Method and kit for determining multiple free fatty acids in body fluid through liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method and a kit for determining various free fatty acids in body fluid by liquid chromatography-tandem mass spectrometry. The method comprises the following steps: pre-treating a biological fluid sample by introducing an isotope internal standard, protein precipitation, liquid-liquid extraction and an automatic pipetting platform, then separating an isomeride and an interferent through liquid chromatography, carrying out mass spectrometry scanning monitored by tandem mass spectrometry multi-reaction, making a standard curve by using a calibrator, and quantifying, and low, medium and high quality control products continuously monitor the performance of the method. The method realizes the purpose of simultaneously analyzing and detecting 22 free fatty acids and 20 D / 13C labeled isotope hormones in a sample by using the UPLC-MS / MS technology without using solid-phase extraction and non-derivatization treatment, has the advantages of rapidness, simplicity and convenience in operation, multiple detection types, high flux, high accuracy and low cost, can be applied to content detection of low-content free fatty acid level, and has a wide application prospect. The research on a lipid metabolism pathway is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of free fatty acid detection, and specifically relates to a method and a kit for measuring multiple free fatty acids in body fluids by using liquid chromatography-mass spectrometry. Background Art

[0002] Free fatty acids (NEFA) refer to fatty acids in serum that are not esterified with glycerol, cholesterol, etc. They are lipids in the human body that are generated by the decomposition of neutral fatty acids. They can provide energy after decomposition and oxidation, and are also called non-esterified fatty acids. NEFA has strong cytotoxicity and can damage cell membranes, mitochondria and lysosome membranes, etc., causing damage to intracellular micro-organisms, and can enhance the toxicity of cytokines. It plays an important role in the pathophysiology of many diseases (such as diabetes, coronary heart disease, metabolic syndrome and peroxisomal diseases).

[0003] Fatty acids can be divided into two categories: saturated fatty acids and unsaturated fatty acids. Excessive saturated fatty acids can make people fat and are the main cause of increased blood cholesterol, triglycerides, and LDL-C, which can subsequently cause arterial stenosis, form atherosclerosis, and increase the risk of cardiovascular disease. Unsaturated fatty acids mainly include monounsaturated fatty acids and polyunsaturated fatty acids, both of which are very beneficial to human health. Unsaturated fatty acids can be divided into ω-3 fatty acids, ω-6 fatty acids, ω-7 fatty acids, ω-9 fatty acids, and trans fats. ω-3 fatty acids are enriched in brain tissue and play an important role in cognitive function, behavioral function, and the normal growth and development of the brain. Balancing the intake of ω-3 and ω-6 is extremely important for maintaining good health. Exceeding a certain content of ω-6 fatty acid: ω-3 fatty acid ratio may increase the probability of some diseases such as chronic heart attack, thrombotic stroke, etc. The optimal ratio is considered to be 4 to 1 or lower. ω-7 fatty acids are raw materials for cardiovascular health products. Omega-9 fatty acids are considered to be a class of fatty acids that can lower cholesterol and promote a healthy immune system, and are important for preventing cardiovascular and stroke diseases. Trans fats have no health benefits and are not nutrients needed by the human body. The intake of trans fatty acids is positively correlated with the occurrence of cardiovascular disease, breast cancer, colon cancer and other cancers.

[0004] The detection methods of free fatty acids in human blood in the prior art mainly include gas chromatography, liquid chromatography and enzymatic methods. The enzymatic method has poor sensitivity and specificity, is easily interfered, and can only quantify total NEFAs. The lipid analysis method based on GCMS usually requires time-consuming hydrolysis and derivatization procedures, which are usually applied after the pre-fractionation of the lipid part. Without hydrolysis and derivatization, many lipids are not suitable for GC. The free fatty acids in the human matrix are low in content, have strong polarity and weak volatility, so it is necessary to first undergo complex sample pretreatment to enrich and purify the free fatty acids, and then methyl esterify the free fatty acids (to improve volatility, thermal stability and chromatographic separation) before GC analysis can be performed. The whole process is very cumbersome and time-consuming, and a large amount of organic solvents are required. In addition, the above method also has the disadvantages of poor stability and low detection sensitivity, and hydrolysis and derivatization may still hinder quantification. Summary of the invention

[0005] In view of the problems existing in the free fatty acid detection methods in the prior art, the first object of the present invention is to provide a method for determining a variety of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry. The method is fast and easy to operate, has a wide range of detection types, high throughput, high accuracy, and low cost. It can be used to detect the level of free fatty acids for non-medical treatment purposes, and is helpful for studying lipid metabolic pathways.

[0006] The second object of the present invention is to provide a kit for determining a variety of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry. The design and optimization of the kit fully consider the convenience of laboratory operation, the efficiency of detection and the reliability of results. The kit integrates key chemical reagents and standards to simplify the pretreatment steps of free fatty acids while ensuring the accuracy and reproducibility of detection.

