Method for detecting various amino acids, hormones and ceramides by liquid chromatography tandem mass spectrometry

Abstract

The present application relates to the technical field of detection, in particular to a method for detecting multiple amino acids, hormones and ceramides in blood and cerebrospinal fluid by liquid chromatography tandem mass spectrometry. Specifically comprising: preparing standard curve equation I and standard curve equation II respectively, pretreating the sample to be tested to obtain sample I for detecting amino acids and hormones and sample II for detecting ceramides; using high performance liquid chromatography mass spectrometry to detect respectively, substituting the detection results of sample I into standard curve equation I and substituting the detection results of sample II into standard curve equation II to obtain the content of multiple amino acids, hormones and ceramides in the sample to be tested. The present application can realize the combined detection of seven kinds of amino acids, eleven kinds of hormones and three kinds of ceramides by collecting blood and cerebrospinal fluid once. The detection method of the present application greatly saves the analysis time and reduces the analysis cost.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for the combined detection of multiple amino acids, hormones and ceramides by liquid chromatography-tandem mass spectrometry. Background Technology

[0002] In clinical disease diagnosis, many endogenous compounds have significant biological importance. Changes in amino acids, hormones, and ceramides in body fluids are particularly crucial, and their levels in various fluids are essential for diagnosing many diseases. Amino acids in the human body are the basic building blocks of enzymes that catalyze metabolism and participate in maintaining the activity of various hormones. Abnormal levels of steroid hormones are closely related to the occurrence of a range of diseases. For example, there are reports of combined detection of multiple gonadal axis hormones in the blood and cerebrospinal fluid of patients with cerebral infarction and comparison with clinicopathological parameters. There are also reports on how hormonal changes in the hypothalamus-pituitary-gonadal axis can lead to changes in blood lipids and blood viscosity. Furthermore, the pathogenesis of diseases such as polycystic ovary syndrome and mood disorders varies, and monitoring changes in hormone and amino acid levels helps in the accurate diagnosis of disease pathogenesis. Ceramides can be used for risk screening and diagnosis of cardiovascular and cerebrovascular diseases in high-risk populations. Therefore, a sensitive and stable detection method is needed to measure the levels of amino acids, steroid hormones, and ceramides in the body fluids of clinical patients, including plasma and cerebrospinal fluid.

[0003] Hormones in the human body are diverse and structurally similar, making them susceptible to cross-interference when using immunological methods. LC-MS / MS, a technique based on the mass-to-charge ratio of analyte ions for accurate quantification, possesses unique sensitivity and specificity, and has gradually become an important choice for hormone detection in medical laboratories, especially for hormones present in extremely low concentrations. However, in specific application scenarios, LC-MS / MS faces many practical challenges. Limited by the polarity, acidity, alkalinity, and other physicochemical properties of substances, as well as the differences in the content of different substances in biological samples from the same source, different sample pretreatment and analytical methods are required. Under these conditions, increasing the speed and quality of testing necessitates increased investment in equipment and personnel. Furthermore, changing a pretreatment method and chromatographic system takes at least two hours, and the constant switching of chromatographic systems severely impacts laboratory efficiency and wastes resources, manpower, and space. This situation prevents LC-MS / MS from fully realizing its speed advantage, wasting significant human, material, and time costs in switching between different methods.

[0004] Because hormones are present in extremely low concentrations in biological samples, they are difficult to detect in conjunction with other substances. Current technologies mostly only allow for the separate detection of certain hormones and other analytes, necessitating multiple assays. Existing detection techniques are generally very complex, requiring derivatization pretreatment, which varies depending on the hormone type, resulting in lengthy and cumbersome derivatization processes. Furthermore, most derivatization reagents are highly harmful to the environment and human health.

[0005] On the other hand, many clinical biological samples, such as cerebrospinal fluid, are limited in quantity due to their characteristics and collection conditions, making it impossible to conduct multiple tests. Collecting more samples would cause greater harm to the patient.

[0006] Therefore, there is a clinical need for a simple, safe, rapid method for the combined detection of multiple amino acids, hormones, and ceramides. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for the combined detection of multiple amino acids, hormones, and ceramides using liquid chromatography-tandem mass spectrometry.

