diseases and abnormal metabolic enzymes in the human body. For example, changes in cholesterol content in the human body are related to Alzheimer's disease, coronary heart disease and other diseases; fatty alcohol content in the human body is related to Sjogren-Larsson syndrome, DHAP synthase deficiency and other diseases are related; the sterol metabolism of fungal cells is the target of antifungal drugs and one of the mechanisms of fungal drug resistance. Therefore, the qualitative and quantitative analysis of alcohol compounds in biological samples (such as plasma, urine, hair, cells) plays a very important role in the study of many physiological changes, disease diagnosis and drug screening.

The analysis methods of alcohols in biological samples (such as urine, hair, cells) are mainly high-performance liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), but the pretreatment is complicated and requires separation Purification cannot meet the needs of high-throughput screening. Matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) has great advantages due to its high resolution, high throughput and high sensitivity. Simplifying tedious sample processing steps, the high resolution of FTMS enables us to detect compounds with very close mass-to-charge ratios in complex samples. MALDI-FT ICR MS has been widely used in the qualitative and quantitative analysis of endogenous substances in various biological samples.

In the reported analysis methods of alcohols in biological samples, alcohols are not easy to ionize due to their weaker proton affinity and weaker acidity, and the signals in MALDI and ESI are weaker and the sensitivity is lower. Easily inhibited by other ionizable substances. In order to improve its mass spectral response, various derivatization reagents have been developed, betaine aldehyde (see Wu, C.P.; Ifa, D.R.; Manicke, N.E.; Cooks, R.G. Anal. Chem. 2009, 81, 7618–7624. ), 2-pyridinecarboxylic acid (see Higashi, T.; Shibayama, Y.J.; Shimada, K.J. Chromatogr. B2007, 846, 195-201.), 2-aminopyrazine (Analyst, 2013, 138, 6270-6276) and dansyl chloride (See Tang, Z.M.; Peter Guengerich, F. Anal. Chem. 2010, 82, 7706-7712.) was used to derivatize alcohols. However, these methods have disadvantages, including complex pretreatment, excess derivatization reagents need to be removed, and derivatization reagents themselves have strong ionization efficiency, which will inhibit the detection of analytes.

Contents of the invention

The problem to be solved by the present invention is to provide a method for the qualitative analysis of alcohol compounds in biological samples based on a quinoline carboxylic acid-based matrix derivatization method combined with MALDI-FT ICR MS, and further to say that it is a method through target esterification reaction to convert these alcohol compounds into quinoline esters, and the excess reagent acts as a matrix, which can greatly improve the analytical sensitivity and specificity of the target compound in the subsequent matrix-assisted laser desorption ionization-Fourier transform mass spectrometry analysis, without the need for The complex sample pretreatment process is used for high-throughput qualitative analysis of alcohol compounds in biological samples. It is a fast, efficient and sensitive analysis method for further research on the content changes of such compounds in the body and their physiological functions and diseases relationship lays the foundation.

In order to achieve the above object, the present invention provides a method for analyzing alcohol compounds in biological samples, which comprises the following steps:

After the pretreated sample is extracted with an organic solvent, the organic layer is taken and dried, and then an acetonitrile solution of quinolinecarboxylic acid and a methylene chloride solution of carbonyl chloride are added to obtain a reaction solution;

Spot 0.5-1 μL of the reaction solution on the target of the laser analysis-Fourier transform ion cyclotron resonance mass spectrometer, react at 15-40°C for 30-60 minutes, and use excess quinoline carboxylic acid as the matrix for matrix-assisted Laser analysis-Fourier transform ion cyclotron resonance mass spectrometry analysis, the molar ratio of the quinoline carboxylic acid to the alcohol compound to be measured is 20:1-50:1;

By comparing the high-resolution data analysis spectrum, the qualitative results of alcohol compounds are obtained;

Wherein, the sample is a biological substance containing alcohol compounds, and the alcohol compounds are cholesterol, fungal ergosterol, androsterone, vitamin D3, cholesterol, dehydrocholesterol, stigmasterol, dehydrooleosterol, A type of lanosterol.

The principle of the invention is: chemically reacting quinoline carboxylic acid and alcohol compound, using the reaction product to analyze the quinoline carboxylic acid derivative product of the alcohol compound through matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry. The reaction formula is shown in Figure 1.

As a preferred version, in the acetonitrile solution of quinoline carboxylic acid, the concentration of quinoline carboxylic acid is 2-5 mg/mL, and the volume is 50-100 μL.