[0007] In order to achieve the above technical objectives, the present invention provides a method for determining a variety of free fatty acids in body fluids by liquid chromatography tandem mass spectrometry, wherein a body fluid sample containing free fatty acids, an isotope internal standard, a precipitant and an extractant are added to a 96-well plate, mixed and then liquid-liquid extraction is performed, and the extract is purified to obtain a sample to be tested; the sample to be tested is detected by UPLC-MS / MS;

[0008] The extractant is at least one of methyl tert-butyl ether, n-hexane (HEX) and ethyl acetate;

[0009] The precipitant is a mixed solution of methanol and normal hexane containing formic acid.

[0010] The technical solution of the present invention does not require the use of complex solid phase extraction and derivatization treatment in the pretreatment process. The key lies in the distinction between fatty acids and other protein components in body fluids during the pretreatment process. A precipitant is first used to quickly precipitate the protein components, and then the target analyte is extracted by an extractant that has good solubility in fatty acids. Combined with the use of 96-well plates and automated pipetting platforms, the pretreatment step operation is greatly simplified and the throughput is improved to meet the detection needs of large quantities of samples. Then, by optimizing the chromatographic conditions, liquid chromatography-mass spectrometry is used to achieve the purpose of simultaneously detecting 22 free fatty acids from C14 to C28 in one experiment.

[0011] The precipitant system used in the present invention can not only quickly achieve protein precipitation, but also improve the response value of free fatty acids in LC-MS, which is beneficial for subsequent detection. It is further preferred that the volume ratio of methanol to n-hexane is 40:10, containing 0.05 vol% formic acid.

[0012] As a preferred embodiment, the body fluid sample includes serum, plasma, urine, amniotic fluid and cerebrospinal fluid.

[0013] As a preferred embodiment, the free fatty acids include at least two of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosanoic acid, cis-8,11,14-eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosatetraenoic acid, docosahexaenoic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, phytanic acid and pristanic acid.

[0014] As a preferred solution, the isotope internal standard includes at least two of 13C3-myristic acid, 13C4-palmitic acid, D13-palmitoleic acid, heptadecanoic acid, D3-stearic acid, 13C18-oleic acid, D17-elaidic acid, 13C18-linoleic acid, D5-linolenic acid, D4-eicosanoic acid, D6-cis-8,11,14-eicosatrienoic acid, D8-arachidonic acid, D5-eicosapentaenoic acid, D4-docosanoic acid, D5-cis-4,7,10,13,16,19-docosahexaenoic acid, D4-lignoceric acid, D18-nervonic acid, D4-cerotic acid, D3-3-methyl-phytanic acid and D3-2-methyl-pristanic acid. The preferred isotope internal standard of the present invention has similar polarity to the target analyte.

[0015] As a preferred solution, the UPLC-MS / MS conditions are:

[0016] Chromatographic conditions:

[0017] Chromatographic column: C8 column, 2.1×50 mm, 1.7 μM; column temperature: 35°C; eluent A: acetonitrile aqueous solution containing ammonium formate and ammonia; eluent B: acetonitrile isopropanol solution containing ammonia, flow rate 0.40 mL / min, injection volume, 3 μL; gradient elution: initial 0% B; 0-0.25 min, 0-31% B; 0.25-3.75 min, 31% B; 3.75-3.8 min, 31-36.5% B; 3.8-6 .8min, 36.5%B; 6.8~8min, 36.5~70%B; 8~10min, 70%B; 10~10.75min, 70~80%B; 10.75~11.75min, 80%B; 11.75~11.76min, 80~90%B; 11.76~12min, 90%B; 12~12.1min, 90~0%B; 12.1~13.5min, 0%B;

[0018] Mass spectrometry conditions: in electrospray negative ion mode; using multiple reaction monitoring mass spectrometry scanning mode, nebulizer gas: 40 kPa, heating gas: 40 kPa, curtain gas: 50 kPa, spray voltage: -4500 V, desolvation temperature: 400 °C, and simultaneously monitoring the target and isotope internal standard.

[0019] As a preferred solution, the purification is to remove the supernatant by centrifugation using an automated platform, nitrogen blowing, re-dissolving, and shaking centrifugation; the re-dissolving uses methanol aqueous solution as the re-dissolving solution.

[0020] As a preferred solution, the automated platform uses a genetic metabolism sample pretreatment system, adopts an air displacement pipetting mode, and a 4-channel automated pipetting device transfers the supernatant from the extraction plate to the upper machine plate. Description of the automated plate transfer program: start pipetting → the pipetting channel takes the tip from the tip position → the pipetting channel aspirates liquid at the extraction position → the pipetting channel spits liquid at the upper machine position → the pipetting channel removes the tip at the waste tank → the whole plate pipetting ends.

[0021] As a preferred solution, the automated platform uses the genetic metabolism sample (liquid chromatography-tandem mass spectrometry) pre-treatment system (Zhehang Machinery No. 20200789) manufactured by Bosheng Intelligent Manufacturing.

[0022] The present invention also provides a kit for determining multiple free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry, the kit comprising an eluent, an isotope internal standard solution, a precipitant, an extractant, a calibration diluent, a quality control product, a reconstitution solution and a chromatographic column; the eluent comprises an eluent A and an eluent B, wherein the eluent A is an acetonitrile aqueous solution containing ammonium formate and ammonia water, and the eluent B is an acetonitrile isopropanol solution containing ammonia water.