[0008] This invention provides a method for the combined detection of multiple amino acids, hormones, and ceramides using liquid chromatography-tandem mass spectrometry. The amino acids include isoleucine, aspartic acid, glutamic acid, methionine, phenylalanine, tryptophan, and kynurenine; the hormones include progesterone, pregnenolone, dihydroprogesterone, tetrahydroprogesterone, allogeneic acid, testosterone, dehydroepiandrosterone, androstenedione (3α,17β), dihydrotestosterone, estriol, and estradiol; the ceramides include ceramide C16 (d18:1 / 16:0), ceramide C18 (d18:1 / 18:0), and ceramide C24 (d18:1 / 24:0); the method includes at least the following steps:

[0009] S1. Standard curve equation I and standard curve equation II are prepared respectively.

[0010] Prepare standard solution I, and use high performance liquid chromatography-mass spectrometry to detect standard solution I to obtain standard curve equation I for calculating hormone and amino acid content;

[0011] Prepare standard solution II, and use high performance liquid chromatography-mass spectrometry to detect standard solution II to obtain standard curve equation II for calculating ceramide content;

[0012] S2. Pre-processing of the sample to be tested, including:

[0013] The sample to be tested is divided into 3 parts: sample a, sample b and sample c;

[0014] Take the mixed amino acid internal standard working solution and the sample to be tested a, add protein precipitant A, mix well and centrifuge, take the supernatant I, add water to dilute to obtain solution I;

[0015] Mix the mixed hormone internal standard working solution and the sample b to be tested, perform solid phase extraction, collect the eluent containing hormone components, dry it and mix it with solution I, and use the supernatant after centrifugation as injection sample I for injection analysis.

[0016] Take the mixed ceramide internal standard working solution and the sample to be tested c, add protein precipitant B, mix well and centrifuge, and take the supernatant as the injection sample II;

[0017] Protein precipitant A is selected from methanol; protein precipitant B is selected from a mixed solvent of methanol and isopropanol in a volume ratio of 1.5 to 2.5:1.

[0018] S3. Detect injection sample I and injection sample II respectively, including:

[0019] High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the contents of sample I and sample II, respectively. The detection results of sample I were substituted into standard curve equation I, and the detection results of sample II were substituted into standard curve equation II to obtain the contents of various amino acids, hormones and ceramides in the test samples.

[0020] Optionally, S1 includes: preparation of internal standard working solution, preparation of standard curve working solution and preparation of standard solution. Preparation of internal standard working solution includes: preparing mixed hormone internal standard working solution, mixed amino acid internal standard working solution and mixed ceramide internal standard working solution respectively. Preparation of standard curve working solution includes: preparing mixed hormone standard curve working solution, mixed amino acid standard curve working solution and mixed ceramide standard curve working solution respectively.

[0021] Optionally, the preparation of standard solution I includes: taking mixed amino acid internal standard working solution and mixed amino acid standard curve working solution of gradient concentration, adding protein precipitant A and mixing well to obtain a mixed solution, adding water to the mixed solution and mixing well to obtain solution I'; taking mixed hormone internal standard working solution and mixed hormone standard curve working solution of gradient concentration, drying and dissolving in solution I'; thus preparing standard solution I of gradient concentration.

[0022] The preparation of standard solution II includes: taking mixed ceramide internal standard working solution, mixed ceramide standard working solution of gradient concentration and protein precipitant B to prepare standard solution II of gradient concentration;

[0023] Standard solution I was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain standard curve equation I; standard solution II was detected using HPLC-MS / MS to obtain standard curve equation II.

[0024] Optionally, in S2, the volume ratio of the mixed amino acid internal standard working solution to the mixed amino acid standard curve working solution is 1:1; the volume ratio of the mixed amino acid internal standard working solution to protein precipitant A is 1:15-20; the volume ratio of the mixed solution to water is 1:8-10; the volume ratio of the mixed hormone internal standard working solution to the mixed hormone standard curve working solution is 1:1; and the volume ratio of the ceramide internal standard working solution, the mixed ceramide standard curve working solution, and protein precipitant B is 1:1:25-35.

[0025] Optionally, in S2, when the sample to be tested is a serum sample, the volume ratio of sample a, sample b, and sample c is 1:20:1.

[0026] After mixing the mixed hormone internal standard working solution and the sample b to be tested, before solid phase extraction, there are steps to add protein precipitant A, mix, centrifuge, and take the supernatant II and add water to reconstitute.

