CN111650286A - Method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry - Google Patents

Method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry Download PDF

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CN111650286A
CN111650286A CN202010252106.6A CN202010252106A CN111650286A CN 111650286 A CN111650286 A CN 111650286A CN 202010252106 A CN202010252106 A CN 202010252106A CN 111650286 A CN111650286 A CN 111650286A
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张赛
曾秋芳
杨超
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Shanghai Applied Protein Technology Co Ltd
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Abstract

The invention aims to provide a method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry, which comprises the steps of adding isotope n-nonadecanoic acid methyl ester into a serum sample as an internal standard, then performing a methyl esterification process, extracting the medium-long chain fatty acid in a sample mixture through a liquid-liquid extraction method, separating 40 kinds of medium-long chain fatty acid by using Agilent DB-23 chromatography, and then performing tandem mass spectrometry by using an internal standard curve method. Has the advantages of good selectivity, high sensitivity, short analysis time and the like.

Description

Method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry
Technical Field
The invention relates to the technical field of metabonomics analysis, in particular to a method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry.
Background
The medium-long chain fatty acid is not only an energy substance, but also a functional substance, influences the growth and development of human beings through mechanisms such as intestinal flora, intestinal morphology, immune function, Ghrelin secretion and the like, and the difference of the influences on the growth performance of human beings is not only related to the content of the medium-long chain fatty acid, but also related to the type of the contained medium-long chain fatty acid, the energy state of the human beings and the health condition of the human beings. In addition, the research of the medium-long chain fatty acid has various physiological effects on preventing and treating chronic diseases and has great significance on maintaining body health.
At present, the GC-MS method is adopted to carry out quantitative analysis on medium-long chain fatty acids, and usually, only less than 37 common medium-long chain fatty acids can be detected, three unsaturated long chain fatty acids, namely docosatetraenoic acid C22:4, docosapentaenoic acid C22:5n-3 and docosapentaenoic acid C22:5n-6, which have important physiological research significance cannot be separated and detected, the development of scientific research is not facilitated, and the achievement is shown, so a new detection method is needed to solve the technical problems.
Disclosure of Invention
The invention aims to provide a method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry, which comprises the steps of adding isotope n-nonadecanoic acid methyl ester into a serum sample as an internal standard, then performing a methyl esterification process, extracting the medium-long chain fatty acid in a sample mixture through a liquid-liquid extraction method, separating 40 kinds of medium-long chain fatty acid by using Agilent DB-23 chromatography, and then performing tandem mass spectrometry by using an internal standard curve method. Has the advantages of good selectivity, high sensitivity, short analysis time and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry comprises the following steps:
s1, preparing an internal standard solution, weighing an isotope internal standard product methyl nonadecanoate, and adding n-hexane to dissolve the isotope internal standard product methyl nonadecanoate to prepare the internal standard solution for later use;
s2, preparing a series of concentration standard substance mixed solution, weighing medium-long chain fatty acid standard substances for gas chromatography-mass spectrometry (GC-MS) after methyl esterification, and preparing a series of concentration gradient standard substance mixed solution by using n-hexane;
step S3, extracting a sample, namely taking a serum sample to be detected, extracting medium-long chain fatty acid in the sample by adopting dichloromethane and methanol, taking a dichloromethane phase, and drying;
step S4, adding an internal standard, redissolving the sample obtained in the step S3 by using normal hexane, adding isotope n-nonadecanoic acid methyl ester (C19:0) as the internal standard, adding potassium hydroxide methanol solution for treatment, adding water, standing for layering, taking supernatant, drying by using nitrogen, and redissolving by using normal hexane to obtain a sample extraction solution;
step S5 gas chromatography-mass spectrometry, wherein the mixed solution of the standard substance with the series concentration gradient obtained in step S2 and the sample extraction solution obtained in step S4 are respectively subjected to gas chromatography, and the mode of the chromatographic temperature gradient is as follows:
heating rate/min Temperature value of Residence time min Run time min
Initial temperature 70 2 2
Procedure 1 20 180 8 15.5
Procedure 2 4 250 3 36
Directly carrying out mass spectrometry on the sample subjected to gas chromatography separation;
and S6, converting the concentration, drawing the mass spectrum data of the standard substance mixed solution with the series of concentration gradients obtained in the step S2 into a standard curve, and converting the mass spectrum data of the sample extraction solution obtained in the step S4 into the corresponding medium-long chain fatty acid concentration through the standard curve.
According to the above scheme, the concentration of the n-nonadecanoic acid methyl ester in the internal standard solution in the step S1 is 1 mg/mL.
