A method for determining 11 polyunsaturated fatty acids based on LC-MS / MS

Abstract

This invention relates to the field of fatty acid detection technology, specifically to a method for determining 11 polyunsaturated fatty acids based on LC-MS / MS. The method includes: preparing a calibration solution; hydrolyzing bound fatty acids in a blood sample, adding a precipitant, centrifuging, and collecting the supernatant as the test sample; detecting the test sample using liquid chromatography-tandem mass spectrometry, and substituting the detection results into the calibration curve equation to obtain the content of the 11 polyunsaturated fatty acids in the test sample. This method can rapidly and accurately detect the content of 11 polyunsaturated fatty acids, providing a more accurate basis for clinical diagnosis and assisting in the early detection and treatment of diseases; at the same time, it can also provide more targeted nutritional advice to individuals, promoting health management.

Description

Technical Field

[0001] This invention relates to the field of fatty acid detection technology, specifically to a method for determining 11 polyunsaturated fatty acids in blood samples based on LC-MS / MS. Background Technology

[0002] Polyunsaturated fatty acids (PUFAs) play a crucial role in regulating human physiological functions. Omega-3 PUFAs, represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), not only help regulate blood lipids, reduce blood viscosity, and lower the risk of cardiovascular disease, but also play a key role in brain development and vision maintenance. Furthermore, PUFAs have a positive effect on immune regulation, helping to alleviate inflammatory responses. Accurate detection of PUFA levels in the blood is of paramount value in many fields, including clinical diagnosis, disease prevention, and nutritional assessment. Among various blood samples, red blood cells, as a key component of human blood, reflect long-term dietary fatty acid intake through their PUFA composition and content, making them a more stable biological sample than serum / plasma. Numerous studies have shown that low levels of omega-3 PUFAs in red blood cells are associated with a significantly increased risk of cardiovascular disease; simultaneously, abnormal PUFA composition in red blood cells has been found in patients with some neurological and inflammatory diseases. Therefore, it is of great significance to develop methods applicable to a variety of blood samples, especially those that can accurately detect total PUFA in red blood cells.

[0003] Currently, there are various techniques for detecting fatty acids, with gas chromatography (GC), gas chromatography-tandem mass spectrometry (GC-MS / MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS) being the most widely used. While GC-MS / MS has been widely applied in fatty acid detection due to its high sensitivity and excellent separation capabilities, this method has certain limitations. In the sample pretreatment stage, fatty acids need to be derivatized to convert them into more volatile substances to meet the separation requirements of gas chromatography. This derivatization process is quite complex, involving multiple chemical reactions. This not only increases the complexity of the operation but also easily introduces errors during the operation, affecting the accuracy of the detection results. Furthermore, GC-MS / MS analysis times are generally long, resulting in low detection efficiency for high-throughput detection scenarios requiring the processing of large numbers of samples. Additionally, GC-MS / MS struggles to achieve ideal detection results for some fatty acids with poor thermal stability or high polarity.

[0004] LC-MS / MS technology combines the powerful separation performance of high-performance liquid chromatography with the advantages of high selectivity, high sensitivity, and the ability to provide information on the molecular structure of substances by mass spectrometry. It can effectively separate and detect more than 85% of polar, thermally unstable natural compounds and samples that GC-MS / MS cannot process. Its applications cover many fields such as biochemistry, environmental monitoring, food processing, and drug development. LC-MS / MS shows particular advantages, especially in the analysis of non-volatile, polar, thermally unstable compounds and high molecular weight substances (such as proteins, peptides, and polymers).

[0005] Despite the numerous advantages of LC-MS / MS in fatty acid detection, a series of challenges remain when detecting PUFAs in blood samples. PUFAs in blood exist in a complex and diverse manner; besides free PUFAs, a significant portion is bound to proteins or exists in esterified forms within higher lipid structures such as triglycerides and phospholipids. Detecting total PUFAs requires first hydrolyzing esterified PUFAs into free fatty acids before subsequent detection, which undoubtedly greatly increases the complexity of the detection process. Currently, due to the convenience of detecting free fatty acids in plasma or serum, most existing LC-MS / MS methods for PUFA detection in the domestic market are for plasma or serum free fatty acids. In contrast, methods specifically for detecting total PUFAs in blood samples, especially erythrocytes, are scarce. Furthermore, some existing fatty acid detection methods suffer from cumbersome pretreatment procedures, high costs, and extremely high requirements for operator skill levels, while also failing to meet the requirements for high-accuracy detection in terms of precision and recovery rate.

[0006] Chinese patent "Method and Application for Detection of Total Fatty Acid Content of 11 Fatty Acids in Blood by High Performance Liquid Chromatography-Tandem Mass Spectrometry" (Publication No.: CN116858976A) employs pretreatment steps of acid hydrolysis, alkaline hydrolysis, and liquid-liquid extraction of serum samples, followed by analysis using a liquid chromatography-mass spectrometry (LC-MS / MS) instrument. However, the pretreatment process is still relatively cumbersome. Another Chinese patent, "A Method for Simultaneous Determination of Multiple Fatty Acid Content in Blood and Its Application" (Publication No.: CN113325100A), invented a method for simultaneously determining the content of multiple fatty acids in blood based on liquid chromatography-tandem mass spectrometry (LC-MS / MS), applicable to whole blood, plasma, or serum samples. However, the fatty acids measured in this patent are free fatty acids, not total fatty acids. Furthermore, this method uses high-concentration ammonium fluoride, a reagent that easily accumulates in the liquid chromatography tubing, corroding the tubing and accumulating in the detector, severely affecting the instrument's sensitivity and noise level. Moreover, under high-temperature environments, ammonium fluoride decomposes to produce hazardous hydrogen fluoride gas, posing a significant safety hazard.