[0027] During the preparation of sample I, the volume ratio of test sample a to mixed amino acid internal standard working solution was 1:1, and the volume ratio of test sample a to protein precipitant A was 1:15–20; the volume ratio of supernatant I to water was 1:8–10; the volume ratio of test sample b to mixed hormone internal standard working solution was 20:1, and the volume ratio of test sample b to protein precipitant A was 1:2–2.5; the volume ratio of supernatant II to water was 1:1–1.2.

[0028] During the preparation of sample II, the volume ratio of test sample c to mixed ceramide internal standard working solution is 1:1, and the volume ratio of test sample c to protein precipitant B is 1:25-35.

[0029] Optionally, when the sample to be tested is a serum sample, the volumes of sample a, sample b, and sample c are 10–20 μL, 200–400 μL, and 10–20 μL, respectively.

[0030] Optionally, when the sample to be tested is a cerebrospinal fluid sample; the volume ratio of sample a, sample b, and sample c is 1:25:5; during the preparation of sample I, the volume ratio of sample a to mixed amino acid internal standard working solution is 2:1, the volume ratio of sample a to protein precipitant A is 2:15-20; the volume ratio of supernatant I to water is 1:8-10; and the volume ratio of sample b to mixed hormone internal standard working solution is 50:1.

[0031] During the preparation of sample II, the volume ratio of test sample c to mixed ceramide internal standard working solution was 10:1, and the volume ratio of test sample c to protein precipitant B was 1:2.5 to 3.5.

[0032] Optionally, the volumes of test sample a, test sample b, and test sample c are 20–30 μL, 500–750 μL, and 100–150 μL, respectively.

[0033] Optionally, when using a high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) system, a pentafluorophenyl column, preferably a Kinetex F5 column, with a packing particle size of 2.6 μm, may be used.

[0034] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0035] This invention enables the simultaneous detection of seven amino acids, eleven hormones, and three ceramides in blood and cerebrospinal fluid, each with significantly different concentrations. Compared to conventional single-method liquid chromatography-mass spectrometry (LC-MS), this invention's combined detection method reduces the number of steps and time required, thus improving detection efficiency. Only one researcher is needed to determine the content of 21 different endogenous compounds through two pretreatment steps, saving on personnel and material costs.

[0036] This invention can detect 21 different endogenous compounds in blood and cerebrospinal fluid in a single sampling process, requiring less sample volume than conventional single liquid chromatography-mass spectrometry (LC-MS) analysis methods, thus increasing patient compliance.

[0037] The standard working solution of this invention requires no pretreatment and has good stability. It is not necessary to prepare a standard working solution for each test; a simple dilution of the standard working solution is sufficient. This can shorten the pretreatment time by at least 30 minutes for each test.

[0038] This invention simplifies the pretreatment steps, eliminating the need for complex techniques such as derivatization, thus shortening the overall pretreatment time. Furthermore, it avoids the use of highly toxic pretreatment reagents, making it safer for the environment and laboratory personnel, and reducing the technical requirements for researchers.

[0039] In the preferred embodiment, this invention uses an isotopic internal standard, which improves the stability and reproducibility of the method. Furthermore, the use of a standard curve for quantification allows for precise quantification of the analyte, improving accuracy and aiding in accurate clinical diagnosis.

[0040] The detection method provided by this invention has high accuracy, with a maximum deviation of no more than ±12% when using known sample concentrations, precision of no more than 12%, and high sensitivity. Attached Figure Description

[0041] Figure 1 This is a chromatogram of hormones and amino acids in the standard solution in an embodiment of the present invention;

[0042] Figure 2 This is a chromatogram of ceramide in the standard solution in an embodiment of the present invention;

[0043] Figure 3 This is a chromatogram of hormones and amino acids in a serum sample from Example 1 of the present invention;

[0044] Figure 4 This is a chromatogram of ceramide in a serum sample from Example 1 of the present invention;

[0045] Figure 5 This is a chromatogram of hormones and amino acids in a cerebrospinal fluid sample from Example 3 of the present invention;

[0046] Figure 6 This is a chromatogram of ceramides in a cerebrospinal fluid sample from Example 3 of the present invention;

[0047] Figure 7 , 8 This is a chromatogram of hormones and amino acids in a serum sample from Example 5 of the present invention;

[0048] Figure 9 , 10 This is a chromatogram of ceramide in a serum sample from Example 6 of the present invention;