According to the scheme, the concentration range of the series concentration standard solution in the step S2 is 1-2500 mg/L.
According to the scheme, the medium-long chain fatty acid standard in the step S2 comprises the following standards: methyl butyrate, methyl hexanoate, methyl octanoate, methyl decanoate, methyl undecanoate, methyl laurate, methyl tridecanoate, methyl myristate, methyl pentadecanoate, methyl palmitate, methyl palmitoleate, methyl heptadecanoate, methyl heptadecenoate, methyl stearate, methyl oleate, methyl elactoate, methyl linoleate, methyl elaidoleate, methyl elanoleate, methyl gama-linolenic acid methyl ester, methyl linolenate, methyl arachidate, methyl eicosenoate, methyl eicosatrienoate, methyl arachidonic acid, methyl eicosapentaenoate, methyl heneicosanoate, methyl docosenoic acid, methyl behenate, methyl erucate, methyl docosedienoate, methyl docosenoic acid, methyl eicosatrinoate, methyl tetracosenoic acid, methyl ester, Methyl tetracosenoic acid, methyl docosatetraenoate, methyl docosapentaenoate and methyl docosapentaenoate.
According to the above scheme, the medium-long chain fatty acid extracted from the sample in step S3 is specifically: adding dichloromethane and methanol into a serum sample to be detected according to a volume ratio of dichloromethane to methanol of 2:1, and uniformly mixing for 1-3min in a vortex manner; oscillating on a shaker for 15-30min at 270rpm at 25 ℃; adding deionized water and anhydrous sodium sulfate, and performing vortex oscillation for 2 min; centrifuging at 2000rpm for 4-6 min; the lower dichloromethane phase was removed and blown dry with nitrogen.
According to the scheme, the detailed steps of adding the internal standard in the step S4 are as follows: adding n-hexane into the sample obtained in the step S3 for redissolving, adding isotope n-nonadecanoic acid methyl ester (C19:0), performing vortex oscillation for 1-3min, adding 0.4mol/L potassium hydroxide methanol solution, performing vortex oscillation for 1-3min, performing oscillation for 20-40min at 37 ℃ by using a shaking table at 180rpm, adding deionized water, performing vortex oscillation for 1-3min, standing for layering, taking the supernatant into a new glass test tube, performing blow-drying by using nitrogen, adding 200 mu L n-hexane, performing vortex oscillation for 2min, standing for layering, and taking the supernatant.
According to the scheme, the medium-long chain fatty acid single-ion detection scanning groups in the mass spectrometry are as follows:
Figure BDA0002435870650000031
the medium-long chain fatty acid single ion detection scanning parameter conditions in the mass spectrometry are as follows:
Figure BDA0002435870650000032
and (4) analyzing the standard substance mixed solution with the series of concentration gradients and the sample extraction solution obtained in the step S4 respectively, and deriving and processing mass spectrum data.
The invention has the beneficial effects that: the pretreatment step of the sample can be completed only by simple liquid-liquid extraction, and meanwhile, the important medium-long chain fatty acid substances in serum can be accurately detected by quantifying through an isotope internal standard, the method has high precision and accuracy, can be used for quantitative analysis of clinical serum samples, is simple in experimental operation and short in experimental period, and provides a reliable detection method for health assessment of the medium-long chain fatty acid level in clinic.
Detailed Description
The technical solution of the present invention will be described with reference to the following examples.