[0007] Therefore, developing a highly efficient, accurate, easy-to-operate, and low-cost liquid chromatography-mass spectrometry (LC-MS) method specifically for detecting polyunsaturated fatty acids in blood samples, especially a method that can meet the needs of red blood cell detection, has become a key issue that urgently needs to be addressed in related fields. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for determining 11 PUFAs in blood samples based on LC-MS / MS, relating to the field of fatty acid detection technology. The method includes: preparing a calibration solution; hydrolyzing bound fatty acids in the blood sample, adding a precipitant, centrifuging, and collecting the supernatant as the test sample; detecting the test sample using liquid chromatography-tandem mass spectrometry, and substituting the detection results into the calibration curve equation to obtain the content of the 11 PUFAs in the test sample. This method is applicable to various blood samples, such as plasma, serum, or erythrocytes, and is particularly suitable for erythrocyte samples. This method can rapidly and accurately detect the content of 11 PUFAs in blood samples, providing a more accurate basis for clinical diagnosis and assisting in the early detection and treatment of diseases; at the same time, it can also provide more targeted nutritional advice to individuals, promoting health management.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] On one hand, this invention provides a method for determining 11 polyunsaturated fatty acids (PUFAs) based on LC-MS / MS. The polyunsaturated fatty acids include α-linolenic acid n3 (ALA), γ-linolenic acid n6 (GLA), linoleic acid n6 (LA), eicosapentaenoic acid n6 (EDA), eicosatrienoic acid n6 (DGLA), arachidonic acid n6 (AA), eicosapentaenoic acid n3 (EPA), docosahexaenoic acid n6 (DTA), docosahexaenoic acid n3 (DPA-n3), docosahexaenoic acid n6 (DPA-n6), and docosahexaenoic acid n3 (DHA). The method involves using a precipitant to precipitate the blood sample to be tested, and then performing liquid chromatography-tandem mass spectrometry (LC-MS / MS) on the supernatant after centrifugation to obtain the PUFA content in the sample. The solute of the precipitant includes either formic acid or acetic acid; the solvent of the precipitant is acetonitrile.

[0011] Furthermore, before precipitation, a hydrolysate is added to the blood sample to be tested for hydrolysis.

[0012] Furthermore, the hydrolysate comprises any concentration of sodium hydroxide aqueous solution with a concentration of 5 to 10 M.

[0013] Furthermore, the hydrolysis temperature is 70~100℃, preferably 90℃; and / or the hydrolysis time is 15~120 min, preferably 30 min.

[0014] Preferably, the hydrolysate is a 10 M sodium hydroxide aqueous solution; the solute of the precipitant is formic acid with a concentration of 1% (v / v); and the solvent of the precipitant is acetonitrile.

[0015] It is important to understand that the acid in the precipitant is to neutralize the alkaline solution in the hydrolysate.

[0016] Furthermore, the mobile phase A in the liquid chromatography-tandem mass spectrometry detection is a 0% to 0.1% aqueous formic acid solution, preferably a 0.1% aqueous formic acid solution.

[0017] Furthermore, the blood sample includes any one of plasma, serum, and red blood cells, preferably red blood cells.

[0018] Specifically, the method includes the following steps: preparing a calibration solution; adding a hydrolysate to the blood sample to be tested sequentially for hydrolysis, using a precipitant to remove proteins, centrifuging, and taking the supernatant as the sample to be tested; performing liquid chromatography-tandem mass spectrometry on the sample to be tested, and substituting the detection results into the calibration curve equation to obtain the content of 11 polyunsaturated fatty acids in the sample to be tested.

[0019] Furthermore, this invention provides a kit for detecting 11 PUFAs, the kit comprising α-linolenic acid n3 (ALA), γ-linolenic acid n6 (GLA), linoleic acid n6 (LA), eicosapentaenoic acid n6 (EDA), eicosatrienoic acid n6 (DGLA), arachidonic acid n6 (AA), eicosapentaenoic acid n3 (EPA), docosapentaenoic acid n6 (DTA), docosapentaenoic acid n3 (DPA-n3), and docosapentaenoic acid n6 (D... The kit uses one or more of the following calibrators: PA-n6, docosahexaenoic acid n3 (DHA); and / or one or more of the following isotopic internal standard raw materials: docosahexaenoic acid-D5 (DHA-D5), eicosapentaenoic acid-D5 (EPA-D5), docosapentaenoic acid-D5 (DPA-D5), and arachidonic acid-D8 (AA-D8), preferably docosahexaenoic acid-D5 (DHA-D5); the kit is used to detect PUFA using the method described above.

[0020] Furthermore, the present invention provides the application of the above-described method and / or the above-described kit in dose-effect studies of fatty acid detection.

[0021] The beneficial effects of this invention include:

[0022] 1. The method provided by this invention can quickly and accurately detect the content of 11 PUFAs in blood samples, which will provide a more accurate basis for clinical diagnosis and help in the early detection and treatment of diseases; in terms of nutritional assessment, it can provide more targeted nutritional advice to individuals and promote health management; at the same time, it will also provide strong technical support for research on related diseases and promote the rapid development and progress of the entire field.

[0023] 2. The method provided by this invention uses the internal standard method for quantification, which effectively corrects matrix effects, pretreatment errors and instrument fluctuations compared to the external standard method. The working calibration solution can be directly analyzed after preparation, simplifying the experimental operation. The concentration difference between adjacent calibration solutions is 2 times, and the preparation process is simple.

[0024] 3. The method of this invention requires a small sample volume, only 50 μL, improving patient compliance in blood collection. The detected fatty acids are 11 PUFAs, a combination of free and bound states. Furthermore, it abandons the complex liquid-liquid extraction steps used in conventional methods, employing only alkaline hydrolysis, simplifying the operation. The hydrolysis temperature is 90℃, providing mild conditions and reducing operator risk. After hydrolysis, the precipitant is added directly, mixed, and centrifuged to complete the pretreatment, further simplifying the experimental steps and increasing operational convenience. The preferred sample type for this invention is red blood cells, and currently there are no related fatty acid detection patents using red blood cells as the preferred sample type. When red blood cells are used as the preferred sample, they can reflect long-term dietary fatty acid intake and have unique clinical application value.

[0025] 4. Conventional fatty acid detection pretreatment steps include acid hydrolysis, alkaline hydrolysis, neutralization, liquid-liquid extraction, concentration, and reconstitution, which are extremely cumbersome and time-consuming. This patented pretreatment process only includes hydrolysis and precipitation, greatly simplifying the process, significantly shortening operation time, and reducing the risk of contamination and human error. Simultaneously, it uses less reagent, keeping costs under control, and is suitable for automated batch processing, meeting the needs of high-throughput clinical testing. Validation has shown that each fatty acid component exhibits good linearity over a wide concentration range, with precision and accuracy meeting requirements. The method is robust and durable, providing reliable technical support for routine screening and disease-related studies of fatty acids in blood-related samples.

[0026] 5. This invention simplifies the process and reduces potential variables introduced during operation, making the detection process more controllable. It is particularly suitable for large-scale population screening and long-term follow-up studies, providing an efficient and stable analytical platform for exploring the association between fatty acid metabolism and chronic diseases. Future optimization of reagent preparation and instrument parameters is expected to achieve fully automated detection, promoting the standardization and intelligent development of clinical lipidomics.