[0049] Figure 11 This is a chromatogram of ceramide in a serum sample from Comparative Example 1 of this invention;

[0050] Figure 12 , Figure 13 The chromatograms of hormones P and E2 in Comparative Example 4 of this invention are shown. Detailed Implementation

[0051] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0052] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0053] This invention proposes a method for the combined detection of multiple amino acids, hormones, and ceramides using liquid chromatography-tandem mass spectrometry. The amino acids include isoleucine, aspartic acid, glutamic acid, methionine, phenylalanine, tryptophan, and kynurenine; the hormones include progesterone, pregnenolone, dihydroprogesterone, tetrahydroprogesterone, allopregnenolone, testosterone, dehydroepiandrosterone, androstenedione (3α,17β), dihydrotestosterone, estriol, and estradiol; the ceramides include ceramide C16 (d18:1 / 16:0), ceramide C18 (d18:1 / 18:0), and ceramide C24 (d18:1 / 24:0). The test samples for this invention can be of various types, including blood samples and cerebrospinal fluid samples. Blood samples include serum samples, plasma samples, and anticoagulated whole blood, with serum samples being preferred. The specific test substances and their abbreviations in this invention are shown in Table 1.

[0054] Table 1

[0055]

[0056] This invention divides the sample into three portions for the extraction of amino acids, hormones, and ceramides, respectively. Innovatively, this invention combines amino acids and hormones with significantly different content and polarity levels into a single injection sample after pretreatment, enabling the simultaneous detection of amino acids and hormones using the same detection system, thus improving detection efficiency. Another sample, after pretreatment, is used for the detection of ceramides. Although ceramide detection is not performed in the same injection sample as amino acids and hormones, the detection of ceramides uses the same chromatographic column as amino acid and hormone detection, eliminating the need for additional time to change the chromatographic system. Therefore, this invention's method for the combined detection of these three substances improves detection efficiency, allowing the same person to perform the detection, thus saving personnel costs.

[0057] The detection method of this invention specifically includes three steps: S1: preparation of standard curve, S2: sample pretreatment, and S3: detection of the processed sample using high performance liquid chromatography-mass spectrometry.

[0058] In this embodiment of the invention, step S1 specifically includes: preparing standard curve equation I and standard curve equation II respectively. Standard curve equation I is used to calculate hormone and amino acid content, and standard curve equation II is used to calculate ceramide content. S1 specifically includes the following steps:

[0059] S11. Solution preparation, specifically including: preparation of internal standard working solution, preparation of standard curve working solution and preparation of standard solution;

[0060] S111. Preparation of internal standard working solutions: Prepare mixed hormone internal standard working solution, mixed amino acid internal standard working solution, and mixed ceramide internal standard working solution respectively.

[0061] Among them, the mixed hormone internal standard working solution contains isotopic internal standards of 10 hormones, the mixed amino acid internal standard working solution contains isotopic internal standards of 7 amino acids, and the mixed ceramide internal standard working solution contains isotopic internal standards of 3 ceramides.

[0062] S112. Preparation of standard curve working solutions: Prepare mixed hormone standard curve working solutions, mixed amino acid standard curve working solutions, and mixed ceramide standard curve working solutions respectively.

[0063] Among them, the mixed hormone standard working solution contains 11 hormone standards, the mixed amino acid standard working solution contains 7 amino acid standards, and the mixed ceramide standard working solution contains 3 ceramide standards.

[0064] In one specific embodiment of the present invention, the preparation method of the internal standard working solution is as follows: Each compound internal standard is accurately weighed, dissolved in an internal standard solution, and stored at -20°C to obtain an internal standard stock solution. Specifically, the amino acid internal standard is prepared using a 65%–75% (preferably 70%) methanol-water solution; the hormone is prepared using methanol; and the ceramide is prepared using a mixed solvent of methanol and dichloromethane in a volume ratio of 3.5–4.5:1 (preferably 4:1). The stock solutions of each compound internal standard are mixed and diluted with a diluent to the concentrations shown in Table 2 to obtain the internal standard working solution. The diluent is a 65%–75% (preferably 70%) methanol-water solution.