The method for detecting medium-long chain fatty acid in human serum based on gas chromatography-mass spectrometry comprises the following steps:
step S1, preparing an internal standard solution, weighing 100mg isotope internal standard product methyl nonadecanoate, adding 1mL of n-hexane to dissolve and prepare 100mg/mL stock solution, taking 10 mu L of stock solution, diluting with n-hexane to a constant volume of 1mL, and preparing 1mg/mL internal standard solution for later use;
s2, preparing a series of concentration standard substance mixed solutions, weighing medium-long chain fatty acid standard substances for gas chromatography-mass spectrometry (GC-MS) after methyl esterification, and preparing a series of concentration gradient standard substance mixed solutions by using n-hexane, wherein the concentration comprises 2500, 1000, 500, 250, 125, 62.5, 25, 10, 5 and 1 mg/L; the medium-long chain fatty acid standard comprises the following standard substances:
methyl butyrate C4:0[623-42-7]2 wt.%;
methyl hexanoate Methyl ester C6:0[106-70-7]2 wt.%;
methyl octanate Methyl octanoate C8:0[111-11-5]2 wt.%;
methyl decanoate Methyl ester C10:0[110-42-9]4 wt.%;
methyl decanoate undecanoate C11:0[1731-86-8]2 wt.%;
methyl dodecanoate Methyl laurate C12:0[111-82-0]4 wt.%;
methyl tricarbacetate tridecanoate C13:0[1731-88-0]2 wt.%;
methyl myrsitate myristate C14:0[124-10-7]2 wt.%;
methyl myristoleate Methyl myristate C14:1, cis-9[56219-06-8]2 wt.%;
methyl pentadecanoate pentadecanoic acid Methyl ester C15:0[7132-64-1]2 wt.%;
methyl cis-10-pentadecenoate pentadecanoic acid Methyl ester C15:1, cis-10[90176-52-6]2 wt.%;
methyl palmitate Methyl ester C16:0[112-39-0]4 wt.%;
methyl palmiteolate palmitoleate C16:1, cis-9[1120-25-8]2 wt.%;
methyl heptadecanoate Methyl ester C17:0[1731-92-6]4 wt.%;
methyl cis-10-heptadecenoate, C17:1, cis-10[75190-82-8]2 wt.%;
methyl stearate C18:0[112-61-8]4 wt.%;
methyl oleate C18:1, cis-9[112-62-9]4 wt.%;
methyl elaidate trans-oleate C18:1T, trans-9[2462-84-2]2 wt.%;
methyl linoleate C18:2, cis-9,12[112-63-0]2 wt.%; (ii) a
Methyl linoleladate Methyl linoleate C18:2TT, trans-9,12[2462-84-2]2 wt.%;
methyl γ -linolenate γ -linolenic acid Methyl ester C18:3, cis-6,9,12[16326-32-2]4 wt.%;
methyl linolenate Methyl ester C18:3, cis-9,12,15[301-00-8]2 wt.%;
methyl arachidate C20:0[1120-28-1]4 wt.%;
cis-11-Eicosenoic acid methyl ester C20:1, cis-11[2390-09-2]2 wt.%;
cis-11,14, 17-eicosarienoic acid methyl ester C20:3, cis-11,14,17[55682-88-7]2 wt.%;
cis-8,11, 14-Eicosaterieno acid methyl ester C20:3, cis-8,11,14[21061-10-9]2 wt.%;
methyl arachidate Methyl arachidonate C20:4, cis-5,8,11,14[2566-89-4]2 wt.%; cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester (EPA-M) C20:5, cis-5,8,11,14,17[2734-47-6]2 wt.%;
methyl hemicosanoate Methyl eicosanoate C21:0[6064-90-0]2 wt.%;
cis-11, 14-Eicosapienoic acid methyl ester C20:2, cis-11,14 [2463-02-7]2 wt.%;
methyl benzoate behenate C22:0[929-77-1]4 wt.%;
methyl erucate Methyl ester C22:1, cis-13[1120-34-9]2 wt.%;
cis-13,16-Docosadienoic acid methyl ester C22:2, cis-13,16 [61012-47-3]2 wt.%;
cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester (DHA-M) C22:6, cis-4,7,10,13,16,19[301-01-9]2 wt.%;
methyl tricosanoate C23:0[2433-97-8]2 wt.%;
methyl tetracosanoate C24:0[2442-49-1]4 wt.%;
methyl cis-15-tetracosenoate Methyl tetracosenoate C24:1, cis-15[2733-88-2]2 wt.%;
METHYL docosateratate DOCOSATETRAENOATE C22:4[13487-42-8]2 wt.%;
METHYL DOCOSAPENTAENOATE (7C,10C,13C,16C,19C) C22:5n-3 [108698-02-8]2 wt.%;
METHYL DOCOSAPENTAENOATE (4C,7C,10C,13C,16C) C22:5n-6[25182-74-5]2 wt.%;
step S3, extracting a sample, namely putting 100 mu L of a human serum sample to be detected into a 150mL glass test tube, adding 6mL of mixed solution (2:1, v/v) of dichloromethane and methanol into the serum sample to be detected, and uniformly mixing for 2min in a vortex manner; shaking again for 20min at 270rpm and 25 deg.C; then adding 2ml of deionized water and 2g of anhydrous sodium sulfate, and carrying out vortex oscillation for 2 min; centrifuging at 2000rpm for 5 min; taking the dichloromethane phase at the lower layer into a new centrifugal tube, and drying by using nitrogen;
step S4, adding an internal standard, redissolving the sample obtained in the step S3 by using 3mL of normal hexane, adding 200 mL of isotope n-nonadecanoic acid methyl ester (C19:0) with the concentration of 10 mug/mL serving as an internal standard, carrying out vortex oscillation for 2min, adding 3mL of 0.4mol/L potassium hydroxide methanol solution, carrying out vortex oscillation for 2min, oscillating for 30min at the temperature of 37 ℃ by using a shaking table at 180rpm, adding 2mL of deionized water, carrying out vortex oscillation for 2min, standing for layering, taking the supernatant to a new glass test tube, carrying out blow-drying by using nitrogen, adding 200 mug L of normal hexane, carrying out vortex oscillation for 2min, standing for layering, and taking the supernatant. (ii) a