[0027] 6. The mobile phase is simple to prepare, with low reagent consumption, high column efficiency, and excellent pressure resistance and durability, maintaining stable separation performance under high-throughput continuous injection conditions; a gradient elution program is used to effectively separate fatty acids from interfering substances, avoiding the impact of interfering substances on the accuracy of quantitative detection; the low injection volume significantly reduces matrix effects, lightens the instrument load, and makes the instrument operation more stable; the instrument analysis time is only 7 minutes, shorter than existing technologies, and also improves detection throughput. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Chromatograms of 11 polyunsaturated fatty acid calibrators

[0030] Figure 2 Chromatograms of GLA and ALA

[0031] Figure 3 Chromatograms of DPA-n3 and DPA-n6

[0032] Figure 4 The standard curve for GLA is: y = 0.08191x + (-0.00182) (r = 0.99970) (weighting: 1 / x)

[0033] Figure 5 The standard curve for LA is: y = 0.03384x + (-0.01848) (r = 0.99988) (weighting: 1 / x)

[0034] Figure 6 EPA standard curve, y = 0.02416x + 0.00635 (r = 0.99971) (weighting: 1 / x)

[0035] Figure 7 The standard curve for AA is: y = 0.01303x + (-0.01432) (r = 0.99966) (weighting: 1 / x)

[0036] Figure 8 The standard curve for DGLA is: y = 0.09870x + 0.01220 (r = 0.99975) (weighting: 1 / x).

[0037] Figure 9 The standard curve for EDA is: y = 0.06636x + (-0.00754) (r = 0.99989) (weighting: 1 / x).

[0038] Figure 10 The standard curve for DHA is: y = 0.03927x + 0.00222 (r = 0.99995) (weighting: 1 / x).

[0039] Figure 11 The standard curve for DPA-n3 is: y = 0.12868x + (-0.00351) (r = 0.99985)(weighting: 1 / x)

[0040] Figure 12 The standard curve for DPA-n6 is: y = 0.02558x + 0.00216 (r = 0.99982) (weighting: 1 / x).

[0041] Figure 13 The standard curve for DTA is: y = 0.00588x + 5.26194e-4 (r = 0.99995) (weighting: 1 / x).

[0042] Figure 14 The standard curve for ALA is: y = 0.04723x + (-0.00285) (r = 0.99986)(weighting: 1 / x)

[0043] Figure 15 The effect of the number of cleaning cycles on ALA results

[0044] Figure 16 The effect of the number of cleaning cycles on GLA results Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the test methods used in the following examples are conventional methods; information on the calibrators, reagents, etc. used is shown in Tables 1 and 2, and unless otherwise specified, they are reagents and materials that are commercially available.

[0047] Table 1. Reagent information used in this invention

[0048]

[0049] Table 2. Calibrator information used in this invention

[0050]

[0051] Example 1: A method for detecting the content of 11 polyunsaturated fatty acids in blood samples using liquid chromatography-tandem mass spectrometry.

[0052] The sample types used for fatty acid detection in patents and literature are mostly serum, plasma, or whole blood. However, fatty acids in these sample types are easily affected by short-term diet or other factors. Fatty acid levels in red blood cells are more stable and do not fluctuate significantly with short-term dietary factors, thus reflecting long-term (3-4 months) dietary fatty acid intake (because red blood cells have a lifespan of approximately 120 days). Therefore, they are more stable biological samples than serum and plasma. The method proposed in this invention is applicable to various blood samples such as plasma, serum, or red blood cells. In particular, when using red blood cells as the detection sample, it provides a more stable analytical tool for long-term dietary fatty acid intake assessment. The specific steps of the method are as follows:

[0053] 1.1 Preparation of calibration solution

[0054] Accurately weigh 100 mg of each of the following analytes into 11 10 mL volumetric flasks: α-linolenic acid n3 (ALA), γ-linolenic acid n6 (GLA), linoleic acid n6 (LA), eicosapentaenoic acid n6 (EDA), eicosatrienoic acid n6 (DGLA), arachidonic acid n6 (AA), eicosapentaenoic acid n3 (EPA), docosatraenoic acid n6 (DTA), docosapentaenoic acid n3 (DPA-n3), docosapentaenoic acid n6 (DPA-n6), and docosahexaenoic acid n3 (DHA). Dissolve each analyte in methanol and dilute to the mark. Mix well to obtain stock solutions for each analyte. Transfer each stock solution to prepare an intermediate mixed stock solution, then dilute it to prepare the highest-level calibration solution S7. Then dilute calibration solution S7 to prepare calibration solutions for each level. The specific mass concentrations are shown in Table 3.

[0055] Table 3. Concentrations (ng / mL) of different fatty acid calibration solutions at each level

[0056] 1.2 Preparation of internal standard working solution

[0057] Transfer DHA-D5 stock solution (1 mg / mL) to a 50 mL volumetric flask and dilute to volume with methanol to bring the internal standard concentration to 1 μg / mL. It should be understood that the DHA-D5 internal standard can correct for all 11 PUFAs; if internal standards for the aforementioned 11 PUFAs are available, they can also be used for calibration.

[0058] 1.3 Preparation of hydrolysate

[0059] Weigh out solid sodium hydroxide and dissolve and dilute it with pure water to a concentration of 10 M.

[0060] 1.4 Preparation of precipitant

[0061] Measure an appropriate amount of acetonitrile and add formic acid to make the formic acid concentration (v / v) 1%.

[0062] 1.5 Preparation of blood samples

[0063] (1) Plasma sample: Collect whole blood in a blood collection tube containing anticoagulant, mix well, centrifuge at 1500 × g for 10 min at 4 ℃, and take the supernatant as plasma.

[0064] (2) Serum sample: Collect whole blood in a blood collection tube without anticoagulant, let it stand at room temperature for 30 min until the blood coagulates, centrifuge at 1500 × g for 10 min at 4℃, and take the supernatant as serum.

[0065] (3) Red blood cell sample: After centrifuging the above plasma sample, discard the supernatant and white membrane layer, take the lower red blood cell precipitate, add an equal volume of physiological saline and gently blow to mix, centrifuge at 1200 × g for 10 min at 4 ℃, discard the supernatant, and repeat the washing twice to obtain the red blood cell sample.

[0066] 1.6 Sample Preprocessing

[0067] Accurately transfer 50 μL of blood sample or calibration solution into a 1.5 mL EP tube, add 100 μL of hydrolysis buffer (i.e., 10 M sodium hydroxide solution) and 200 μL of internal standard working solution, vortex to mix, and hydrolyze at 90℃ for 30 min. After hydrolysis, remove the tube and cool to room temperature, add 800 μL of precipitant (i.e., 1% formic acid-acetonitrile solution), vortex to mix, centrifuge at 1200 g for 5 min, and transfer the supernatant for analysis. The calibration solution can be analyzed directly without hydrolysis.