[0023] Further preferably, the eluent A is an acetonitrile aqueous solution containing 1-10 mmol / L ammonium formate and 0.05-0.2 vol% ammonia; the eluent B is an acetonitrile isopropanol solution containing 0.05-0.2 vol% ammonia. In the present invention, the alkaline eluent is more conducive to the ionization of the compound than the acidic eluent, and the response is significantly higher by 2-3 orders of magnitude. However, with the increase of the concentration of ammonia, the retention time drifts backward, which has no obvious effect on the response. The retention time drifts backward to a certain extent, which is conducive to the adjustment of the elution gradient. Too much drift backward will prolong the elution time.

[0024] As a preferred solution, the calibrator dilution solution is prepared by adding a calibration line matrix to a mixed standard working solution to dilute seven concentrations of mixed standard solutions; the calibration line matrix is ​​a substitute matrix composed of bovine serum albumin and phosphate buffered saline solution; the dilution multiples of the seven concentrations of the mixed standard solutions corresponding to the mixed standard S7 working solution are 1, 2, 5, 10, 25, 50, and 100 times, respectively.

[0025] As a preferred scheme, in the mixed standard working solution, the concentrations of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosanoic acid, cis-8,11,14-eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosatetraenoic acid, docosahexaenoic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, phytanic acid and pristanic acid are 50, 1500, 100, 1500, 750, 50, 500, 100, 50, 10, 15, 200, 15, 15, 10, 50, 10, 5, 15, 1, 15, 100 μg / ml, respectively.

[0026] As a preferred embodiment, in the internal standard mixed solution, 13C3-myristic acid, 13C4-palmitic acid, D13-palmitoleic acid, heptadecanoic acid, D3-stearic acid, 13C18-oleic acid, D17-elaidic acid, 13C18-linoleic acid, D5-linolenic acid, D4-eicosanoic acid, D6-cis-8,11,14-eicosatrienoic acid, D8-arachidonic acid, D5-eicosapentaenoic acid, D4-docosahexaenoic acid, D5- The concentrations of cis-4,7,10,13,16,19-docosahexaenoic acid, D4-lignoceric acid, D18-neuroerotic acid, D4-cerotic acid, D3-3-methyl-phytanic acid and D3-2-methyl-pristanic acid were 100, 200, 200, 120, 200, 500, 100, 1000, 200, 15, 20, 200, 36, 30, 200, 20, 10, 20, 30, and 750 μg / ml, respectively.

[0027] As a preferred solution, the quality control product contains quality control serum at three levels of low, medium and high concentrations, and the quality control product is prepared by adding mixed free fatty acid standard substances to serum blank samples, matrix-free serum or matrix-free plasma purified by activated carbon, and the target value is determined by detection.

[0028] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0029] (1) The present invention provides a method for determining 22 free fatty acids (NEFA) in body fluids by liquid chromatography-tandem mass spectrometry. The method has a quick and simple pre-treatment process, does not require the use of complex solid phase extraction and derivatization treatment, and can effectively extract 22 free fatty acids and 20 isotope-labeled internal standards only through protein precipitation and liquid-liquid extraction. Combined with the use of 96-well plates and automated pipetting platforms, the pre-treatment step operation is greatly simplified and the throughput is improved to meet the detection needs of large quantities of samples.

[0030] (2) The detection method of the present invention achieves the purpose of simultaneously detecting 22 kinds of free fatty acids from C14 to C28 in body fluids, including saturated fatty acids, ω-3 polyunsaturated fatty acids, ω-6 polyunsaturated fatty acids, ω-7 monounsaturated fatty acids, ω-9 monounsaturated fatty acids, ω-9 polyunsaturated fatty acids, trans fatty acids, and fatty acid metabolic intermediates. It involves many indicators and can meet the requirements of comprehensive detection and tracking of C14 to C28 NEFA indicators in one experiment.

[0031] (3) The method of the present invention has high specificity and high accuracy. By optimizing the chromatographic conditions, the present invention performs baseline separation on 8 groups of isomers and other interfering substances, effectively avoiding the influence of isomers and other interfering substances on the index determination results; and the treated analyte is directly detected according to the inherent mass / charge ratio, rather than the traditional indirect detection of derivatives, and the method has strong specificity; finally, the corresponding 20 D / 13C labeled isotopes are introduced as internal standard quantification, which reduces the influence of the matrix effect existing between different matrices on the quantitative results.

[0032] (4) The kit provided by the present invention uses a calibrator to prepare a standard curve for quantification, and three levels of quality control products, low, medium and high, continuously monitor the performance of the method. It can more conveniently provide stable and reliable free fatty acid detection for non-medical treatment purposes, which is helpful for studying the pathways of lipid metabolism and sugar metabolism and the pathogenesis of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the LC-MS / MS ion chromatogram of Example 1 for simultaneously detecting 22 free fatty acids, C14 to C28, in plasma.

[0034] Figure 2Graph showing the ion currents of isomers C20, PHA, D6-DGLA and D8-AA in the plasma sample of Example 1.

