CN112748195A

CN112748195A - Method for simultaneously detecting fatty acid, amino acid and multifunctional group organic acid by GC-NCI-MS

Info

Publication number: CN112748195A
Application number: CN202011537173.9A
Authority: CN
Inventors: 白冰; 刘笑男; 赵楠; 颜廷才; 张旋; 李春强; 程顺昌; 魏宝东; 周倩; 赵瑛博
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2021-05-04

Abstract

The invention provides a method for simultaneously detecting fatty acid, amino acid and polyfunctional group organic acid by using GC-NCI-MS, belonging to the technical field of analysis and detection. The method utilizes the chloroformate derivatization agent to simultaneously derivatize fatty acid, amino acid and polyfunctional group organic acid, has the advantages of quick derivatization, good reproducibility and high stability of the derivatized product under the normal temperature condition, and can realize simultaneous derivatization of short-chain fatty acid carboxyl, short-chain polyfunctional group organic acid carboxyl and amino and carboxyl of branched chain amino acid. And the GC-NCI-MS method has small matrix interference and simple and clean mass spectrum, can retain molecular ion information, utilizes the obtained molecular/quasi-molecular ion information to carry out manual spectrum resolution and qualification, and combines a corresponding standard curve to realize accurate quantification of fatty acid, amino acid and polyfunctional group organic acid. The example data shows that: the method provided by the invention has the advantages of strong anti-interference capability, high sensitivity, wide application range and stable detection result.

Description

Method for simultaneously detecting fatty acid, amino acid and multifunctional group organic acid by GC-NCI-MS

Technical Field

The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting fatty acid, amino acid and organic acid by using GC-NCI-MS.

Background

Gas chromatography-mass spectrometry (GC-MS) has been used as a standard detection method for detecting volatile, low boiling, thermally stable substances with its high sensitivity, high resolution, and high reproducibility. Meanwhile, the conditions limit the detection and analysis of polar, high boiling point and heat unstable substances by the method. Fatty acid, amino acid and polyfunctional organic acid are common micromolecular organic matters in organisms, and have stronger polarity and thermal instability due to the fact that the structures of the organic acids all have carboxyl, amino, hydroxyl and other groups. In order to apply the GC-MS method to the detection of fatty acid/amino acid/multifunctional organic acid, derivatization treatment of the sample is essential.

Pre-column derivatization is an effective method for rendering polar, thermally unstable, high boiling molecules sufficiently volatile and reducing boiling points. Common derivatization methods include a sulfuric acid-methanol method, a potassium hydroxide-methanol method, an acetyl chloride-methanol method, an ammonia-ethanol method and the like, and the derivatization principle of the methods is that saponification is carried out under different acid-base conditions, and the long-chain fatty acid is converted into esters by using esterification reaction of alcohol and carboxyl of the long-chain fatty acid. These conventional derivatization methods have a disadvantage that the carboxyl groups of short-chain fatty acids and short-chain organic acids and the amino groups of amino acids cannot be derivatized, and thus the derivatization treatment of amino acids and polyfunctional organic acids cannot be achieved.

At present, methods for measuring fatty acid, amino acid and multifunctional organic acid mainly include High Performance Liquid Chromatography (HPLC), Ion Chromatography (IC), high performance liquid chromatography-mass spectrometry (HPLC-MS), automatic amino acid analyzer method, colorimetric method and the like. The structure of the object to be detected is complex, fatty acid is divided into long chain, medium chain and short chain, polyfunctional group organic acid has a plurality of carboxyl and hydroxyl, amino acid has a plurality of amino and carboxyl, and possibly has branched chain and annular structure, etc., which increases the difficulty of detection, and the simultaneous detection can be realized by matching a plurality of methods.

Therefore, the development of a method which has wide application range, high sensitivity and good reproducibility and can realize simultaneous qualitative and quantitative determination of fatty acid, amino acid and polyfunctional organic acid has important theoretical and practical significance.

Disclosure of Invention

In view of the above, the present invention provides a method for simultaneously detecting fatty acids, amino acids, and multifunctional organic acids by using GC-NCI-MS, which can simultaneously derivatize fatty acids, amino acids, and multifunctional organic acids, and simultaneously determine the nature and quantity of fatty acids, amino acids, and multifunctional organic acids by combining GC-NCI-MS.

In order to achieve the purpose, the invention provides the following scheme:

a method for simultaneously detecting fatty acid, amino acid and multifunctional organic acid by using GC-NCI-MS comprises the following steps:

mixing a solution to be detected, an alkaline reagent, an alkylating reagent and a chloroformate derivative, and carrying out a derivative reaction to obtain a derivative solution;

carrying out GC-NCI-MS detection on the derivative liquid to obtain molecular ion information and peak area of a derivative substance in the derivative liquid, and carrying out qualitative determination on the types of fatty acid, amino acid and multifunctional organic acid based on the obtained molecular ion information of the derivative substance;

establishing a corresponding fatty acid concentration-peak area standard curve, an amino acid concentration-peak area standard curve and a multifunctional group organic acid concentration-peak area standard curve based on qualitative fatty acid, amino acid and multifunctional group organic acid results;

substituting the peak area of the derivative substance in the derivative liquid into the peak area-fatty acid concentration standard curve, the peak area-amino acid concentration standard curve and the peak area-polyfunctional group organic acid concentration standard curve to obtain the contents of fatty acid, amino acid and polyfunctional group organic acid in the solution to be detected;