[0068] 1.7 Detection conditions for liquid chromatography-tandem mass spectrometry

[0069] Instrument Model: AB SCIEX Triple Quad™ 4500MD

[0070] Chromatographic conditions: Mobile phase A was 0.1% (v / v) aqueous solution of formic acid; mobile phase B was methanol; the chromatographic column was Waters ACQUITY UPLC HSS T3 1.8 μm 2.1*100 mm; the injector temperature was 5~15℃ (preferably 10℃); the column temperature was 50℃; the injection volume was 0.5~2 μL (preferably 1 μL); the injection solution was 50% methanol-water; the elution program is shown in Table 4.

[0071] Table 4 Elution procedures for chromatography

[0072]

[0073] Mass spectrometry conditions: ionization mode: ESI-; scan type: MRM; Curtain Gas: 30; Collision Gas: 9; IS: -4500 V; Temperature: 600℃; GS1: 55; GS2: 55; ion pair parameters are shown in Table 5, and valve switching time and corresponding valve position are shown in Table 6.

[0074] Table 5 Ion pair parameters of mass spectrometry

[0075]

[0076] Table 6 Mass Spectrometer Switching Valves

[0077]

[0078] 1.8 Calculation of Sample Concentration

[0079] A calibration curve was established using the instrument's built-in analysis software, with a weighting of 1 / x. A linear regression equation was used, and the internal standard method was employed for quantification. The calculated concentration value displayed by the instrument is the actual measured concentration of the sample. See the attached PUFA standard curves. Figures 4-14 .

[0080] Example 2: Specificity Evaluation

[0081] Blood sample matrices are complex, and some PUFAs are isomers with similar retention times, which can easily cause detection interference. This invention optimizes chromatographic gradients and mass spectrometry multiple reaction monitoring parameters to achieve baseline separation of each component, significantly improving identification capabilities. Experimental results show that the resolution between each PUFA and its isomers or unknown chromatographic peaks is above 1.5, meeting the requirements for accurate quantification. Simultaneously, internal standard correction effectively eliminates matrix effects, ensuring consistency between different batches and providing stable and reliable quantitative results.

[0082] TIC chromatograms of each analyte are shown below. Figure 1 The chromatogram of PUFA, which is susceptible to interference, is shown in [the image]. Figure 2 and Figure 3 GLA and ALA are isomers of each other. Figure 2 The results showed that GLA and ALA achieved baseline separation, with symmetrical peak shapes and no obvious tailing, meeting the quantitative requirements; DPA-n3 and DPA-n6 are isomers, from Figure 3 It can be seen that the two have been completely separated.

[0083] Example 3: Precision Evaluation

[0084] In this embodiment, three analytical batches were tested according to the method described in Example 1. Each batch of mixed red blood cell samples (sample source: Hangzhou Weidu Huimei Medical Laboratory Co., Ltd.) was tested 10 times in parallel to examine the intra-batch and inter-batch coefficients of variation. The results showed (Table 7) that the intra-batch coefficients of variation for each PUFA component were less than 8.76% and the inter-batch coefficients of variation were less than 7.39%.

[0085] Table 7 Precision Evaluation

[0086]

[0087] Example 4: Accuracy Evaluation

[0088] This embodiment validated the assay using NIST SRM 1950 standard material (serum matrix) and spiked clinical mixed red blood cell samples (samples sourced from Hangzhou Weidu Huimei Medical Laboratory Co., Ltd.). The results showed that the accuracy of detecting each fatty acid in the NIST SRM 1950 standard material ranged from 87.88% to 110.77%, and the recovery rates of each PUFA in the blood samples ranged from 92.61% to 113.38%, meeting the accuracy requirements for clinical testing. Detailed data are shown in Tables 8 and 9.

[0089] Table 8. NIST SRM 1950 Accuracy

[0090]

[0091] Table 9 Recovery rate of spiked red blood cell samples

[0092]

[0093] Example 5: Reference Interval Verification

[0094] This embodiment analyzed the PUFA profiles of 200 healthy adult red blood cell samples according to the method described in Example 1. Statistical validation yielded reference intervals, as shown in Table 10. These reference intervals meet clinical application requirements, providing reliable detection data for clinical laboratories. This further validates the accuracy and stability of the methodology, ensuring that the test results have practical guiding significance in individualized nutritional assessment and disease risk warning.

[0095] Table 10 Reference Interval

[0096]

[0097] Example 6: Validation of the applicability of conventional serum / plasma detection methods in erythrocyte samples

[0098] This embodiment aims to verify whether conventional methods for detecting PUFA in serum or plasma are applicable to erythrocyte samples, reveal methodological differences between different matrices, and demonstrate the applicability and necessity of the method of the present invention in erythrocyte samples.

[0099] 1. Selection of comparison methods

[0100] This embodiment selects two representative PUFA detection methods, where Method A is derived from patent CN113325100A and Method B is derived from patent CN116858976A, respectively, for detecting red blood cell samples, and compares them with the method of this invention. Information on the comparison methods is shown in Table 11.

[0101] Table 11 Comparison Method Information

[0102]

[0103] 2. Experimental Samples and Grouping

[0104] Fresh whole blood was collected from the same donor, and red blood cells and plasma samples were separated. Red blood cell samples were washed twice with physiological saline before use. All samples were tested in triplicate, with three replicates per batch.

[0105] 3. Applicability verification of Method A

[0106] Method A involves direct precipitation of proteins with organic solvents followed by detection of free fatty acids (including free saturated fatty acids, monounsaturated fatty acids, and PUFAs). It does not include a hydrolysis step, and the chromatographic conditions are a C18 column and a mobile phase containing ammonium fluoride. When applied to erythrocyte samples, the detection results are compared with those of the method of this invention (Table 12). Since Method A's detection results include both saturated and monounsaturated fatty acids, this comparison only lists the PUFA portion, which overlaps with the method of this invention.

[0107] Table 12 Comparison of detection results (μmol / L) between Method A and the method of the present invention

[0108]

[0109] The results showed that free PUFAs accounted for only 3.2% to 3.8% of the total PUFAs in erythrocytes, with the vast majority of PUFAs existing in bound form within the erythrocyte membrane phospholipids. Method A only detects free PUFAs, and its detection results differ from the total PUFAs detected by the method of this invention by more than an order of magnitude. If Method A is used directly to detect erythrocytes, the detected values ​​cannot reflect the true total amount of PUFAs in erythrocytes, leading to a serious underestimation.