[0035] Figure 3 This is the ion current diagram of isomers OA and EA in the plasma sample of Example 1.

[0036] Figure 4 This is the ion current diagram of isomers PRA and 13C18-LA in the plasma sample of Example 1.

[0037] Figure 5 This is the ion current diagram of isomers GLA and ALA in the plasma sample of Example 1.

[0038] Figure 6 This is the ion current diagram of NA and interfering substances in the plasma sample of Example 1.

[0039] Figure 7 This is the ion current diagram of DHA and interfering substances in the plasma sample of Example 1.

[0040] Figure 8 This is the ion current diagram of C26 and interfering substances in the plasma sample of Example 1.

[0041] Fig. 9 This is the ion current diagram of C28 and interfering substances in the plasma sample of Example 1.

[0042] Fig.10 This is a standard curve diagram of C24 established based on a standard substance according to an embodiment of the present invention.

[0043] Fig.11 1 is a comparative ion current graph of a normal human plasma sample and an adrenoleukodystrophy plasma sample according to an embodiment of the present invention. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0046] The method and kit of the present invention are further described by the following examples in conjunction with the accompanying drawings, but these examples do not constitute any limitation to the present invention.

[0047] The abbreviations of various free fatty acids involved in the present invention are shown in Table 1 below.

[0048] Table 1 Abbreviations of free fatty acids

[0049]

[0050]

[0051] Example 1

[0052] This embodiment is a method and kit for determining multiple free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry, as follows:

[0053] A method for determining various free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry.

[0054] 1. Preparation of Calibrators

[0055] The dissolution of the standards and the preparation of concentrated stock solutions are shown in Table 2.

[0056] Add 244 μl of 80% methanol to the mixed solution prepared by pipetting 1 ml of the working solution in Table 2 to obtain a concentrated stock solution.

[0057] Free fatty acid calibrators were prepared by diluting concentrated stock solutions with a calibration line matrix consisting of a surrogate matrix consisting of bovine serum albumin and phosphate-buffered saline.

[0058] The seven concentration points S1-S7 of the calibrator were obtained by serially diluting the high-concentration calibrator with the calibration line matrix. The dilution multiples were 100, 50, 25, 10, 5, 2, and 1, respectively. The concentrations of the calibration points S1-S7 are shown in Table 3.

[0059] Table 2 Concentrations of free fatty acid stock solutions and mixed standards

[0060]

[0061]

[0062] Table 3 Concentrations of free fatty acids at different concentration levels at calibration points S1 to S7

[0063] serial number Substance (μg / ml) S1 S2 S3 S4 S5 S6 S7 1 MA 0.5 1 2 5 10 25 50 2 PA 15 30 60 150 300 750 1500 3 PLA 1 2 4 10 20 50 100 4 S 15 30 60 150 300 750 1500 5 OA 7.5 15 30 75 150 375 750 6 EA 0.5 1 2 5 10 25 50 7 LA 5 10 20 50 100 250 500 8 ALA 1 2 4 10 20 50 100 9 GLA 0.5 1 2 5 10 25 50 10 C20 0.1 0.2 0.4 1 2 5 10 11 DGLA 0.15 0.3 0.6 1.5 3 7.5 15 12 AA 2 4 8 20 40 100 200 13 EPA 0.15 0.3 0.6 1.5 3 7.5 15 14 C22 0.15 0.3 0.6 1.5 3 7.5 15 15 ADA 0.1 0.2 0.4 1 2 5 10 16 DHA 0.5 1 2 5 10 25 50 17 C24 0.1 0.2 0.4 1 2 5 10 18 NA 0.05 0.1 0.2 0.5 1 2.5 5 19 C26 0.15 0.3 0.6 1.5 3 7.5 15 20 C28 0.01 0.02 0.04 0.1 0.2 0.5 1 21 PHA 0.15 0.3 0.6 1.5 3 7.5 15 22 PRA 1 2 4 10 20 50 100

[0064] 2. Preparation of quality control products

[0065] Quality control spike concentration setting and preparation process: The quality control products contain three levels of low, medium and high concentration quality control serum. The quality control products are prepared by adding mixed free fatty acid standard substances to serum blank samples purified by activated carbon or serum / plasma without free fatty acids. The concentrated stock solution is mixed with the serum blank sample and diluted 25, 10, and 2 times respectively to obtain three levels of low, medium and high quality control products. The low, medium and high quality control products are represented by L, M, and H respectively. The concentrations of free fatty acids are shown in Table 4.