[0065] Table 2

[0066]

[0067] In one specific embodiment of the present invention, the standard curve working solution is prepared by: accurately weighing each compound standard, using a standard solution to a certain concentration, and storing it as a standard stock solution at -20°C. The solution for dissolving the amino acid standard can be an aqueous solution containing 65%–75% (preferably 70%) methanol by volume; the hormone standard is prepared using methanol as the stock solution; and the ceramide standard is prepared using a mixed solvent of methanol and dichloromethane at a volume ratio of 3.5–4.5:1 (preferably 4:1). The stock solutions of each compound standard are mixed and diluted to the concentrations shown in Tables 3–5 to obtain three standard curve working solutions. The diluent is an aqueous solution of methanol with a volume percentage of 65%–75% (preferably 70%).

[0068] Table 3

[0069]

[0070] Table 4

[0071] level Glu / Asp / Phe / Ile / Try / Met(μg / mL) Kyn (μg / mL) L7 80 5 L6 40 2.5 L5 20 1.25 L4 8 0.5 L3 4 0.25 L2 2 0.125 L1 0.8 0.05

[0072] Table 5

[0073] level C16(d18:1 / 16:0), C18(d18:1 / 18:0), and C24(d18:1 / 24:0) (ng / mL) L9 5000 L8 2000 L7 1000 L6 500 L5 200 L4 100 L3 50 L2 20 L1 10

[0074] S113. The preparation of standard solution I includes: taking mixed amino acid internal standard working solution and mixed amino acid standard curve working solutions of different concentrations, adding protein precipitant A and mixing well to obtain a mixed solution, adding water to the mixed solution and mixing well to obtain solution I'; taking mixed hormone internal standard working solution and mixing with mixed hormone standard curve working solutions of different concentrations, drying and then mixing with solution I'; preparing standard solution I with gradient concentrations;

[0075] The preparation of standard solution II includes: taking mixed ceramide internal standard working solution, mixed ceramide standard working solutions of different concentrations and protein precipitant B to prepare standard solution II with gradient concentrations.

[0076] S12. Standard solution I was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of standard solutions with gradient concentrations and internal standard solutions. The peak areas of standard solution I and internal standard were obtained from the chromatograms. The ratio of the peak area of ​​standard solution I to the peak area of ​​internal standard was used as the ordinate y1 of the standard curve equation, and the ratio of the concentration in the standard working solution to the concentration in the internal standard working solution was used as the abscissa x1 of the standard curve equation. Linear regression was performed on the data of different concentrations obtained above to fit the standard curve equation y1=a×x1+b, and the linear equation coefficients a and b were obtained.

[0077] Standard solution II was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of standard solutions with gradient concentrations and internal standard. The peak areas of standard solution II and internal standard were obtained from the chromatograms. The ratio of the peak area of ​​standard solution II to the peak area of ​​internal standard was used as the ordinate (y1) of the standard curve equation, and the ratio of the concentration of the standard working solution to the concentration of the internal standard working solution was used as the abscissa (x1) of the standard curve equation. Linear regression was performed on the data of different concentrations obtained above to fit the standard curve equation y1 = c × x1 + d, and the linear equation coefficients c and d were obtained.

[0078] As a preferred embodiment of the present invention, in S113, the mixing conditions are vortex mixing at 1000-2500 rpm for 30 s to 1 min. According to this step, the standard curve preparation method of the present invention does not require pretreatment, only vortex mixing, thus simplifying the standard curve preparation steps and shortening the standard curve preparation time.

[0079] As a preferred embodiment of the present invention, in S113, in the preparation of standard solution I: the volume ratio of the mixed amino acid internal standard working solution to the mixed amino acid standard curve working solution is 1:1; the volume ratio of the mixed amino acid internal standard working solution to protein precipitant A is 1:15-20, preferably 1:18; the volume ratio of the mixed solution to water is 1:8-10, preferably 1:9; the volume ratio of the mixed hormone internal standard working solution to the mixed hormone standard curve working solution is 1:1. Protein precipitant A is selected from methanol.

[0080] As a preferred embodiment of the present invention, in S113, the volume ratio of the mixed ceramide internal standard working solution, the mixed ceramide standard working solution, and the protein precipitant B in the preparation of standard solution II is 1:1:25-35, preferably 1:1:30. The protein precipitant B is selected from a mixed solvent of methanol and isopropanol with a volume ratio of 1.5-2.5:1 (preferably 2:1).

[0081] As a specific embodiment of the present invention, the standard solution can be calibrated using the following method:

[0082] Take 10 μL of the mixed hormone internal standard working solution and 10 μL of the mixed hormone standard working solution and place them in a 1.5 mL centrifuge tube 1, and blow them dry with nitrogen.