[0110] In addition, ammonium fluoride is added to the mobile phase of Method A. This reagent tends to deposit in the mass spectrometry ion source when erythrocyte samples are processed continuously, which leads to a rapid decrease in sensitivity and further affects the detection accuracy.

[0111] 4. Applicability verification of method B

[0112] Method B involves multiple steps including acid hydrolysis, alkaline hydrolysis, neutralization, liquid-liquid extraction, nitrogen blowing concentration, and reconstitution. The chromatographic conditions are a C18 column and an ammonium acetate-water-acetonitrile mobile phase system. It was originally applicable to the detection of 11 PUFAs in serum. Table 13 shows the comparison results between this method and the method of this invention when applied to erythrocyte samples.

[0113] Table 13 Comparison of detection results (μmol / L) between Method B and the method of the present invention

[0114]

[0115] The results showed that the detection results of Method B were generally lower than those of the method of this invention, and the precision was also poor. The reasons for this include: ① Red blood cell samples contain a large amount of hemoglobin and membrane proteins, and the liquid-liquid extraction step in Method B is prone to emulsification, leading to incomplete PUFA extraction; ② The dense structure of red blood cell membranes requires more thorough hydrolysis conditions to completely release bound PUFA; ③ Multiple operations (nitrogen blowing, reconstitution) increase the risk of PUFA oxidative degradation; ④ The C18 column used in Method B has insufficient separation capability for isomers (such as GLA / ALA, DPA-n3 / DPA-n6) in red blood cell samples, and some chromatographic peaks overlap and interfere; ⑤ Method B optimized the conditions for the serum matrix, without considering the matrix interference specific to red blood cells.

[0116] 5. Effects of different matrices on the detection performance of Method B

[0117] To further verify the applicability of Method B in erythrocytes and serum matrix, Method B was used to simultaneously test serum and erythrocyte samples from the same donor. The results are shown in Table 14.

[0118] Table 14 Comparison of detection performance of Method B in different matrices

[0119]

[0120] The results showed that Method B had good detection performance in serum samples, but when applied directly to red blood cell samples, the detection performance decreased significantly, the isoform separation decreased to below 1.0, baseline separation could not be achieved, and emulsification was prone to occur during the pretreatment process.

[0121] 6. Comprehensive comparison of various methods

[0122] The applicability of methods A, B, and the method of the present invention in red blood cell samples was comprehensively compared, and the results are shown in Table 15.

[0123] Table 15. Overall Comparison of the Applicability of Different Methods in Red Blood Cell Samples

[0124]

[0125] Based on the above results, the following conclusions can be drawn:

[0126] (1) Method A only detects free PUFAs, while fatty acids in erythrocytes mainly exist in bound form, with free PUFAs accounting for less than 4%. This method cannot reflect the total PUFA level in erythrocytes, and the detected value is seriously low. In addition, the ammonium fluoride added to the mobile phase poses a safety hazard and is prone to deposition in the mass spectrometer ion source, affecting the stability of the instrument.

[0127] (2) Although Method B includes a hydrolysis step, it is optimized for serum matrix and has problems such as low recovery rate (average 81.6%), poor precision (average CV% 10.8%), insufficient isoform separation (<1.0) and easy emulsification (pretreatment success rate 84.4%) when applied to red blood cell samples. It cannot meet the requirements of clinical testing for accuracy and stability.

[0128] (3) The method of the present invention adopts a one-step alkaline hydrolysis combined with formic acid-acetonitrile precipitation pretreatment method. The hydrolysis conditions, chromatographic column and mobile phase system are optimized for the characteristics of red blood cell matrix, and baseline separation of each PUFA is achieved. The average recovery rate is 98.6%, the average intra-batch CV% is 3.1%, the pretreatment success rate is 100%, and it is superior to the comparative method in terms of safety and detection efficiency.

[0129] Therefore, conventional methods for detecting PUFA in serum or plasma cannot be directly applied to red blood cell samples, while the method of this invention exhibits good precision, accuracy, and clinical applicability in red blood cell samples. The following comparative examples aim to systematically examine the influence of key parameters in the method of this invention on the detection results, providing experimental basis for the selection of optimal conditions for this method.

[0130] Comparative Example 1: The impact of pretreatment method on test results

[0131] This comparative example compares the effects of the pretreatment method of this patent embodiment (one-step alkaline hydrolysis + formic acid-acetonitrile precipitation) and the conventional PUFA detection pretreatment method (acid hydrolysis + alkaline hydrolysis + neutralization + liquid-liquid extraction + nitrogen blowing concentration + resolution) on the detection results of 11 PUFAs in red blood cell samples.

[0132] The detection steps of the conventional detection method are as follows:

[0133] Transfer 10 μL of red blood cell sample to a 2 mL centrifuge tube, add 10 μL of internal standard, mix at 2500 r / min for 30 s, add 250 μL of hydrolysis solution 1 (acetonitrile: 6 mol / L hydrochloric acid (v:v) = 9:1), mix at 2500 r / min for 30 s, heat at 85℃ for 30 min, shaking the centrifuge tube every 10 min to incorporate the solids adhering to the tube wall into the solution. After cooling to room temperature, add 250 μL of hydrolysis solution 2 (methanol: 10 mol / L sodium hydroxide (v:v) = 9:1), mix at 2500 r / min for 30 s, heat at 85℃ for 30 min, shaking the centrifuge tube every 10 min to incorporate the solids adhering to the tube wall into the solution. After cooling to room temperature, add 45 μL of 6 mol / L hydrochloric acid, mix at 2500 r / min for 1 min, add 1 mL of n-hexane, and heat at 2500 r / min for 1 min. Mix at 10 rpm for 10 min, centrifuge at 14000 rpm and 4℃ for 5 min, transfer 950 μL of supernatant to a new 2 mL centrifuge tube, dry under nitrogen, add 220 μL of reconstitution solution (acetonitrile (containing 0.1% ammonia)), mix at 2500 rpm for 2 min, centrifuge at 14000 rpm and 4℃ for 10 min to obtain the sample to be tested, and inject the supernatant for analysis. The remaining steps are the same as in Example 1.

[0134] The test results are shown in Tables 16 and 17.