the solution to be detected comprises fatty acid, amino acid and polyfunctional organic acid, wherein the polyfunctional organic acid is organic acid which does not comprise fatty acid and amino acid;

the parameters detected by the GC-NCI-MS comprise:

gas chromatography conditions:

a chromatographic column: HP-5MS or a comparable type of chromatographic column;

sample introduction amount: 1 mu L of the solution;

the split ratio is as follows: 1-50: 1;

sample inlet temperature: 250 ℃;

carrier gas: helium, flow rate: 1 mL/min;

temperature rising procedure: keeping the temperature at 80 ℃ for 0-5 min, heating to 180 ℃ at 1-10 ℃/min, and keeping the temperature for 0-5 min; heating to 200 ℃ at a speed of 1-10 ℃/min, keeping for 0-5 min, and then operating at 230 ℃ and keeping for 0-5 min;

NCI-MS conditions:

an ionization mode: NCI;

ionization energy: 50 eV;

ion source temperature: 180 ℃;

emission current: 200 muA;

transmission line temperature: 250 ℃;

solvent retardation: 0-10 min;

reaction gas: methane;

scanning range: m/z is 50 to 650.

Preferably, the chloroformate-derived reagent is methyl chloroformate, ethyl chloroformate or propyl chloroformate; the alkaline reagent is one or more of sodium hydroxide, potassium hydroxide, pyridine and ammonium salt; the alkylating agent is an alcohol.

Preferably, the ratio of the total amount of fatty acid, amino acid and multifunctional organic acid, the amount of alkylating agent and the amount of chloroformate derivative in the solution to be detected is (1-50): (10-100): (1-10).

Preferably, the temperature of the derivatization reaction is 50-90 ℃, the pH value is more than or equal to 10, and the time is 30-60 min.

Preferably, after the derivatization reaction, the method further comprises: subjecting the derived system to a post-treatment comprising the steps of: mixing the obtained derivative system with an organic extracting agent, extracting, and controlling the pH value in the extraction process to be 10-14; standing and layering to obtain an organic extractant layer as a derivative liquid; the organic extracting agent comprises one or more of dichloromethane, trichloromethane, carbon tetrachloride, normal hexane and ethyl acetate.

Preferably, the solution to be detected is prepared from a sample to be detected; the sample to be detected is fermented milk or shiitake mushroom;

the method for preparing the solution to be detected by the fermented milk comprises the following steps:

mixing fermented milk and ammonia water, and heating; mixing the obtained feed liquid with ethanol and diethyl ether, and performing first ultrasonic treatment; mixing the obtained first ultrasonic feed liquid with petroleum ether, performing second ultrasonic treatment, and repeating the second ultrasonic treatment for 3-5 times; centrifuging the obtained second ultrasonic feed liquid, and combining organic layers; after the organic solvent in the organic layer was distilled off under reduced pressure, the resulting liquid was used as a test solution.

Preferably, the volume concentration of the ammonia water is 25-28%; the volume ratio of the fermented milk to the ammonia water to the ethanol to the diethyl ether to the petroleum ether is (10-50): (2-20): (10-20): (25-50): (25-50).

Preferably, the ethanol is absolute ethanol.

Preferably, the temperature of the heating treatment is 60-80 ℃, and the time is 15-30 min; the first ultrasonic treatment time is 2-10 min; the second ultrasonic treatment time is 2-10 min; the centrifugal rotating speed is 2000-10000 r/min, and the time is 15-30 min.

Preferably, the preparation of the solution to be tested by the shiitake mushrooms comprises the following steps:

drying Lentinus Edodes, crushing, and sieving to obtain Lentinus Edodes powder;

mixing the mushroom powder with water, and carrying out ultrasonic treatment to obtain the solution to be detected; the using amount ratio of the mushroom powder to water is (0.1-10) g: (10-200) mL; the ultrasonic time is 10 min.

The invention provides a method for simultaneously detecting fatty acid, amino acid and polyfunctional group organic acid by using GC-NCI-MS, which comprises the following steps: mixing a solution to be detected, an alkaline reagent, an alkylating reagent and a chloroformate derivative, and carrying out a derivative reaction to obtain a derivative solution; carrying out GC-NCI-MS detection on the derivative liquid to obtain molecular ion information and peak area of a derivative substance in the derivative liquid, and carrying out qualitative determination on the types of fatty acid, amino acid and multifunctional organic acid based on the obtained molecular ion information of the derivative substance; establishing a corresponding fatty acid concentration-peak area standard curve, an amino acid concentration-peak area standard curve and a multifunctional group organic acid concentration-peak area standard curve based on qualitative fatty acid, amino acid and multifunctional group organic acid results; substituting the peak area of the derivative substance in the derivative liquid into the peak area-fatty acid concentration standard curve, the peak area-amino acid concentration standard curve and the peak area-polyfunctional group organic acid concentration standard curve to obtain the contents of fatty acid, amino acid and polyfunctional group organic acid in the solution to be detected. The method utilizes the chloroformate derivatization agent to realize simultaneous derivatization of fatty acid, amino acid and polyfunctional group organic acid, has the advantages of quick derivatization, good reproducibility and high stability of the derivatized product at normal temperature, and can realize simultaneous derivatization of short chain fatty acid carboxyl, short chain polyfunctional group organic acid carboxyl and amino and carboxyl of branched chain amino acid; molecular ion information of the derived substances is obtained by combining GC-NCI-MS, and fatty acid, amino acid and polyfunctional group organic acid can be simply and accurately determined by manual spectrum decomposition; and then combining with a corresponding acid-peak area standard curve to realize the quantification of fatty acid, amino acid and polyfunctional organic acid. The data of the examples show that: the method provided by the invention has excellent minimum detection Limit (LOD), minimum quantification Limit (LOQ), repeatability, reproducibility, accuracy and precision.