As a preferred version, the quinoline carboxylic acid is quinoline-3-carboxylic acid, quinoline-4-carboxylic acid, quinoline-5-carboxylic acid, quinoline-6-carboxylic acid, quinoline-7-carboxylic acid or quinoline-8-carboxylic acid.

As a preferred solution, in the methylene chloride solution of phosgene, the volume fraction of phosgene is 10-40%, and the volume is 50-100 μL.

As a preferred solution, the pretreatment process of the sample is: add 1-2 mL of potassium hydroxide/ethanol solution with a volume ratio of 1:1 to the sample, and saponify and hydrolyze it at 60-70°C for 1-2 hours.

As a preferred solution, the conditions for the matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry analysis are: laser energy 20-30%, scanning range: m/z 100-1500, frequency: 1000 Hz, spot diameter 500-1000 μm, data Acquisition was carried out in positive ion mode; product ion scanning conditions: collision energy 10-30eV.

As a preferred solution, the sample is hair or yeast cells.

Compared with the prior art, the present invention has the following beneficial effects:

1. It is used to specifically derivatize alcoholic hydroxyl groups on the MALDI target, which is more targeted in the process of mass spectrometry analysis, improves the ionization efficiency of the analyte, and enhances the analytical sensitivity (detection limit 0.2-0.5ng/mL);

2. No complicated sample pretreatment process is required after the on-target reaction, and the reaction is directly analyzed to achieve high-throughput analysis. The operation is simple and the high resolution enables us to detect compounds with very close mass-to-charge ratios in complex samples;

3, the used derivatization reagent of this invention is simple, easy to get, and labeling part is relatively small (173Da), and structure is also relatively simple, and property is relatively stable, can produce and take off a molecular quinolone under the impact of collision energy in mass spectrometry product ion scanning analysis. Phenyl carboxylate fragment ion [M-172]+, which improves selectivity and helps to enhance the accuracy of qualitative analysis.

The present invention is applied to the qualitative analysis of alcohol compounds in various biological samples, such as cholesterol, cholestanol, fungal ergosterol, androsterone, vitamin D3, dehydrocholesterol, stigmasterol, sitosterol, campesterol, dehydrogenated rapeseed oil Sterols, lanosterol, etc. Through this method, rapid, efficient and sensitive detection of alcohol compounds can be realized.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 is the chemical reaction formula of middle alcohol compound of the present invention and quinoline carboxylic acid;

Figure 2 is the matrix-assisted laser desorption-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) spectrum of the cholesterol standard after the esterification reaction on the quinoline-3-carboxylic acid/carbonyl chloride target, the parent ion m/z 542.4 , product ion m/z 369.4;

Figure 3 is a schematic diagram of the qualitative analysis process of alcohol compounds in biological samples by derivatization-matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS);

Figure 4 is the underivatized MALDI-FT ICR MS spectrum of the hair sample;

Figure 5 is the MALDI-FT ICR MS spectrum of the hair sample derivatized on the quinoline-3-carboxylic acid/carbonyl chloride target, m/z 542.4.

Figure 6 is the underivatized MALDI-FT ICR MS spectrum of yeast cells;

Figure 7 is the MALDI-FT ICR MS spectrum of yeast cells derivatized on the quinoline-3-carboxylic acid/carbonyl chloride target, m/z 542.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

The structural formula of the quinoline carboxylic acid of the present invention and the alcohol compound to be tested is shown in 1, and the process flow of the present invention is shown in Figure 3.

Qualitative Analysis of Cholesterol in Example 1 Hair Sample

The operation steps are as follows:

Wash the hair with distilled water, dichloromethane and isopropanol, dry at 50-60°C, cut the hair into 1-2 mm, weigh 0.3-0.4 mg, add 1-2 mL of potassium hydroxide: ethanol (volume ratio 1:1), Saponify and hydrolyze at 60-70°C for 1-2 hours, add 2.5-5 mL of diethyl ether to extract twice, combine the organic layers, dry with KOH, blow dry with nitrogen, add 50-100 μL of 2-5 mg/mL quinoline-3-carboxylic acid/ Mix acetonitrile solution with 50-100μL 10-40v/v% phosgene/dichloromethane solution; spot 0.5-1μL of the reaction solution on the target, react at 15-40°C for 30-60 minutes, matrix-assisted laser analysis- Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) analysis, the spectrum is shown in Figure 2, and the MALDI-FT ICR MS spectrum of the hair sample before and after derivatization is shown in Figures 4 and 5. It can be seen that after the hair sample is reacted, the mass spectrum peak of the derivatized product of cholesterol can be obtained at m/z 542.4, while in the unreacted hair sample, no cholesterol peak is detected.