[0135] Table 16 Comparison of detection results for different pretreatment methods (μmol / L)

[0136]

[0137] Table 17 Comparison of Precision and Efficiency of Different Pretreatment Methods

[0138]

[0139] The results showed no significant difference in the PUFA detection results obtained by the two pretreatment methods, indicating that the method of the present invention has good consistency with the conventional method in terms of detection accuracy. However, the method of the present invention exhibits significant advantages in terms of precision, operational efficiency, and method robustness. Conventional methods involve multiple transfer steps and nitrogen blowing concentration, resulting in a higher average intra-batch coefficient of variation (CV%) compared to the present invention. The conventional method involves seven pretreatment steps, taking approximately 180 minutes, while the present invention has only two steps, taking approximately 35 minutes. The liquid-liquid extraction step in the conventional method is prone to emulsification, resulting in a pretreatment success rate of only 87.2%, while the present invention has no emulsification risk and a 100% pretreatment success rate. The average recovery rate of the present invention is 98.6%, superior to the 84.8% of the conventional method. Therefore, by simplifying the pretreatment steps, the present invention reduces the introduction of errors, achieves higher detection precision than conventional methods, and improves the accuracy and reliability of the detection results. Furthermore, due to its simpler and faster operation, it significantly shortens the sample pretreatment time, improves detection efficiency, and is more suitable for large-scale clinical sample testing.

[0140] Comparative Example 2: The Influence of Hydrolysis Method on Detection Results

[0141] This comparative example, under the optimal conditions of the method of the present invention (Example 1) in all other aspects, systematically investigated the effects of different hydrolysis methods on the detection results of PUFAs in red blood cell samples. The effects of four hydrolysis methods—acid hydrolysis, alkaline hydrolysis (the present invention), acid-then-alkaline hydrolysis, and alkaline-then-acid hydrolysis—on the detection results of 11 PUFAs in red blood cell samples were compared.

[0142] The acid hydrolysis step is as follows: transfer 50 μL of red blood cell sample, add 100 μL of 6 M hydrochloric acid solution and 200 μL of internal standard working solution, vortex mix, hydrolyze at 90 ℃ for 30 min, and then remove and cool to room temperature. The remaining steps are the same as in Example 1.

[0143] The acid-then-alkali step is as follows: Transfer 50 μL of red blood cell sample, add 100 μL of 6 M hydrochloric acid solution and 200 μL of internal standard working solution, hydrolyze at 90 ℃ for 30 min, cool, add 100 μL of 10 M sodium hydroxide solution, and continue hydrolyzing at 90 ℃ for 30 min. After completion, remove and cool to room temperature. The remaining steps are the same as in Example 1.

[0144] The step of first alkali and then acid is as follows: Transfer 50 μL of red blood cell sample, add 100 μL of 10 M sodium hydroxide solution and 200 μL of internal standard working solution, hydrolyze at 90 ℃ for 30 min, cool, add 100 μL of 6 M hydrochloric acid solution, continue hydrolysis at 90 ℃ for 30 min, and after completion, remove and cool to room temperature. The remaining steps are the same as in Example 1.

[0145] The test results are shown in Table 18.

[0146] Table 18 Effect of different hydrolysis methods on the recovery rate of each PUFA (%)

[0147]

[0148] Experimental results show that the average recovery rate of each PUFA by acid hydrolysis is only 56.1%, significantly lower than other hydrolysis methods. This is because acid cannot effectively break the ester bonds in triglycerides and phospholipids. The average recovery rate of acid-then-alkali hydrolysis is 83.8%, about 13.5% lower than that of this invention. The average recovery rate of alkali-then-acid hydrolysis is 77.9%, lower than that of acid-then-alkali hydrolysis, because the alkali has fully hydrolyzed the ester bonds, and adding acid afterwards leads to the degradation or re-esterification of the released PUFA. The one-step alkali hydrolysis method of this invention achieves an average recovery rate of 97.3%, which is significantly better than other hydrolysis methods.

[0149] Comparative Example 3: The Influence of Alkaline Solution Type on Detection Results

[0150] This comparative example, under the optimal conditions of the method of the present invention (Example 1) in all other aspects, systematically investigated the effects of different types of alkaline solutions on the detection results of PUFA in red blood cell samples. The hydrolysis effects of four alkaline solutions—sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH), and ammonia (NH3·H2O)—were compared.

[0151] 10 M NaOH, KOH, and LiOH aqueous solutions, as well as 12.5 M concentrated ammonia, were prepared as hydrolysis solutions. All other pretreatment and detection conditions remained consistent with those in Example 1. The effects of each alkaline solution on the recovery rate of each PUFA were compared. The results are shown in Table 19.

[0152] Table 19 Effect of different alkaline solutions on the recovery rate of each PUFA (%)

[0153]

[0154] Experimental results show that NaOH and KOH at a concentration of 10 M achieved recoveries of over 95% for each PUFA, with no significant difference in average recovery rate. LiOH had an average recovery rate of 86.9%, slightly lower than NaOH and KOH; ammonia had an average recovery rate of only 56.6%, failing to effectively saponify ester bonds. Therefore, NaOH and KOH showed the best hydrolysis effect at a concentration of 10 M, with no significant difference between them, and can be substituted for each other as hydrolysis reagents in this invention. Considering hydrolysis efficiency, cost, and ease of operation, this invention preferentially uses NaOH as the hydrolysis solution.

[0155] This invention also investigated the effect of different concentrations of NaOH on the detection results of PUFAs. The results showed that when the NaOH concentration was below 5 M, the erythrocyte membrane structure was not completely destroyed, and the release of bound PUFAs was insufficient, with the recoveries of DPA-n6 and DPA-n3 below 82% and 80%, respectively. When the concentration was above 10 M, the recoveries of EPA and AA decreased to below 85%, indicating that strongly alkaline conditions easily induce the oxidative degradation of PUFAs. When the NaOH concentration was 10 M, the recoveries of all PUFAs were above 95%, showing the best overall effect.

[0156] Comparative Example 4: The effect of hydrolysis conditions on test results

[0157] This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effects of hydrolysis temperature and hydrolysis time on the detection results of PUFAs in erythrocyte samples. Since different PUFAs have different sensitivities to hydrolysis temperature, PUFAs with higher polyunsaturated double bond content, such as DHA and EPA, are more susceptible to oxidative degradation at high temperatures, while DGLA and DHA are more sensitive to incomplete hydrolysis. Therefore, this comparative example selected DGLA, EDA, EPA, and DHA as representative PUFAs for investigation to comprehensively reflect the trend of the influence of hydrolysis temperature on PUFA recovery. The results are shown in Table 20.

[0158] Table 20 Effect of different hydrolysis temperatures on the recovery rate of various PUFAs (%)

[0159]

[0160] Experimental results show that as the hydrolysis temperature gradually increases from 60℃ to 90℃, the recovery rates of each PUFA show an upward trend, indicating that within this temperature range, increasing the temperature can effectively promote the complete hydrolysis of PUFAs in red blood cell samples and significantly improve the extraction recovery rate. When the temperature continues to rise to 100℃, the recovery rates of each PUFA decrease, with EPA and DHA showing a more significant decrease, consistent with their polyunsaturated double bond structure's tendency to undergo oxidative degradation at high temperatures. In summary, under the conditions of this invention, 90℃ is the optimal temperature for the hydrolysis of PUFAs in red blood cell samples, balancing complete hydrolysis with structural stability, and achieving the highest recovery rates for various PUFAs.