Drawings

FIG. 1 is a total ion flow diagram of a decanoic acid gradient solution;

FIG. 2 is a total ion flow diagram of a fumaric acid gradient solution;

FIG. 3 is a total ion flow diagram of a succinic acid gradient solution;

FIG. 4 is a total ion flow graph of an alanine gradient solution;

FIG. 5 is a total ion flow graph of a valine gradient solution;

FIG. 6 is a total ion flow graph of an isoleucine gradient solution;

FIG. 7 is a total ion flow diagram of a glycine gradient solution;

FIG. 8 is a capric acid concentration-peak area standard curve;

FIG. 9 is a fumaric acid concentration-peak area standard curve;

FIG. 10 is a succinic acid concentration-peak area standard curve;

FIG. 11 is an alanine concentration-peak area standard curve;

FIG. 12 is a valine concentration-peak area standard curve;

FIG. 13 is an isoleucine concentration-peak area standard curve;

FIG. 14 is a glycine concentration-peak area standard curve;

FIG. 15 is a total ion flow graph of fermented milk detected by GC-NCI-MS;

FIG. 16 is a mass spectrum of

substances

1 and 2 obtained by detecting fermented milk by GC-NCI-MS;

FIG. 17 is a total ion flow graph obtained by detecting shiitake mushrooms by a GC-NCI-MS method;

FIG. 18 is a mass spectrum of substances 1-8 obtained by detecting shiitake mushrooms by a GC-NCI-MS method;

FIG. 19 is a graph comparing the total ion current obtained by testing mixed standard solutions by sulfuric acid-methanol derivatization/GC-EI-MS and chloroformate derivatization/GC-NCI-MS.

Detailed Description

The invention provides a method for simultaneously detecting fatty acid, amino acid and polyfunctional group organic acid by using GC-NCI-MS, which comprises the following steps:

substituting the peak area of the derivative substance in the derivative liquid into the peak area-fatty acid concentration standard curve, the peak area-amino acid concentration standard curve and the peak area-polyfunctional group organic acid concentration standard curve to obtain the contents of fatty acid, amino acid and polyfunctional group organic acid in the solution to be detected.

The method comprises the steps of mixing a solution to be detected, an alkaline reagent, an alkylating reagent and a chloroformate derivative, and carrying out a derivative reaction to obtain a derivative solution. In the invention, the solution to be detected comprises fatty acid, amino acid and multifunctional organic acid, wherein the multifunctional organic acid is organic acid which does not comprise fatty acid and amino acid. In the present invention, the chloroformate-based derivatizing agent is Methyl Chloroformate (MCF) or Ethyl Chloroformate (ECF) or Propyl Chloroformate (PCF); the alkaline reagent is preferably one or more of sodium hydroxide, potassium hydroxide, pyridine and ammonium salt, and is further preferably sodium hydroxide; the sodium hydroxide is preferably used in the form of an aqueous sodium hydroxide solution, the concentration of which is preferably 1 mol/L; the alkylating agent is preferably an alcohol, and more preferably n-propanol. In the invention, the ratio of the total amount of fatty acid, amino acid and multifunctional organic acid, the amount of alkylating reagent and the amount of chloroformate derivative in the solution to be detected is preferably (1-50): (10-100): (1-10), more preferably 1: 50: 2. in the invention, the total amount of the fatty acid, the amino acid and the multifunctional organic acid in the solution to be detected is preferably obtained by searching the amount of the fatty acid, the amino acid and the multifunctional organic acid in the sample to be detected reported in the literature to determine the sample amount of the detection solution; the amounts of fatty acids, amino acids and multifunctional organic acids in the sample solution can be determined by analogy with respect to the whole new sample solution. The amount of the alkaline agent used in the present invention is not particularly limited as long as the pH of the derivatization reaction can be a desired value. In the invention, the temperature of the derivatization reaction is preferably 50-90 ℃, and more preferably 80 ℃; the pH value is preferably not less than 10, more preferably 12; the time is preferably 30-60 min, and more preferably 60 min; the derivatization reaction is preferably carried out in a water bath.

After the derivatization reaction, the present invention preferably further comprises a post-treatment, which preferably comprises the following steps: mixing the obtained derivative system with an organic extracting agent, extracting, and controlling the pH value in the extraction process to be 10-14; standing for layering, and obtaining an organic extractant layer as a derivative liquid. In the invention, the organic extractant comprises one or more of dichloromethane, trichloromethane, carbon tetrachloride, n-hexane and ethyl acetate, and dichloromethane is further preferable. In the present invention, the pH of the extraction process is preferably 12. In the invention, the volume ratio of the solution to be detected to the organic extracting agent is preferably (1-10): (1-10), more preferably 1: 1; the reagent for controlling the pH value of the extraction process is preferably sodium bicarbonate, and the concentration of the sodium bicarbonate is preferably 50 mmol/L.