Step S5 gas chromatography-mass spectrometry, wherein the mixed solution of the standard substance with the series concentration gradient obtained in step S2 and the sample extraction solution obtained in step S4 are respectively subjected to gas chromatography, and the mode of the chromatographic temperature gradient is as follows:
heating rate/min Temperature value of Residence time min Run time min
Initial temperature 70 2 2
Procedure 1 20 180 8 15.5
Procedure 2 4 250 3 36
Directly carrying out mass spectrometry on the sample subjected to gas chromatography separation;
the medium-long chain fatty acid single ion detection scanning group in the mass spectrometry is as follows:
Figure BDA0002435870650000061
the medium-long chain fatty acid single ion detection scanning parameter conditions in the mass spectrometry are as follows:
Figure BDA0002435870650000062
Figure BDA0002435870650000071
respectively analyzing the standard substance mixed solution with the series of concentration gradients and the sample extraction solution obtained in the step S4, and deriving mass spectrum data for processing;
and S6, converting the concentration, drawing the mass spectrum data of the standard substance mixed solution with the series of concentration gradients obtained in the step S2 into a standard curve, and converting the mass spectrum data of the sample extraction solution obtained in the step S4 into the corresponding medium-long chain fatty acid concentration through the standard curve.
The test results of this example were further analyzed to verify the feasibility of the method provided by the present invention:
and establishing a standard curve by adopting an isotope internal standard quantitative method and taking the concentration of the medium-long chain fatty acid standard as an x axis and the peak area ratio of the amino acid standard to the internal standard as a y axis. The concentration of medium-long chain fatty acids in serum was calculated from this curve and the results were as follows:
Figure BDA0002435870650000072
Figure BDA0002435870650000081
the above embodiments are only used for illustrating but not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.

Claims (7)

1. The method for detecting medium-long chain fatty acids in human serum based on gas chromatography-mass spectrometry is characterized by comprising the following steps:
s1, preparing an internal standard solution, weighing an isotope internal standard product methyl nonadecanoate, and adding n-hexane to dissolve the isotope internal standard product methyl nonadecanoate to prepare the internal standard solution for later use;
s2, preparing a series of concentration standard substance mixed solution, weighing a medium-long chain fatty acid standard substance for gas chromatography-mass spectrometry after methyl esterification, and preparing a series of concentration gradient standard substance mixed solution by using n-hexane;
step S3, extracting a sample, namely taking a serum sample to be detected, extracting medium-long chain fatty acid in the sample by adopting dichloromethane and methanol, taking a dichloromethane phase, and drying;
step S4, adding an internal standard, redissolving the sample obtained in the step S3 by using normal hexane, adding isotope n-nonadecanoic acid methyl ester as the internal standard, adding a potassium hydroxide methanol solution for treatment, adding water, standing for layering, taking supernatant, drying by blowing with nitrogen, and redissolving by using normal hexane to obtain a sample extraction solution;
step S5 gas chromatography-mass spectrometry, wherein the mixed solution of the standard substance with the series concentration gradient obtained in step S2 and the sample extraction solution obtained in step S4 are respectively subjected to gas chromatography, and the mode of the chromatographic temperature gradient is as follows:
heating rate/min Temperature value of Residence time min Run time min Initial temperature 70 2 2 Procedure 1 20 180 8 15.5 Procedure 2 4 250 3 36
Directly carrying out mass spectrometry on the sample subjected to gas chromatography separation;
and S6, converting the concentration, drawing the mass spectrum data of the standard substance mixed solution with the series of concentration gradients obtained in the step S2 into a standard curve, and converting the mass spectrum data of the sample extraction solution obtained in the step S4 into the corresponding medium-long chain fatty acid concentration through the standard curve.
2. The method for detecting medium-chain fatty acids in human serum based on gas chromatography-mass spectrometry according to claim 1, wherein the concentration of the n-nonadecanoic acid methyl ester in the internal standard solution of step S1 is 1 mg/mL.
3. The method for detecting medium-and long-chain fatty acids in human serum based on gas chromatography-mass spectrometry according to claim 1, wherein the concentration range of the serial concentration standard solution in step S2 is 1-2500 mg/L.