[0083] Take 10 μL of the mixed amino acid internal standard working solution, 10 μL of the mixed amino acid standard curve working solution, and 180 μL of methanol and place them in a 1.5 mL centrifuge tube 2. Vortex at 1000–2500 rpm for 30 s–1 min. Then, take 20 μL of the mixture and add 180 μL of water. Vortex at 1000–2500 rpm for 30 s–1 min. Finally, take 50 μL of the mixture and place it in a dried 1.5 mL centrifuge tube 1. Mix to prepare 3–10 standard solutions of the same concentration, preferably 7 concentration gradients.

[0084] Take 10 μL of mixed ceramide internal standard working solution, 10 μL of mixed ceramide standard working solution and 300 μL of protein precipitant B into a 1.5 mL centrifuge tube 3, and mix to prepare 5 to 12 standard solutions of the same concentration, preferably 9 different concentration standard solutions.

[0085] As a preferred technical solution for the pretreatment of the sample to be tested in the embodiments of the present invention, it specifically includes:

[0086] S21. Divide the sample to be tested into 3 parts: sample a, sample b and sample c;

[0087] S22. Take the mixed amino acid internal standard working solution and the sample to be tested a, add protein precipitant A, mix well and centrifuge, take the supernatant I, add water to dilute to obtain solution I containing amino acids; take the mixed hormone internal standard working solution and the sample to be tested b, mix well, perform solid phase extraction, collect the eluent containing hormone components, dry it and mix it with solution I, centrifuge, take the supernatant as injection sample I, and use it for injection analysis;

[0088] S23. Take the mixed ceramide internal standard working solution and the sample to be tested c, add protein precipitant B, mix well and centrifuge, and take the supernatant as the injection sample II.

[0089] Similar to the standard curve preparation process, protein precipitant A is selected from methanol; protein precipitant B is selected from a mixed solvent of methanol:isopropanol with a volume ratio of 1.5 to 2.5:1, and more preferably, the volume ratio of methanol:isopropanol is 2:1.

[0090] Specifically, in S21, this embodiment of the invention divides the sample to be tested into three parts: sample a, sample b, and sample c. Sample a is used to detect amino acids in serum and cerebrospinal fluid, sample b is used to detect hormones in serum and cerebrospinal fluid, and sample c is used to detect ceramides in serum and cerebrospinal fluid. Because the content and properties of the three types of analytes differ significantly in the sample, separate extraction is employed to ensure effective detection.

[0091] As a further preferred embodiment of the pretreatment step, in the preparation of sample I, the mixing conditions are 2000-2500 rpm for 3-5 min, and the centrifugation conditions are 12000-14000 rpm for 5-10 min. Preferably, the mixing conditions are 2500 rpm for 5 min, and the centrifugation conditions are 14000 rpm for 10 min.

[0092] As a further preferred embodiment of the pretreatment step, solid-phase extraction (SPE) is performed using an SPE plate loaded with an Oasis PRiME pretreatment column during the preparation of sample I. Through research, this embodiment of the invention found that due to the low hormone content, other extraction methods such as protein precipitation and liquid-liquid extraction cannot meet the sensitivity and accuracy requirements. Therefore, this embodiment of the invention employs solid-phase extraction. Research has shown that acetonitrile as the eluent can completely elute the hormone. Before collecting the eluent, impurities are removed by sequential rinsing with water, a 20%–40% (v / v) methanol aqueous solution, and a 5%–15% (v / v) acetonitrile aqueous solution. This combination of rinsing and elution steps achieves the maximum extraction recovery rate. More preferably, before collecting the eluent, rinsing is performed sequentially with water, a 30% (v / v) methanol aqueous solution, and a 10% (v / v) acetonitrile aqueous solution.

[0093] As a further preferred embodiment of the pretreatment step, in the preparation of sample II, the mixing conditions are 2000-2500 rpm for 3-5 min, and the centrifugation conditions are 12000-14000 rpm for 5-10 min. Preferably, the mixing conditions are 2500 rpm for 5 min, and the centrifugation conditions are 14000 rpm for 5 min.