In the invention, the solution to be detected is preferably prepared from a sample to be detected; the sample to be tested is preferably a substance containing fatty acid, amino acid and multifunctional organic acid, and is particularly preferably, but not limited to, fermented milk or shiitake mushroom. In the present invention, the preparation of the test solution for fermented milk preferably comprises the following steps:

mixing fermented milk and ammonia water, and heating; mixing the obtained feed liquid, ethanol and diethyl ether, and performing first ultrasonic treatment; mixing the obtained first ultrasonic feed liquid with petroleum ether, performing second ultrasonic treatment, and repeating the second ultrasonic treatment for 3-5 times; centrifuging the obtained second ultrasonic feed liquid, and combining organic layers; after the organic solvent in the organic layer was distilled off under reduced pressure, the resulting liquid was used as a test solution. In the present invention, the volume concentration of the ammonia water is preferably 25%; the ethanol is preferably absolute ethanol; the volume ratio of the fermented milk to the ammonia water to the ethanol to the diethyl ether to the petroleum ether is preferably (10-50): (2-20): (10-20): (25-50): (25-50), more preferably 50: 2: 10: 25: 25; the temperature of the heating treatment is preferably 60-80 ℃, further preferably 60 ℃, and the time is preferably 15-30 min, further preferably 15 min; the first ultrasonic treatment time is preferably 2-10 min, and more preferably 2 min; the second ultrasonic treatment time is preferably 2-10 min, and further preferably 2 min; the rotation speed of the centrifugation is preferably 2000-10000 r/min, more preferably 2000r/min, the time is preferably 15-30 min, more preferably 15 min; the method of the present invention for distillation under reduced pressure is not particularly limited, and may be any method known to those skilled in the art, as long as the organic solvent in the organic layer can be removed.

In the invention, the preparation of the solution to be detected by the shiitake mushrooms comprises the following steps:

drying Lentinus Edodes, crushing, and sieving to obtain Lentinus Edodes powder; and mixing the mushroom powder with water, and carrying out ultrasonic treatment to obtain the solution to be detected. In the present invention, the shiitake mushroom is preferably washed with water before being dried. In the invention, the drying temperature is preferably 150-250 ℃, and the drying temperature is not particularly limited as long as the shiitake mushrooms can be dried; the crushing mode is not particularly limited, and technical means well known to those skilled in the art can be adopted, so long as the crushed material can pass through a 50-mesh sieve. In the invention, the ratio of the mushroom powder to the water is preferably (0.1-10) g: (10-200) mL, more preferably 3 g: 90 mL; the time of the ultrasound is preferably 10 min. After the ultrasonic treatment, the invention preferably also comprises the step of separating the material liquid obtained by the ultrasonic treatment, wherein the obtained liquid is the solution to be detected.

According to the invention, the chloroformate derivatization agent is utilized to simultaneously derivatize fatty acid, amino acid and polyfunctional group organic acid; in particular, the chloroformate derivatization agent is an excellent reagent which can be rapidly derivatized with amino and/or carboxyl without heating, and has the advantages of rapid derivatization under normal temperature, good reproducibility and high stability of the derivatized product; and can realize simultaneous derivatization of short-chain fatty acid carboxyl, short-chain polyfunctional organic acid carboxyl and amino and carboxyl of branched-chain amino acid.

After the derivative liquid is obtained, the GC-NCI-MS detection is carried out on the derivative liquid to obtain the molecular ion information and the peak area of the derivative substance in the derivative liquid. In the present invention, the parameters for the GC-NCI-MS detection include:

gas chromatography conditions:

sample introduction amount: 1 mu L of the solution;

the split ratio is as follows: 1-50: 1, preferably 10: 1;

sample inlet temperature: 250 ℃;

carrier gas: helium, flow rate: 1 mL/min;

NCI-MS conditions:

an ionization mode: NCI;

ionization energy: 50 eV;

ion source temperature: 180 ℃;

emission current: 200 muA;

transmission line temperature: 250 ℃;

solvent retardation: 0-10 min, preferably 3 min;

reaction gas: methane;

scanning range: m/z is 50 to 650.

And obtaining the molecular ion information and the peak area of the derivative substance in the derivative liquid through GC-NCI-MS detection, and determining the types of the fatty acid, the amino acid and the multifunctional group organic acid based on the obtained molecular ion information of the derivative substance. In the present invention, the type of fatty acid, amino acid and polyfunctional organic acid is preferably determined by manual resolution. In the invention, NCI is used as a soft ionization technology, which bombards an object to be detected by using methane macromolecules (relative to electrons), has low speed and low energy and can reserve most of molecules/quasi-molecule ions of the substance to be detected; secondly, the bombardment energy is low, the fragments of the object to be detected are few, the spectrum is simple, the molecular ion information is kept, the mass spectrum is easy to manually analyze, and the qualitative accuracy is greatly improved; thirdly, the NCI source has high selectivity and high sensitivity for analytes containing electronegative groups (e.g., -N ═ Cl, -F, C ═ O, -O-, -P ═), and can effectively reduce matrix interference and improve sensitivity.