[0161] Similarly, the effect of hydrolysis time on PUFA recovery also varied. PUFAs with higher polyunsaturated double bond content, such as DHA, DPA-n3, and EPA, were more prone to degradation under prolonged hydrolysis conditions. Therefore, this comparative example selected DHA, DPA-n3, and EPA as representative PUFAs to investigate the effect of hydrolysis time on PUFA recovery. The results are shown in Table 21.

[0162] Table 21 Effect of different hydrolysis times on the recovery rate of each PUFA (%)

[0163]

[0164] Experimental results showed that as the hydrolysis time was gradually extended from 10 min to 30 min, the recovery rates of each PUFA showed a significant upward trend, indicating that within this time range, extending the hydrolysis time could effectively promote the complete hydrolysis of PUFA in erythrocyte samples and significantly improve the extraction recovery rate. When the hydrolysis time was extended to 60 min, the recovery rates of each PUFA only decreased slightly, and remained at a high level overall. When the hydrolysis time was extended to 120 min, the recovery rates of each PUFA declined significantly, which is completely consistent with the characteristic that highly unsaturated PUFA is prone to oxidative degradation under long-term hydrolysis conditions. In summary, under the conditions of this invention, 30 min is the optimal time for PUFA hydrolysis in erythrocyte samples, which can achieve the highest recovery rate of various PUFAs, while maintaining a good recovery rate level within 60 min, thus ensuring the operational tolerance of the method.

[0165] Comparative Example 5: The Influence of Precipitant Components on Detection Results

[0166] The precipitant serves a dual purpose: neutralizing pH and precipitating proteins. The choice of precipitant directly affects the protein precipitation effect and the stability of the target analyte. In this comparative example, under the optimal conditions of the method of this invention (Example 1), the influence of precipitant components on the detection results of PUFAs in erythrocyte samples was systematically investigated. The effect of different precipitants on PUFA stability is mainly reflected in their sensitivity to oxidative degradation. PUFAs with high polyunsaturated double bond content, such as ALA, LA, EPA, and DHA, are more susceptible to degradation by strong oxidizing precipitants (such as perchloric acid). Therefore, this comparative example selected ALA, LA, EPA, and DHA as representative PUFAs to evaluate the impact of different precipitants on PUFA recovery. The results are shown in Table 22.

[0167] Table 22 Effect of different precipitants on the recovery rate of each PUFA (%)

[0168]

[0169] Experimental results showed that while saturated ammonium sulfate effectively precipitated proteins, it significantly reduced the recovery rate of some fatty acids (such as ALA and LA) (<80%). Using 10% perchloric acid as a precipitant, although it rapidly precipitated proteins, the strong oxidizing properties of perchloric acid led to the oxidative degradation of unsaturated fatty acids, especially EPA and DHA, reducing their recovery rate to below 75%, and the experimental reproducibility was poor. However, using formic acid-acidified acetonitrile as a precipitant not only achieved efficient protein precipitation but also preserved the stability of various fatty acids to the greatest extent. Therefore, considering the protein precipitation effect, fatty acid recovery rate, and experimental reproducibility, formic acid-acidified acetonitrile was ultimately determined as the optimal precipitant. Systematic investigation revealed that using acetonitrile containing 1%–2% (v / v) (preferably 1%) formic acid as a precipitant can efficiently remove proteins while minimizing fatty acid loss. Under these conditions, protein precipitation was good, and the supernatant after centrifugation was clear and transparent, suitable for high-throughput liquid chromatography-mass spectrometry (LC-MS) analysis. The method reproducibility and sensitivity both met clinical testing requirements.

[0170] Comparative Example 6: The effect of the number of times red blood cell samples are washed on the test results

[0171] The method of the present invention does not include red blood cell washing in the pretreatment step of Example 1. In order to verify whether residual plasma will interfere with the detection results of PUFA in red blood cells, this comparative example systematically investigated the effect of the number of red blood cell sample washings on the detection results.

[0172] The number of erythrocyte sample washes plays a crucial role in the pretreatment of erythrocyte fatty acid (PUFA) detection, primarily to remove residual plasma after whole blood centrifugation. This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effect of the number of erythrocyte sample washes on the PUFA detection results in erythrocytes. Physiological saline was used as the wash solution; approximately 2-3 times the volume of erythrocytes was added each time, followed by centrifugation (1200 g, 10 min), and the supernatant was discarded. The erythrocyte precipitate was retained for subsequent processing. The number of erythrocyte washes mainly affects the interference of PUFAs in residual plasma. GLA and ALA show the greatest concentration difference between plasma and erythrocytes, and are therefore most sensitive to the number of washes. This comparative example selected GLA and ALA as representative PUFAs to evaluate the impact of different erythrocyte wash counts on PUFA detection results.

[0173] The results are as follows Figure 15 , 16As shown, unwashed samples (0 washes) had a higher residual plasma content, leading to significant deviations in the detection results of fatty acids (such as GLA and ALA) in erythrocytes, affecting quantitative accuracy. After one wash, the deviation decreased significantly. With two or more washes, the erythrocyte fatty acid detection results tended to stabilize. Considering both measurement accuracy and operational efficiency, two washes were ultimately determined to be the optimal condition.

[0174] Comparative Example 7: The Influence of Flow Relative Detection Results

[0175] The mobile phase plays a crucial role in liquid chromatography-tandem mass spectrometry (LC-MS / MS), not only carrying the target analyte through the column but also affecting the separation efficiency and detection sensitivity. This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the influence of different mobile phase compositions on the detection results of PUFA in erythrocyte samples. The influence of mobile phase composition on PUFA detection is mainly reflected in the isomer resolution. GLA and ALA, and DPA-n3 and DPA-n6 are isomers with similar retention times and are most sensitive to mobile phase composition. Therefore, this comparative example selected the resolution of GLA / ALA and DPA-n3 / DPA-n6 as evaluation indicators to assess the separation effect of different mobile phase compositions. The results are shown in Table 23.