4. The method for detecting medium-long chain fatty acids in human serum based on gas chromatography-mass spectrometry according to claim 1, wherein the medium-long chain fatty acid standard in step S2 comprises the following standards: methyl butyrate, methyl hexanoate, methyl octanoate, methyl decanoate, methyl undecanoate, methyl laurate, methyl tridecanoate, methyl myristate, methyl pentadecanoate, methyl palmitate, methyl palmitoleate, methyl heptadecanoate, methyl heptadecenoate, methyl stearate, methyl oleate, methyl elactoate, methyl linoleate, methyl elaidoleate, methyl elanoleate, methyl gama-linolenic acid methyl ester, methyl linolenate, methyl arachidate, methyl eicosenoate, methyl eicosatrienoate, methyl arachidonic acid, methyl eicosapentaenoate, methyl heneicosanoate, methyl docosenoic acid, methyl behenate, methyl erucate, methyl docosedienoate, methyl docosenoic acid, methyl eicosatrinoate, methyl tetracosenoic acid, methyl ester, Methyl tetracosenoic acid, methyl docosatetraenoate, methyl docosapentaenoate and methyl docosapentaenoate.
5. The method for detecting medium-long chain fatty acids in human serum based on gas chromatography-mass spectrometry according to claim 1, wherein the step of extracting medium-long chain fatty acids from the sample in step S3 specifically comprises: adding dichloromethane and methanol into a serum sample to be detected according to a volume ratio of dichloromethane to methanol of 2:1, and uniformly mixing for 1-3min in a vortex manner; oscillating on a shaker for 15-30min at 270rpm at 25 ℃; adding deionized water and anhydrous sodium sulfate, and performing vortex oscillation for 2 min; centrifuging at 2000rpm for 4-6 min; the lower dichloromethane phase was removed and blown dry with nitrogen.
6. The method for detecting medium-long chain fatty acids in human serum based on gas chromatography-mass spectrometry as claimed in claim 1, wherein the step S4 is detailed by adding an internal standard: adding n-hexane into the sample obtained in the step S3 for redissolving, adding isotope n-nonadecanoic acid methyl ester, performing vortex oscillation for 1-3min, adding 0.4mol/L potassium hydroxide methanol solution, performing vortex oscillation for 1-3min, performing oscillation for 20-40min at 37 ℃ by using a shaking table at 180rpm, adding deionized water, performing vortex oscillation for 1-3min, standing for layering, taking the supernatant into a new glass test tube, drying by using nitrogen, adding 200 mu L n-hexane, performing vortex oscillation for 2min, standing for layering, and taking the supernatant.
7. The method for detecting medium-long chain fatty acids in human serum based on gas chromatography-mass spectrometry as claimed in claim 1, wherein the medium-long chain fatty acid single ion detection scans in mass spectrometry are grouped as follows:
Figure FDA0002435870640000021
the medium-long chain fatty acid single ion detection scanning parameter conditions in the mass spectrometry are as follows:
Figure FDA0002435870640000031
and (4) analyzing the standard substance mixed solution with the series of concentration gradients and the sample extraction solution obtained in the step S4 respectively, and deriving and processing mass spectrum data.
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CN112763608A (en) * 2020-12-25 2021-05-07 北京金城泰尔制药有限公司 Method for determining content of medium-chain triglyceride in promestrene cream
CN113030360A (en) * 2021-02-23 2021-06-25 上海百趣生物医学科技有限公司 Free fatty acid high-throughput target detection method and application
CN113156015A (en) * 2021-04-25 2021-07-23 云南锡业锡化工材料有限责任公司 Method for analyzing content of industrial coconut oil component by internal standard method without derivatization treatment
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CN112763608A (en) * 2020-12-25 2021-05-07 北京金城泰尔制药有限公司 Method for determining content of medium-chain triglyceride in promestrene cream
CN113030360A (en) * 2021-02-23 2021-06-25 上海百趣生物医学科技有限公司 Free fatty acid high-throughput target detection method and application
WO2022188746A1 (en) * 2021-03-08 2022-09-15 江成鸿 Application of composition in preparation of drug for diagnosing infantile hemangioma and/or monitoring progress and prognosis thereof, and kit and drug
CN113156015A (en) * 2021-04-25 2021-07-23 云南锡业锡化工材料有限责任公司 Method for analyzing content of industrial coconut oil component by internal standard method without derivatization treatment
CN114740107A (en) * 2022-03-25 2022-07-12 武汉生物样本库有限公司 Method for determining content of short-chain fatty acids in feces, plasma and urine samples based on GC-MS or GC-MS/MS
CN114965764A (en) * 2022-05-18 2022-08-30 陕西安宁云生生物技术有限公司 Diagnosis and treatment of constipation

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