After the fatty acid, the amino acid and the multifunctional group organic acid are qualitatively determined by manual spectrum decomposition, corresponding fatty acid concentration-peak area standard curve, amino acid concentration-peak area standard curve and multifunctional group organic acid-peak area standard curve are established according to qualitative results. In the specific embodiment of the invention, after the fermented milk is subjected to manual spectrum decomposition and contains caprylic acid and capric acid, a standard curve of caprylic acid concentration-peak area and a standard curve of capric acid concentration-peak area are established; when the mushroom is subjected to manual decolourization, the mushroom is found to contain fumaric acid, succinic acid, alanine, valine, isoleucine, leucine, glycine and proline; establishing a fumaric acid concentration-peak area standard curve, a succinic acid concentration-peak area standard curve, an alanine concentration-peak area standard curve, a valine concentration-peak area standard curve, an isoleucine concentration-peak area standard curve, a leucine concentration-peak area standard curve, a glycine concentration-peak area standard curve and a proline concentration-peak area standard curve. In the present invention, the method for establishing the corresponding fatty acid concentration-peak area standard curve, amino acid concentration-peak area standard curve and multifunctional organic acid-peak area standard curve preferably comprises the steps of:

respectively preparing standard solutions of fatty acid, amino acid and polyfunctional group organic acid with gradient concentration and determined qualitatively;

respectively mixing the standard solution with an alkaline reagent, an alkylating reagent and a chloroformate derivative for derivative reaction to obtain derivative solutions of the standard solutions;

carrying out GC-NCI-MS detection on the derivative liquid to obtain peak areas of derivative substances in the derivative liquid;

and respectively carrying out linear fitting on the concentrations of the fatty acid, the amino acid and the multifunctional group organic acid and corresponding peak areas to obtain a fatty acid concentration-peak area standard curve, an amino acid concentration-peak area standard curve and a multifunctional group organic acid-peak area standard curve.

In the present invention, the kind and the amount ratio of the alkaline reagent, the alkylating reagent and the chloroformate-derived reagent are preferably the same as those in the above technical scheme, and are not described herein again. In the present invention, the conditions of the derivatization reaction are preferably the same as those described above, and are not described herein.

In the present invention, the parameters of the GC-NCI-MS are preferably consistent with the above technical solutions, and are not described herein again.

The linear fitting method is not particularly limited in the present invention, and a linear fitting method known to those skilled in the art may be adopted.

After GC-NCI-MS detection, the peak area of a derivative substance in the derivative liquid is also obtained; substituting the peak area of the derivative substance in the derivative liquid into the peak area-fatty acid concentration standard curve, the peak area-amino acid concentration standard curve and the peak area-polyfunctional group organic acid concentration standard curve to obtain the contents of fatty acid, amino acid and polyfunctional group organic acid in the solution to be detected.

The method provided by the invention can realize simultaneous qualitative and quantitative analysis of fatty acid, amino acid and polyfunctional organic acid, and has the advantages of wide application range, concise derivatization operation, stable derivative, comprehensive fatty acid/organic acid/amino acid detection, good linearity and reproducibility and high sensitivity.

The method for simultaneously detecting fatty acids, amino acids and multifunctional organic acids by GC-NCI-MS provided in the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

The parameters of the GC-NCI-MS measurements described in the following examples are: the gas chromatography conditions include: a chromatographic column: HP-5MS chromatographic column; sample introduction amount: 1 mu L of the solution; the split ratio is as follows: 10: 1; sample inlet temperature: 250 ℃; carrier gas: helium, flow rate: 1 mL/min; temperature rising procedure: keeping the temperature at 80 ℃ for 3min, heating to 180 ℃ at 10 ℃/min, and keeping the temperature for 4 min; heating to 200 deg.C at 10 deg.C/min, maintaining for 1min, and maintaining at 230 deg.C for 3 min; the NCI-MS conditions include: an ionization mode: NCI; ionization energy: 50 eV; ion source temperature: 180 ℃; emission current: 200 muA; transmission line temperature: 250 ℃; solvent retardation: 3 min; reaction gas: methane; scanning range: m/z is 50 to 650.

The GC-EI-MS: the gas chromatography conditions included: a chromatographic column: HP-5MS chromatographic column; sample introduction amount: 1 mu L of the solution; the split ratio is as follows: 10: 1; sample inlet temperature: 250 ℃; carrier gas: helium (purity 99.999%), flow rate: 1 mL/min; temperature rising procedure: keeping at 80 deg.C for 3min, heating to 180 deg.C at speed of 10 deg.C/min for 4min, heating to 200 deg.C at speed of 10 deg.C/min for 1min, and keeping at 230 deg.C for 3 min. The EI-MS conditions include: an ionization mode: EI; electron energy: 70 ev; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; solvent retardation: 3 min; reaction gas: helium (purity is more than or equal to 99.999%); scanning range: m/z is 50-650.