[0094] In this embodiment of the invention, the sample to be tested can be a serum sample or a cerebrospinal fluid sample. Since serum samples are characterized by a high content of protein impurities, while cerebrospinal fluid samples have a low protein content and a low concentration of the analyte, this invention further improves the specific pretreatment conditions according to the characteristics of different samples.

[0095] When the sample to be tested is a serum sample, the specific pretreatment conditions are as follows:

[0096] The volume ratio of test sample a, test sample b, and test sample c is 1:20:1. Since the concentration of hormones in serum is low, this invention increases the sample volume used for hormone detection, allowing for the collection of more analytes and improving detection sensitivity. In one specific embodiment, the volumes of test sample a, test sample b, and test sample c can be 10–20 μL, 200–400 μL, or 10–20 μL, preferably 10 μL, 200 μL, and 10 μL.

[0097] Because serum contains many protein impurities, it is necessary to add protein precipitant A not only to the test sample a used for amino acid detection, but also to the test sample b used for hormone detection for protein precipitation. The specific steps are as follows: take the mixed hormone internal standard working solution and the test sample b, mix them well, add protein precipitant A, mix well, centrifuge, take the supernatant II and add water to mix well.

[0098] Specifically, in the preparation of sample I, the volume ratio of test sample a to mixed amino acid internal standard working solution is 1:1, the volume ratio of test sample a to protein precipitant A is 1:15-20, preferably 1:16-19, more preferably 1:18; the volume ratio of supernatant I to water is 1:8-10, preferably 1:9. In a specific embodiment of sample I preparation, the volumes of test sample a, mixed amino acid internal standard working solution, and protein precipitant A are 10 μL, 10 μL, and 180 μL, respectively; the volumes of supernatant I and water are 20 μL and 180 μL, respectively.

[0099] The volume ratio of test sample b to mixed hormone internal standard working solution is 20:1, and the volume ratio of test sample b to protein precipitant is 1:2 to 2.5, preferably 1:2.25; the volume ratio of supernatant II to water is 1:1 to 1.2, preferably 1:1. In a specific embodiment of sample preparation, the volumes of test sample b, mixed hormone internal standard working solution, and protein precipitant are 200 μL, 10 μL, and 450 μL, respectively; the volumes of supernatant II and water are 450 μL and 450 μL, respectively. Specifically, in the preparation of sample II, the volume ratio of test sample c to mixed ceramide internal standard working solution is 1:1, and the volume ratio of test sample c to protein precipitant B is 1:25 to 35, preferably 1:30. In a specific embodiment of sample II preparation, the volumes of test sample c, mixed ceramide internal standard working solution, and protein precipitant B are 10 μL, 10 μL, and 300 μL, respectively.

[0100] When the sample to be tested is a cerebrospinal fluid sample, the specific pretreatment conditions are as follows:

[0101] Based on the content characteristics of various substances in cerebrospinal fluid, this application implements a volume ratio of test sample a, test sample b, and test sample c of 1:25:5. Since the content of hormones in serum is low and the content of ceramides is also low, this invention increases the sample volume used for hormone detection, and also increases the sample volume used for ceramide detection to a certain extent, thereby collecting more test substances and improving detection sensitivity. In one specific embodiment, the volume ratio of test sample a, test sample b, and test sample c is 20–30 μL, 500–750 μL, and 100–150 μL, preferably 20 μL, 500 μL, and 100 μL.

[0102] In the preparation of sample I, the volume ratio of test sample a to mixed amino acid internal standard working solution is 2:1, the volume ratio of test sample a to protein precipitant A is 2:15-20, preferably 2:16-18, more preferably 2:17; the volume ratio of supernatant I to water is 1:8-10, preferably 1:9. Compared with the pretreatment conditions for serum samples, since the content of the analyte in cerebrospinal fluid is low, this invention, through repeated experiments, has determined that at this specific ratio, the added volumes of internal standard and protein precipitant A are both low, which not only ensures the protein precipitation effect but also avoids further dilution of the test sample, ensuring it is under relatively concentrated conditions. In a specific embodiment, the volumes of test sample a, mixed amino acid internal standard working solution, and protein precipitant A are 20 μL, 10 μL, and 170 μL, respectively; the volumes of supernatant I and water are 20 μL and 180 μL, respectively.