[0066] Table 4 Concentration of free fatty acids in quality control products

[0067] serial number Substance (μmol / l) L M H 1 MA 8.76 21.89 109.47 2 PA 233.99 584.98 2924.89 3 PLA 15.72 39.31 196.53 4 S 210.91 527.28 2636.39 5 OA 106.21 265.52 1327.62 6 EA 7.08 17.70 88.51 7 LA 71.31 178.28 891.42 8 ALA 14.37 35.92 179.58 9 GLA 7.18 17.96 89.79 10 C20 1.28 3.20 16.00 11 DGLA 1.96 4.89 24.47 12 AA 26.28 65.69 328.45 13 EPA 1.98 4.96 24.80 14 C22 1.76 4.40 22.02 15 ADA 1.20 3.01 15.04 16 DHA 6.09 15.22 76.11 17 C24 1.09 2.71 13.56 18 NA 0.55 1.36 6.82 19 C26 1.51 3.78 18.91 20 C28 0.09 0.24 1.18 21 PHA 1.92 4.80 24.00 22 PRA 13.40 33.50 167.50

[0068] 3. Liquid chromatography and mass spectrometry conditions

[0069] Chromatographic conditions:

[0070] Eluent A: 0.1 vol% ammonia + 5 mM ammonium formate + 10 vol% acetonitrile + 90 vol% water;

[0071] Eluent B: 0.1 vol% ammonia water + 10 vol% isopropanol + 90 vol% acetonitrile;

[0072] Chromatographic column: ACQUITY UPLC BEH C8 column, 50 mm × 2.1 mm, 1.7 μm; flow rate: 0.4 mL / min; column temperature: 35 °C; injection volume: 3 μL; gradient elution was performed, and the gradient elution program is shown in Table 5.

[0073] Table 5 Gradient elution program

[0074] Time (min) Flow rate (ml / min) ％A ％B initial 0.4 100 0 0.25 0.4 69 31 3.75 0.4 69 31 3.8 0.4 63.5 36.5 6.8 0.4 63.5 36.5 8 0.4 30 70 10 0.4 30 70 10.75 0.4 20 80 11.75 0.4 20 80 11.76 0.4 0 90 12 0.4 0 90 12.1 0.4 100 0 13.5 0.4 100 0

[0075] Mass spectrometry conditions:

[0076] In the electrospray (ESI) negative ion mode, the mass spectrometry scanning mode of multiple reaction monitoring (MRM) was adopted, the nebulizer gas: 40 kPa, the heating gas: 40 kPa, the curtain gas: 50 kPa, the spray voltage: -4500 V, the desolvation temperature: 400 ° C. The target and the isotope internal standard were monitored simultaneously.

[0077] The mass spectrometry parameters for detecting 22 free fatty acids in multiple reaction monitoring mode are shown in Table 6.

[0078] The mass spectrometry parameters for detecting 20 isotope-labeled free fatty acids in multiple reaction monitoring mode are shown in Table 7.

[0079] Table 6 Mass spectrometry parameters of free fatty acids

[0080]

[0081]

[0082] Table 7 Mass spectrometry parameters of isotope-labeled free fatty acids

[0083] serial number substance Q1Mass(Da) Q3Mass(Da) DP(volts) CE(volts) 1 C17 269 269 -80 -20 2 13C4-PA 259.1 259.1 -82 -20 3 D4-C22 343.1 343.1 -70 -20 4 D4-C24 371.2 371.2 -70 -20 5 D4-C26 399.2 399.2 -10 -20 6 13C18-OA 299 299 -73 -20 7 D5-DHA 331.9 331.9 -20 -8 8 D3-C18 286.1 286.1 -109 -21 9 13C3-C14MA 229.9 229.9 -82 -17 10 D17-t-C18:1EA 298 298.01 -80 -20 11 13C18-C18:2LA 296.9 296.91 -80 -30 12 D5-A-C18:3ALA 282 282 -72 -23 13 D4-C20 315 315.01 -96 -27 14 D6-C20:3DGLA 311.01 311 -75 -16 15 D8-C20:4AA 310.9 310.9 -72 -30 16 D18-C24:1NA 383.1 383.1 -10 -22 17 D3-PHA 314 314 -10 -23 18 D13-C16:1PLA 265.9 265.9 -75 -25 19 D5-C20:5EPA 306 306 -75 -13 20 D3-PRA 299.9 299.9 -75 -19

[0084] 4. Experimental process

[0085] Preparation of isotope internal standard solution

[0086] Dissolve and prepare the internal standard mixed solution of the internal standard according to the preparation steps of the standard solution, [13C3]-myristic acid (1,2,3-13C3), [13C4]-palmitic acid (1,2,3,4-13C4), D13-palmitoleic acid, C17, D3-stearic acid, 13C18-oleic acid, D17-elaidic acid, 13C18-linoleic acid, D5-linolenic acid (17,17,18,18,18-D5), D4-eicosanoic acid (arachidic acid) (12,12,13,13-D4), D6-cis-8,11,14-eicosatrienoic acid, D8-arachidonic acid The concentrations of D5-eicosapentaenoic acid, D4-docosapentaenoic acid (behenic acid), cis-4,7,10,13,16,19-docosahexaenoic acid-[21,21,22,22,22-D5], D4-lignoceric acid, D18-neuroceric acid, D4-cerotic acid, D3-[3-methyl-2H3]-phytanic acid, and [2-methyl-2H3]-pristanic acid were 100, 200, 200, 120, 200, 500, 100, 1000, 200, 15, 20, 200, 36, 30, 200, 20, 10, 20, 30, and 750 μg / ml, respectively.

[0087] Selection of precipitant: The precipitant is a mixed solution of methanol containing formic acid and n-hexane, wherein the volume ratio of formic acid, methanol and n-hexane is 0.05 vol%:40:10.