Example 1

Establishment of a Standard Curve

Preparing 0.1mmol/L, 0.2mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L and 5mmol/L decanoic acid standard solutions; meanwhile, preparing the same series of fumaric acid standard solutions, succinic acid standard solutions, alanine standard solutions, valine standard solutions, isoleucine standard solutions and glycine standard solutions;

mixing 1mL series capric acid standard solution with 0.6mL 1mol/L sodium hydroxide, 3.2mL n-propanol and 0.2mL methyl chloroformate, shaking, heating in 80 deg.C water bath for 60min, and cooling to room temperature; adding 1mL of dichloromethane for extraction, adjusting the pH value to 12 by 50mmoL/L of sodium bicarbonate, standing for layering, sucking the dichloromethane layer by using a glass syringe, and filtering the dichloromethane layer by using a 0.22 mu m organic phase filter membrane to obtain a series of derivative solutions;

detecting the series of derived liquids by adopting the GC-NCI-MS detection parameters to obtain the total ion current of the series of capric acid standard liquids, wherein the result is shown in figure 1; detecting other acid standard solutions according to the method, wherein the obtained total ion flow diagram is shown in figures 2-7; wherein, fig. 1 is a total ion flow diagram of a capric acid gradient solution; FIG. 2 is a total ion flow diagram of a fumaric acid gradient solution; FIG. 3 is a total ion flow diagram of a succinic acid gradient solution; FIG. 4 is a total ion flow graph of an alanine gradient solution; FIG. 5 is a total ion flow graph of a valine gradient solution; FIG. 6 is a total ion flow graph of an isoleucine gradient solution; figure 7 is a total ion flow graph of a glycine gradient solution.

Meanwhile, peak areas of decanoic acids with different concentrations are obtained; linearly fitting the concentration and the peak area of the decanoic acid to obtain a decanoic acid concentration-peak area standard curve, wherein the result is shown in figure 8, fitting the concentrations and the peak areas of other acid standard liquids according to the method to obtain other acid concentration-standard curves, the result is shown in figures 9-14, and figure 8 is the decanoic acid concentration-peak area standard curve; FIG. 9 is a fumaric acid concentration-peak area standard curve; FIG. 10 is a succinic acid concentration-peak area standard curve; FIG. 11 is an alanine concentration-peak area standard curve; FIG. 12 is a valine concentration-peak area standard curve; FIG. 13 is an isoleucine concentration-peak area standard curve; FIG. 14 is a glycine concentration-peak area standard curve.

1. LOD and LOQ

After derivatization of 7 series of acid standard solutions, the assay was repeated 3 times using GC-NCI-MS. LOD is calculated according to the signal-to-noise ratio of 3 times, and LOQ is calculated according to the signal-to-noise ratio of 10 times. The results are as follows: capric acid: LOD is 0.05mmol/L, LOQ is 0.16 mmol/L; fumaric acid: LOD is 0.05mmol/L, LOQ is 0.26 mmol/L; succinic acid: LOD is 0.03mmol/L, LOQ is 0.15 mmol/L; alanine: LOD is 0.04mmol/L, LOQ is 0.10 mmol/L; valine: LOD is 0.03mmol/L, LOQ is 0.11 mmol/L; isoleucine: LOD is 0.04mmol/L, LOQ is 0.08 mmol/L; glycine: LOD was 0.02mmol/L and LOQ was 0.12 mmol/L.

2. Repeatability of

The concentrations of 0.2mmol/L, 1mmol/L and 5mmol L in 7 series of acid standard solutions were selected and detected in triplicate to obtain peak areas as shown in Table 1.

TABLE 1 Peak area of three concentrations of standard substance measured in three parallel experiments

The Relative Standard Deviation (RSD) of the peak areas detected at each concentration was calculated, and the reproducibility was expressed as the average of these relative standard deviations, and the value corresponding to 0.95% for capric acid, 0.87% for fumaric acid, 0.09% for succinic acid, 0.10% for alanine, 0.28% for valine, 0.26% for isoleucine, and 0.34% for glycine.

3. Reproducibility of

The peak areas of the standard solutions of 7 acids at 1mmol/L were determined in triplicate by the same investigator on three different days as shown in Table 2.

TABLE 2 Peak area of triplicate determinations of standards

The average value of the relative standard deviations of the peak areas was calculated, and the values were 0.96% for the standard deviation of capric acid, 1.53% for the standard deviation of fumaric acid, 2.16% for the standard deviation of succinic acid, 0.20% for the standard deviation of alanine, 1.00% for the standard deviation of valine, 2.73% for the standard deviation of isoleucine and 0.11% for the standard deviation of glycine.

4. Accuracy and precision

Pretreatment of mushroom: removing roots of the mushrooms, cleaning the mushrooms with clear water, placing the mushrooms into an oven for drying and mashing at 150 ℃, and sieving the mushrooms with a 50-mesh sieve for later use; weighing 3g of shiitake powder by using a ten-thousandth balance, adding 90mL of distilled water, performing ultrasonic extraction for 10min, centrifuging, and taking supernate as a solution to be detected.

The accuracy is expressed by recovery rate, and seven standard substances of 5 mug, 10 mug and 15 mug are respectively added to three same solutions to be detected; the accuracy is expressed as the coefficient of variation (CV%) which is the ratio of the standard deviation to the mean of three replicates of the same sample to which 7 standards were added; the calculated values of recovery and CV% are shown in table 3.

TABLE 3 accuracy (recovery) and precision (coefficient of variation,%) of the GC-NCI-MS method

As can be seen from table 3: the method presents excellent accuracy and precision, and through adding a known amount of standard substance into a shiitake mushroom sample derived from methyl chloroformate, the recovery rate of the added decanoic acid is calculated to be between 91% and 103%, and the coefficient of variation is calculated to be between 3.13% and 3.58%; the recovery rate of the added fumaric acid is between 96% and 105%, and the coefficient of variation is between 3.09% and 3.25%; the recovery rate of the added succinic acid is between 91% and 109%, and the coefficient of variation is between 2.81% and 3.93%; the recovery rate of the added alanine is between 95% and 105%, and the coefficient of variation is between 2.11% and 3.41%; the recovery rate of the added valine is between 104% and 116%, and the coefficient of variation is between 3.16% and 4.31%; the recovery rate of added isoleucine is between 96% and 102%, and the coefficient of variation is between 3.48% and 3.58%; the recovery rate of the added glycine is between 94% and 106%, and the coefficient of variation is between 2.5% and 2.93%; the recovery rate of the method meets 80-120%, and all the variation coefficients meet 0-5%.