Qualitative analysis of ergosterol in embodiment 2 yeast cells

Weigh 200-500 mg of yeast cells, add 1-2 mL of potassium hydroxide: ethanol (volume ratio 1:1), saponify and hydrolyze at 60-70 °C for 1-2 hours, add 2.5-5 mL of ether to extract twice, and combine the organic layers Dry with KOH, dry with nitrogen, add 50-100μL 2-5mg/mL quinoline-3-carboxylic acid/acetonitrile solution, and mix with 50-100μL 10-40v/v% carbonyl chloride/dichloromethane solution; the reaction solution Spot 0.5-1 μL on the target, react at 15-40°C for 30-60 minutes, and analyze by matrix-assisted laser desorption-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS).

The MALDI-FT ICR MS spectra of yeast cells before and after derivatization are shown in Figures 6 and 7. It can be seen that after the reaction of the yeast cell sample, the mass spectrum peak of the derivative product of ergosterol m/z 536.4 can be obtained, while the peak of ergosterol was not detected in the unreacted yeast cell sample.

The present invention adopts the accurate mass measurement technology of high-resolution FTMS, takes the error less than 5ppm as the standard, compares the mass-to-charge ratio in the spectrum with the accurate mass-to-charge ratio of the derivatized product of the target compound, and further calculates the derivatized product of the target compound element composition. In the experiment, accurate mass determination of cholesterol-derived products and ergosterol-derived products was carried out. By comparing the high-resolution mass spectrometry data after derivatization of hair samples or yeast cell samples, the corresponding elemental composition was obtained. Ergosterol is carried out qualitative analysis, find out from table 1, the theoretical mass-to-charge ratio of derivative product and experimental value error are within 5ppm.

Table 1. Analysis results of sterols in hair and yeast cells after on-target derivatization and mass spectrometry detection

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (7)

1. an analytical method for alcohol compounds in a biological sample, characterized in that, comprising the steps: After the pretreated sample is extracted with an organic solvent, the organic layer is taken and dried, and then an acetonitrile solution of quinolinecarboxylic acid and a methylene chloride solution of carbonyl chloride are added to obtain a reaction solution; Spot 0.5-1 μL of the reaction solution on the target of the laser analysis-Fourier transform ion cyclotron resonance mass spectrometer, and react at 15-40°C for 30-60 minutes, so that the molar ratio to the alcohol compound to be measured is 20 :1-50:1 quinoline carboxylic acid as a matrix for matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry analysis; By comparing the high-resolution data analysis spectrum, the qualitative analysis results of alcohol compounds are obtained; Wherein, the sample is a biological substance containing alcohol compounds, and the alcohol compounds are cholesterol, fungal ergosterol, androsterone, vitamin D3, cholesterol, dehydrocholesterol, stigmasterol, dehydrooleosterol, A type of lanosterol. 2. analysis method as claimed in claim 1 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 method as claimed in claim 1 or 2, is characterized in that, described quinoline carboxylic acid is quinoline-3-carboxylic acid, quinoline-4-carboxylic acid, quinoline-5-carboxylic acid, quinoline line-6-carboxylic acid, quinoline-7-carboxylic acid or quinoline-8-carboxylic acid. 4. The analysis method according to claim 1, characterized in that, in the methylene chloride solution of phosgene, the volume fraction of phosgene is 10-40%, and the volume is 50-100 μL. 5. The analysis method according to claim 1, wherein the pretreatment process of the sample is: adding 1 to 2 mL of potassium hydroxide/ethanol solution with a volume ratio of 1:1 to the sample, Saponification and hydrolysis for 1 to 2 hours. 6. The analysis method according to claim 1, wherein the conditions for the matrix-assisted laser analysis-Fourier transform ion cyclotron resonance mass spectrometry analysis are: laser energy 20-30%, scanning range: m/z100-1500, Frequency: 1000Hz, spot diameter 500-1000μm, data acquisition is carried out in positive ion mode; product ion scanning conditions: collision energy 10-30eV. 7. The analysis method according to claim 1, wherein the sample is hair or yeast cells.