[0088] Selection of extraction agent: The extraction agent is n-hexane.

[0089] Selection of reconstitution solution: The reconstitution solution is 85 vol% methanol + 15 vol% aqueous solution.

[0090] (4) Sample processing

[0091] 1) Take 50 μl of plasma into a 1 ml 96-well plate, add 200 μl of precipitant solution containing isotope internal standard, cover with 96-well silica cover and mix thoroughly;

[0092] 2) Remove the 96-well silica gel cover, pipette 400 μL of the extractant into the 96-well plate, cover the 96-well silica gel cover, vortex and mix thoroughly, and then centrifuge at 3000 rpm and 10°C for 10 min;

[0093] 3) Turn on the pipetting platform and software, wait for the device self-check to end, open the pipetting platform door, pull out the sample processing rack (pipetting module), place the clean 400μl 96-well plate, the 1ml 96-well plate to be pipetted, and a sufficient amount of 300μl pipetting platform gun tips on the upper plate, extraction plate, and gun tip positions respectively, return the sample processing rack (pipetting module) to its original position, close the door, and click Start on the software.

[0094] 4) After the pipetting platform operation is completed, place the 400 μl 96-well plate under a gentle nitrogen flow to remove the solvent, discard the 1 ml 96-well plate and the disposable gun holder of the gun tip, and turn off the software and the power of the pipetting platform.

[0095] 5) Redissolve the residue in 100 μL of reconstitution solution and load the sample after it is fully dissolved. The injection volume is 3 μL.

[0096] The LC-MS / MS ion chromatograms of 22 free fatty acids from C14 to C28 in plasma were obtained. Figure 1 , the ion flow diagrams of each compound and their isomers are shown in Figure 2 to Figure 9 .

[0097] The method of this embodiment is used to detect normal human plasma samples and adrenal leukoencephalopathy plasma samples to obtain comparative ion current graphs. Fig.11 ,It can be seen from the figure that the indicators of very long-chain saturated fatty acids related to adrenoleukodystrophy are significantly increased.

[0098] Method performance verification:

[0099] After the sample to be tested enters the chromatographic column for separation in the UPLC gradient elution mode, the multiple ion reaction monitoring mode (MRM) of negative ion electrospray ionization (ESI) is used to perform qualitative and / or quantitative MS / MS detection on the substance to be tested. A standard curve is prepared using a mixed standard solution of seven concentrations, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, and the corresponding peak area of ​​the substance to be tested is substituted into the regression equation to calculate the concentration of the substance to be tested in the sample. The linear results are shown in Table 8. The standard curve diagram of C24 in the embodiment based on the standard is shown in Table 8. Fig.10 .

[0100] Table 8 Linearity results of tested substances

[0101]

[0102] Using the method of the present invention, the linear range of the 22 detection indicators is greater than or equal to 2 orders of magnitude, with a wide linear interval, which can meet the accurate quantitative requirements of samples of different concentrations. The r value of the linear equation of each indicator of the set 7 calibration points is ≥0.99, and the linear performance is excellent.

[0103] The present invention prepares quality control products of three levels of concentration, L, M, and H, by adding a standard of known concentration to a serum blank sample, and then tests each level of concentration five times. The theoretical value is the sum of the concentration of the endogenous analyte in the sample and the added standard. The ratio of the measured value to the theoretical value can be used to evaluate the accuracy. The detected value is within ±20% of the target value, and the method has good accuracy. The range of spiked recovery of specific substances is shown in Table 9.

[0104] Table 9 Sample spike recovery results

[0105]

[0106] Note: L0 in Table 9 represents the base value of the spiked sample.

[0107] Using the method of the present invention, the detection limit and quantification limit of 22 detection indicators are evaluated together with the precision and correctness. At least 3 analysis batches are inspected for at least two days, and 6 parallel tests are performed on each sample. The RE and coefficient of variation (CV) are used to determine that the RE is within ±20% and the CV is ≤20%. The detection limit and quantification limit are shown in Table 10 below.

[0108] Using the method of the present invention, three different concentrations of quality control products were selected: low, medium and high. To evaluate the intra-batch precision, each concentration was measured 6 times. To evaluate the inter-batch precision, three analytical batches should be prepared and measured continuously in different batches. The specific results are shown in Table 10. The requirements that the low value CV near the LLMI concentration should be ≤20% and the CV of other concentrations should be ≤15% were met.

[0109] Table 10

[0110]

[0111]

[0112] Six batches of blank matrices of different individuals were investigated. By comparing the signals of the analytes spiked into the extracted samples with the signals of the analytes spiked into the standard curve matrix after extraction, the matrix factors of each analyte (low, medium and high quality control concentrations) and internal standard were calculated by calculating the ratio of the peak area response in the presence of matrix (measured by adding analytes and internal standards after extraction of the blank matrix) to the corresponding peak area response without matrix (pure solutions of analytes and internal standards). The matrix factors normalized by the internal standard were calculated. The results are shown in Table 11.