The evaluation indexes are summarized to obtain table 4.

TABLE 4 summary of GC-NCI-MS detection methodological indexes

Example 2

Pretreatment of fermented milk: putting 50mL of fermented milk into a 250mL conical flask, adding 2mL of ammonia water solution with the volume fraction of 25%, plugging, shaking up, heating in water bath at 60 ℃ for 15min, and cooling to room temperature; adding 10mL of absolute ethyl alcohol and 25mL of diethyl ether, adding a plug, shaking up, and performing ultrasonic treatment for 2 min; adding 25mL of petroleum ether, and performing ultrasonic treatment for 2 min; centrifuging at 2000r/min for 15min, repeating the above extraction process for 2 times, mixing the extracted organic layers, and distilling under reduced pressure to remove solvent to obtain solution to be tested.

Derivatization of test solutions

Taking 1mL of solution to be tested, sequentially adding 0.6mL of 1mol/L sodium hydroxide, 3.2mL of n-propanol and 0.2mL of methyl chloroformate, shaking up, heating in a water bath at 80 ℃ for 60min, and cooling to room temperature; adding 1mL of dichloromethane for extraction, adjusting the pH value to 12 by 50mmoL/L of sodium bicarbonate, standing for layering, sucking the dichloromethane layer by using a glass syringe, and filtering the dichloromethane layer by using a 0.22 mu m organic phase filter membrane to obtain a derivative solution;

detecting the derivative liquid obtained from the fermented milk according to the GC-NCI-MS detection parameters to obtain molecular ion information of derivative substances in the derivative liquid, qualitatively determining substances in the fermented milk by adopting an artificial spectrum resolving mode based on the molecular ion information, and determining that the fermented milk contains caprylic acid and capric acid;

establishing a caprylic acid concentration-peak area standard curve and a capric acid concentration-peak area standard curve, and bringing the peak areas of corresponding acids into the standard curves to obtain the contents of caprylic acid and capric acid in the fermented milk.

FIG. 15 is a total ion flow diagram of fermented milk detected by GC-NCI-MS method, and FIG. 16 is a mass spectrum diagram of

substances

1 and 2 detected by GC-NCI-MS method. As can be seen from FIGS. 15-16: two substances, namely caprylic acid and capric acid, are detected in the fermented milk.

Detailed analysis was performed using NIST standard library and manual review, see table 5 for details of 1 and 2 in fig. 15 and fig. 16 (molecular information, retention time and peak area).

TABLE 5 summary of the results of the GC-NCI-MS method for testing fermented milk samples

Detecting the derived liquid of the mushrooms according to the GC-NCI-MS detection parameters to obtain molecular ion information of derived substances in the derived liquid, qualitatively determining the substances in the mushrooms by adopting an artificial spectrum resolving mode based on the molecular ion information, and determining that the mushrooms contain fumaric acid, succinic acid, alanine, valine, isoleucine, leucine, glycine and proline;

establishing a fumaric acid concentration-peak area standard curve, a succinic acid concentration-peak area standard curve, an alanine concentration-peak area standard curve, a valine concentration-peak area standard curve, an isoleucine concentration-peak area standard curve, a leucine concentration-peak area standard curve, a glycine concentration-peak area standard curve and a proline concentration-peak area standard curve, and substituting the peak areas of the corresponding acids into the standard curves to obtain the content of each acid in the lentinus edodes.

FIG. 17 is a total ion flow diagram obtained by detecting shiitake mushrooms by a GC-NCI-MS method, and FIG. 18 is a mass spectrum diagram of substances 1-8 obtained by detecting shiitake mushroom samples by the GC-NCI-MS method.

The 8 species molecular information, retention times and peak areas of fig. 17 and 18 were summarized using NIST standard library and manual review for analysis to give table 6. As can be seen from FIGS. 17-18 and Table 6: the GC-NCI-MS method identifies 8 substances, 2 of which are organic acids, and 6 substances are amino acids, and the specific conditions are shown in Table 6.

TABLE 6 summary of the results of the GC-NCI-MS method for detecting Lentinus edodes samples

As can be seen from tables 5 to 6: the GC-NCI-MS method can realize simultaneous qualitative and quantitative detection of fatty acid, amino acid and polyfunctional group organic acid in fermented milk and mushroom samples. 10 substances, namely fatty acid (caprylic acid and capric acid), polyfunctional group organic acid (fumaric acid and succinic acid) and amino acid (alanine, valine, leucine, isoleucine, glycine and proline) are detected in the fermented milk and the mushroom sample, so that the fatty acid, the organic acid and the amino acid can be simultaneously qualitatively and quantitatively determined.