[0176] Table 23 Effect of different mobile phase compositions on isomer separation degree

[0177]

[0178] Experimental results showed that while methanol-water or acetonitrile-water could achieve partial separation of fatty acids using the mobile phase, the separation effect was not ideal, especially for isomers with similar retention times (such as DPA-n3 and DPA-n6, GLA and ALA), where baseline separation was difficult to achieve, and batch-to-batch drift in retention times was observed. Adding formic acid to the aqueous phase significantly improved the peak shape of the target chromatographic peaks and enhanced the stability of retention times between batches. Simultaneously, the separation of isomers with similar retention times also improved. However, with the addition of formic acid, the sensitivity of each fatty acid decreased; by reducing the proportion of formic acid, the response of each fatty acid gradually increased. Adding ammonium acetate (5 mM) as a buffer salt to the aqueous phase worsened the separation of ALA and GLA. Regarding the choice of organic phase, methanol and acetonitrile showed no significant difference in fatty acid chromatographic separation. Considering all factors, an aqueous solution containing 0.1% formic acid was used as the aqueous phase, and methanol was used as the organic phase to construct the mobile phase of the detection system.

[0179] Comparative Example 8: The Influence of Chromatographic Column on Detection Results

[0180] The chromatographic column is a core component in liquid chromatography-tandem mass spectrometry (LC-MS / MS), and its performance directly affects the separation efficiency, peak shape, and detection sensitivity of analytes. This comparative example, under the optimal conditions of the method of this invention (Example 1) in all other aspects, systematically investigated the effects of different types of chromatographic columns (T3 column: Waters ACQUITY UPLC HSS T3 1.8μm 2.1*100mm, C8 column: Yuexu Ultimate UHPLC XB-C8 1.8μm 2.1*30mm, biphenyl column: Feinome Kinetex Biphenyl 2.6 µm 2.1 x 100 mm) on the detection results of PUFA in red blood cell samples. The influence of column type on PUFA detection is mainly reflected in the isomer resolution. Therefore, this comparative example also selected the resolution of GLA / ALA and DPA-n3 / DPA-n6 as evaluation indicators to assess the separation effect of different columns. The results are shown in Table 24.

[0181] Table 24 Effect of different chromatographic columns on isomer resolution

[0182]

[0183] Experimental results showed that the T3 column exhibited the best performance in fatty acid separation. Its unique bonded phase structure effectively separated various polyunsaturated fatty acids, especially for isomers with similar retention times (such as DPA-n3 and DPA-n6, GLA and ALA), achieving effective baseline separation with symmetrical and sharp peaks and no tailing. In contrast, while the C8 column could also separate some fatty acids, its separation effect on some polyunsaturated fatty acids (such as DPA-n3 and DPA-n6) was poor, and the peak shape was broader; the biphenyl column was not ideal for separating GLA and ALA. Therefore, considering separation efficiency, peak shape, and detection sensitivity, the T3 column was ultimately determined to be the best choice.

[0184] Comparative Example 9: The Influence of the Detection Object on the Detection Results

[0185] This comparative example aims to compare the impact of different detection matrices (plasma, erythrocytes) on the stability of PUFA detection results. Since the concentration distribution of different PUFAs in the two matrices and their degree of influence from diet vary, this comparative example presents the detection results for all 11 PUFAs to comprehensively reflect the differences between matrices.

[0186] Peripheral whole blood from the same donor was used, and plasma and erythrocytes obtained after separation were used as detection matrices. PUFAs from plasma and erythrocytes from the same donor were detected at two time points, day 0 (baseline) and day 15. Pretreatment was performed according to the method described in Example 1, followed by LC-MS / MS analysis to analyze changes in PUFA content in the two matrices. The results are shown in Tables 25 and 26.

[0187] Table 25 Detection bias of PUFA in plasma

[0188]

[0189] Table 26 Deviation in PUFA detection in erythrocytes

[0190]

[0191] The results showed that the concentrations of various PUFAs in plasma samples were significantly affected by recent diet, with an average absolute deviation of 37.52% between day 0 and day 15, and some PUFAs (such as GLA and EPA) showing deviations exceeding 70%. In contrast, the concentrations of various PUFAs in erythrocytes were more stable than those in plasma, with an average absolute deviation of only 10.04%, far lower than that in plasma samples. This confirms that PUFAs in erythrocytes can reflect long-term dietary PUFA intake and are a more stable biological sample than plasma. Therefore, erythrocytes were selected as the preferred detection matrix in the method of this invention.

[0192] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for determining 11 polyunsaturated fatty acids based on LC-MS / MS, characterized in that, The method refers to using a precipitant to precipitate the blood sample to be tested, and then performing liquid chromatography-tandem mass spectrometry on the supernatant after centrifugation to obtain the content of polyunsaturated fatty acids in the sample; the solute of the precipitant includes either formic acid or acetic acid; the solvent of the precipitant is acetonitrile.

2. The method as described in claim 1, characterized in that, The blood sample includes any one of plasma, serum, or red blood cells.

3. The method as described in claim 1, characterized in that, Before precipitation, hydrolysate is added to the blood sample to be tested.

4. The method as described in claim 3, characterized in that, The hydrolysis temperature is 70~100℃; and / or the hydrolysis time is 15~120 min.

5. The method as described in claim 3, characterized in that, The hydrolysate is an aqueous solution of sodium hydroxide with a concentration of 5-10 M.

6. The method as described in claim 1, characterized in that, In liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection, mobile phase A is an aqueous solution of formic acid, with a formic acid concentration of any value between 0% and 1%.

7. The method as described in claim 1, characterized in that, The chromatographic column used in liquid chromatography-tandem mass spectrometry detection is a T3 column.

8. The method as described in claim 1, characterized in that, The 11 polyunsaturated fatty acids include any one or more of α-linolenic acid n3, γ-linolenic acid n6, linoleic acid n6, eicosadienoic acid n6, eicosatrienoic acid n6, arachidonic acid n6, eicosapentaenoic acid n3, docosatraenoic acid n6, docosapentaenoic acid n3, docosapentaenoic acid n6, and docosahexaenoic acid n3.

9. A kit for detecting 11 polyunsaturated fatty acids, characterized in that, The kit includes any one or more calibrators selected from α-linolenic acid n3, γ-linolenic acid n6, linoleic acid n6, eicosapentaenoic acid n6, eicosatrienoic acid n6, arachidonic acid n6, eicosapentaenoic acid n3, docosatraenoic acid n6, docosapentaenoic acid n3, docosapentaenoic acid n6, and docosahexaenoic acid n3; and / or any one or more of docosahexaenoic acid-D5, eicosapentaenoic acid-D5, docosapentaenoic acid-D5, and arachidonic acid-D8 as isotopic internal standards; the kit uses the method described in any one of claims 1 to 8 to detect polyunsaturated fatty acids.

10. The use of the method according to any one of claims 1 to 8 and / or the kit according to claim 9 in a dose-effect study of fatty acid detection.

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