[0113] Table 11

[0114]

[0115] Results As shown in the table above, the coefficient of variation of the matrix factor (ME) of 6 different random blood matrix samples is ≤15%, and the RE of the matrix factor ME% normalized by the internal standard is within ±20%. The introduction of the corresponding 20 D / 13C labeled isotopes as internal standard quantification reduces the influence of the matrix effect between different matrices on the quantitative results. The matrix effect is manifested as no obvious inhibition or enhancement, with quantitative consistency.

[0116] The present embodiment provides a kit for determining multiple free fatty acids in body fluids by liquid chromatography tandem mass spectrometry, which is composed of an eluent, an isotope internal standard solution, a precipitant, an extractant, a calibration diluent, a quality control product, a re-solution and a chromatographic column, wherein the eluent includes an eluent A and an eluent B, wherein the eluent A is an acetonitrile aqueous solution containing ammonium formate and ammonia water, and the eluent B is an acetonitrile isopropanol solution containing ammonia water.

[0117] Example 2: Optimization of eluent additives and buffer salt concentration

[0118] Alkaline eluent is more conducive to the ionization of compounds than acidic eluent, and the response is significantly higher by 2 to 3 orders of magnitude. However, with the increase of ammonia concentration, the retention time drifts backward, which has no obvious effect on the response. A certain degree of backward drift of the retention time will be conducive to the adjustment of the elution gradient. Too much backward drift will prolong the elution time. The best choice is 0.1 vol% NH3H2O. At the same time, with the increase of the concentration of buffer salt ammonium formate, the retention time drifts backward, and the separation of C18:3 isomers will become better. 10mM ammonium formate is the best. Adding isopropanol (IPA) to the eluent has no obvious effect on the peak shape of substances before C24, but has a more obvious effect on the peak shape of C26 and C28. The elution effect is better and the peak shape is greatly improved. The best choice for eluent A is: 0.1 vol% ammonia water + 10 mM ammonium formate + 10 vol% acetonitrile + 90 vol% water; the best choice for eluent B is: 0.1 vol% ammonia water + 90 vol% acetonitrile + 10 vol% isopropanol.

[0119] Example 3: Selection and optimization of precipitation agent and precipitation process

[0120] With other conditions and steps unchanged, six precipitants were compared: methanol, acetonitrile, methanol+ZnSO4, methanol+n-hexane+phosphoric acid, methanol+n-hexane+formic acid, and methanol+acetonitrile+DMSO.

[0121] The results show that when other types of precipitants are used, the response of some free fatty acids will be low or the uniformity of the solution will be affected. Only when the methanol+n-hexane+formic acid system of the present invention is used, the response values ​​of all target analytes are the highest.

[0122] Example 4: Is centrifugation required after protein precipitation?

[0123] If other conditions and steps remain unchanged, the response of multiple indicators will be low if the protein is precipitated and then centrifuged and extracted. Therefore, the protein can be directly extracted without centrifugation after precipitation.

[0124] Example 5: Selection and optimization of 96-well plates and automated pipetting platforms

[0125] In the pretreatment process of conventional body fluids, especially in the method with liquid-liquid extraction step, centrifuge tubes are often used for operation. During the pretreatment process, the centrifuge tubes are repeatedly removed, which greatly limits the detection throughput and increases the incidence of erroneous operations. In the method of the present invention, a 96-well plate is used in combination with the use of an automated pipetting platform, which greatly simplifies the pretreatment step operation and improves the throughput, meets the detection needs of large batches of samples, and reduces the pretreatment process of 96 samples before loading to about 1 hour.

[0126] The automated pipetting platform uses the genetic metabolism sample (liquid chromatography-tandem mass spectrometry) pretreatment system (Zhejiang-Hangzhou Machinery No. 20200789) manufactured by Bosheng Intelligent Manufacturing. The automated plate transfer program description: using air displacement pipetting mode, the 4-channel automated pipetting device transfers the supernatant from the extraction plate to the upper plate, automatically replaces the gun tip, and some program parameter settings are as follows:

[0127] Liquid aspiration position: The center of the plate hole is 9.8mm from the bottom of the plate hole

[0128] Aspiration volume setting: 300μl

[0129] Aspiration speed: 80μl / s

[0130] Aspirate air after aspirating liquid: 5μl

[0131] Dispensing volume setting: 300μl

[0132] Discharge position: The center of the plate hole is 5mm from the bottom of the plate hole

[0133] Dispensing setting: Liquid level following mode (when dispensing, the pipette rises with the liquid level)

[0134] Dispensing speed: 200μl / s

[0135] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry, characterized in that: A body fluid sample containing free fatty acids, an isotope internal standard, a precipitant and an extractant are added to a 96-well plate and mixed to perform liquid-liquid extraction, and the extract is purified to obtain a sample to be tested; the sample to be tested is detected by UPLC-MS / MS; The extractant is at least one of methyl tert-butyl ether, n-hexane and ethyl acetate; The precipitant is a mixed solution of methanol and normal hexane containing formic acid.

2. The method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The body fluid samples include serum, plasma, urine, amniotic fluid and cerebrospinal fluid; The free fatty acids include at least two of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosanoic acid, cis-8,11,14-eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosatetraenoic acid, docosahexaenoic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, phytanic acid and pristanic acid.