Example 3

Preparing mixed standard substance solutions with the concentrations of heptanoic acid, octanoic acid, sorbic acid, fumaric acid, decanoic acid, benzoic acid, succinic acid, salicylic acid, leucine and glycine of 5mmol/L respectively;

sulfuric acid-methanol derivatization/GC-EI-MS method:

taking 1mL of mixed standard solution, adding 2mL of 20% sulfuric acid-methanol, heating in 80 ℃ water bath for 60min, taking 1mL of reacted solution, adding 0.5mL of distilled water and 1mL of trichloromethane for extraction, standing for layering, sucking the trichloromethane layer by using a glass syringe, and filtering through a 0.22 mu m organic phase filter membrane to obtain a comparative derivative solution.

Chloroformate derivatization/GC-NCI-MS method:

taking 1mL of mixed standard solution, sequentially adding 0.6mL of 1mol/L sodium hydroxide, 3.2mL of n-propanol and 0.2mL of methyl chloroformate, shaking up, heating in a water bath at 80 ℃ for 60min, and cooling to room temperature; adding 1mL of dichloromethane for extraction, adjusting the pH value to 12 by 50mmoL/L of sodium bicarbonate, standing for layering, sucking the dichloromethane layer by using a glass syringe, and filtering the dichloromethane layer by using a 0.22 mu m organic phase filter membrane to obtain a derivative solution;

and detecting the comparative derivative liquid and the derivative liquid according to GC-EI-MS and GC-NCI-MS detection conditions respectively. The effect of the chloroformate-derived/GC-NCI-MS method on the mixed standard solutions was finally compared with the sulfuric acid-methanol-derived/GC-EI-MS method, as shown in fig. 19.

The specific result information of the sulfuric acid-methanol derivatization/GC-EI-MS method is shown in Table 7, and the specific result information of the chloroformate derivatization/GC-NCI-MS method is shown in Table 8.

TABLE 7 summary of sulfuric acid-methanol derivatization/GC-EI-MS method for detecting material components

TABLE 8 summary of chloroformate derivative/GC-NCI-MS method for detecting substance components

As can be seen from fig. 19 and tables 7 to 8: the fatty acid, the amino acid and the organic acid detected in the detection sample by the sulfuric acid-methanol derivatization/GC-EI-MS method are all less than the results of the chloroformate derivatization/GC-NCI-MS method in types and contents, and only trace contents are detected for malic acid and citric acid because the derivatization of the organic acid by the derivatization agent is incomplete. The chloroformate derivative/GC-NCI-MS method has the result that the obtained fatty acid, amino acid and organic acid are more than those derived by the sulfuric acid-methanol derivative/GC-EI-MS method in type and content, and the chloroformate derivative/GC-NCI-MS method has more comprehensive and stable detection on the fatty acid, the amino acid and the organic acid.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for simultaneously detecting fatty acid, amino acid and multifunctional organic acid by using GC-NCI-MS is characterized by comprising the following steps:

the parameters detected by the GC-NCI-MS comprise:

gas chromatography conditions:

sample introduction amount: 1 mu L of the solution;

the split ratio is as follows: 1-50: 1;

sample inlet temperature: 250 ℃;

carrier gas: helium, flow rate: 1 mL/min;

NCI-MS conditions:

an ionization mode: NCI;

ionization energy: 50 eV;

ion source temperature: 180 ℃;

emission current: 200 muA;

transmission line temperature: 250 ℃;

solvent retardation: 0-10 min;

reaction gas: methane;

scanning range: m/z is 50 to 650.

2. The method of claim 1, wherein said chloroformate-derivatizing agent is methyl chloroformate, ethyl chloroformate, or propyl chloroformate; the alkaline reagent is one or more of sodium hydroxide, potassium hydroxide, pyridine and ammonium salt; the alkylating agent is an alcohol.

3. The method according to claim 1 or 2, wherein the ratio of the total amount of the fatty acid, the amino acid and the multifunctional organic acid, the amount of the alkylating agent and the amount of the chloroformate-type derivatizing agent in the solution to be tested is (1-50): (10-100): (1-10).

4. The method according to claim 1, wherein the temperature of the derivatization reaction is 50-90 ℃, the pH value is more than or equal to 10, and the time is 30-60 min.

5. The method of claim 1 or 4, further comprising, after the derivatizing reaction: subjecting the derived system to a post-treatment comprising the steps of: mixing the obtained derivative system with an organic extracting agent, extracting, and controlling the pH value in the extraction process to be 10-14; standing and layering to obtain an organic extractant layer as a derivative liquid; the organic extracting agent comprises one or more of dichloromethane, trichloromethane, carbon tetrachloride, normal hexane and ethyl acetate.

6. The method according to claim 1, wherein the test solution is prepared from a test sample; the sample to be detected is fermented milk or shiitake mushroom;

7. The method according to claim 6, wherein the volume concentration of the ammonia water is 25-28%; the volume ratio of the fermented milk to the ammonia water to the ethanol to the diethyl ether to the petroleum ether is (10-50): (2-20): (10-20): (25-50): (25-50).

8. The method of claim 6, wherein the ethanol is absolute ethanol.

9. The method according to claim 6, 7 or 8, wherein the temperature of the heat treatment is 60 to 80 ℃ and the time is 15 to 30 min; the first ultrasonic treatment time is 2-10 min; the second ultrasonic treatment time is 2-10 min; the centrifugal rotating speed is 2000-10000 r/min, and the time is 15-30 min.

10. The method as claimed in claim 6, wherein the preparation of the solution to be tested by the shiitake mushrooms comprises the following steps:

drying Lentinus Edodes, crushing, and sieving to obtain Lentinus Edodes powder;