3. The method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 2, characterized in that: The isotope internal standard includes at least two of 13C3-myristic acid, 13C4-palmitic acid, D13-palmitoleic acid, heptadecanoic acid, D3-stearic acid, 13C18-oleic acid, D17-elaidic acid, 13C18-linoleic acid, D5-linolenic acid, D4-eicosanoic acid, D6-cis-8,11,14-eicosatrienoic acid, D8-arachidonic acid, D5-eicosapentaenoic acid, D4-docosanoic acid, D5-cis-4,7,10,13,16,19-docosahexaenoic acid, D4-lignoceric acid, D18-neuroceric acid, D4-cerotic acid, D3-3-methyl-phytanic acid and D3-2-methyl-pristanic acid.

4. The method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to any one of claims 1 to 3, characterized in that: The conditions of the UPLC-MS / MS are: Chromatographic conditions: Chromatographic column: C8 column, 2.1×50 mm, 1.7 μM; column temperature: 35°C; eluent A: acetonitrile aqueous solution containing ammonium formate and ammonia; eluent B: acetonitrile isopropanol solution containing ammonia, flow rate 0.40 mL / min, injection volume, 3 μL; gradient elution: initial 0% B; 0-0.25 min, 0-31% B; 0.25-3.75 min, 31% B; 3.75-3.8 min, 31-36.5% B; 3.8-6 .8min, 36.5%B; 6.8~8min, 36.5~70%B; 8~10min, 70%B; 10~10.75min, 70~80%B; 10.75~11.75min, 80%B; 11.75~11.76min, 80~90%B; 11.76~12min, 90%B; 12~12.1min, 90~0%B; 12.1~13.5min, 0%B; Mass spectrometry conditions: in electrospray negative ion mode; using multiple reaction monitoring mass spectrometry scanning mode, nebulizer gas: 40 kPa, heating gas: 40 kPa, curtain gas: 50 kPa, spray voltage: -4500 V, desolvation temperature: 400 °C, and simultaneously monitoring the target and isotope internal standard.

5. The method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that: The purification comprises the following steps: removing the supernatant by centrifugation using an automated platform, nitrogen blowing, re-dissolving, and shaking centrifugation; The re-dissolution adopts methanol aqueous solution as the re-dissolution solution.

6. The method for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 5, characterized in that: The automated platform uses a genetic metabolism sample pre-treatment system, an air displacement pipetting mode, and a 4-channel automated pipetting device to transfer the supernatant from the extraction plate to the upper machine plate.

7. A kit for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to any one of claims 1 to 6, characterized in that: Including eluent, isotope internal standard solution, precipitant, extractant, calibration diluent, quality control product, reconstitution solution and chromatographic column; The eluent includes eluent A and eluent B, wherein the eluent A is an acetonitrile aqueous solution containing ammonium formate and ammonia water, and the eluent B is an acetonitrile isopropanol solution containing ammonia water.

8. The kit for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that: The calibration dilution solution is prepared by adding a calibration line matrix to a mixed standard working solution to dilute a mixed standard solution of seven concentrations; The calibration line matrix is ​​a substitute matrix consisting of bovine serum albumin and phosphate buffered saline; The seven concentrations of the mixed standard solution corresponding to the dilution multiples of the mixed standard S7 working solution are 1, 2, 5, 10, 25, 50, and 100 times respectively; In the mixed standard working solution, the concentrations of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosanoic acid, cis-8,11,14-eicosatrienoic acid, arachidonic acid, eicosapentaenoic acid, docosanoic acid, docosatetraenoic acid, docosahexaenoic acid, lignoceric acid, nervonic acid, cerotic acid, montanic acid, phytanic acid and pristanic acid are 50, 1500, 100, 1500, 750, 50, 500, 100, 50, 10, 15, 200, 15, 15, 10, 50, 10, 5, 15, 1, 15, 100 μg / ml, respectively.

9. The kit for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 8, characterized in that: In the internal standard mixed solution, 13C3-myristic acid, 13C4-palmitic acid, D13-palmitoleic acid, heptadecanoic acid, D3-stearic acid, 13C18-oleic acid, D17-elaidic acid, 13C18-linoleic acid, D5-linolenic acid, D4-eicosanoic acid, D6-cis-8,11,14-eicosatrienoic acid, D8-arachidonic acid, D5-eicosapentaenoic acid, D4-docosahexaenoic acid, D5-cis-4,7 The concentrations of ,10,13,16,19-docosahexaenoic acid, D4-lignoceric acid, D18-neuroerotic acid, D4-cerotic acid, D3-3-methyl-phytanic acid, and D3-2-methyl-pristanic acid were 100, 200, 200, 120, 200, 500, 100, 1000, 200, 15, 20, 200, 36, 30, 200, 20, 10, 20, 30, and 750 μg / ml, respectively.

10. The kit for determining a plurality of free fatty acids in body fluids by liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that: The quality control product contains quality control serum at three levels of low, medium and high concentrations, and the quality control product is prepared by adding mixed free fatty acid standard substances to serum blank samples purified by activated carbon, matrix-free serum or matrix-free plasma.

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