[0103] The volume ratio of test sample b to the mixed hormone internal standard working solution is 50:1. During cerebrospinal fluid pretreatment, due to the low protein content and even lower hormone levels in the cerebrospinal fluid, this embodiment of the invention found that, in the above solution system, no protein precipitant needs to be added to test sample b for hormone detection; simply mixing the mixed hormone internal standard working solution with test sample b and then performing solid-phase extraction is sufficient. In one specific embodiment, the volumes of test sample b and the mixed hormone internal standard working solution are 500 μL and 10 μL, respectively.

[0104] Specifically, in the preparation of sample II, the volume ratio of the test sample c to the mixed ceramide internal standard working solution is 10:1, and the volume ratio of the test sample c to protein precipitant B is 1:2.5 to 3.5; preferably 1:3. In one specific embodiment, the volumes of the test sample c and the mixed ceramide internal standard working solution are 100 μL and 10 μL, respectively, and the volumes of the test sample c and the protein precipitant B are 100 μL and 300 μL, respectively.

[0105] As a preferred technical solution for sample detection in this invention, it specifically includes:

[0106] S3. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the injected sample I and the injected sample II respectively. The detection results of the injected sample I were substituted into the standard curve equation I to obtain the amino acid and hormone content in the sample to be tested. The detection results of the injected sample II were substituted into the standard curve equation II to obtain the ceramide content in the sample to be tested.

[0107] As a further preferred implementation method for on-machine detection, a pentafluorophenyl column, preferably a Kinetex F5 column, is used when analyzing with a high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) system. The packing material has a particle size of 2.6 μm and a diameter of 3.0 × 50 mm. Compared with other chromatographic systems, the pentafluorophenyl column can simultaneously meet the analysis requirements for amino acids, hormones, and ceramides, ensuring detection results without the need to change the chromatographic system, thus improving detection efficiency.

[0108] As a further preferred implementation method for on-machine detection, in S1 and S3, when using a high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument to detect standard solution I and injected sample I:

[0109] The chromatographic analysis conditions are as follows:

[0110] Mobile phase: Phase A: Water containing 0.4–0.5 mmol / L ammonium fluoride; Phase B: Methanol;

[0111] The flow rate is 0.4–0.5 mL / min, preferably 0.5 mL / min;

[0112] Gradient elution conditions:

[0113] 0.00–1.00 min, phase A uses concentration A1, and phase B uses concentration B1;

[0114] 1.01–3.00 min, phase A uses concentration A2, and phase B uses concentration B2;

[0115] 3.00–3.50 min, phase A changes from concentration A2 to concentration A3 at a constant rate, and phase B changes from concentration B2 to concentration B3 at a constant rate.

[0116] 3.50–12.00 min, concentration A3 changes uniformly to concentration A4; Phase B: concentration B3 changes uniformly to concentration B4.

[0117] From 12.01 to 12.50 min, the concentration of phase A4 was changed at a constant rate to 0; in phase B, the concentration of phase B4 was changed at a constant rate to 100%.

[0118] 12.50–13.00 min, Phase A is 0%; Phase B is 100%;

[0119] From 13.01 to 15.00 min, phase A uses concentration A1, and phase B uses concentration B1;

[0120] Concentration A1 is selected from 95% to 90%, concentration B1 is selected from 5% to 10%, and concentration A1 + concentration B1 = 100%;

[0121] Concentration A2 is selected from 85% to 75%, concentration B2 is selected from 15% to 25%, and concentration A2 + concentration B2 = 100%;

[0122] Concentration A3 is selected from 60% to 65%, concentration B3 is selected from 35% to 40%, and concentration A3 + concentration B3 = 100%;

[0123] Concentration A4 is selected from 10% to 20%, concentration B4 is selected from 80% to 90%, and concentration A4 + concentration B4 = 100%.

[0124] The mass spectrometry analysis conditions were as follows: electrospray ionization (ESI) source, simultaneous acquisition in positive and negative ion modes, and multiple reaction monitoring (MRM). Ion source parameters are shown in Table 5, and ion pair parameters are shown in Table 6.

[0125] Table 5

[0126]

[0127] Table 6

[0128]

[0129]

[0130] As a further preferred implementation method for on-machine detection, in S1 and S3, the chromatographic analysis conditions for detecting standard solution II and injected sample II using high-performance liquid chromatography-mass spectrometry are as follows:

[0131] Mobile phase: Phase A: an aqueous solution containing 0.1% to 0.2% formic acid and 1 to 2 mmol / L ammonium acetate by volume; Phase B: a mixed solvent of methanol and isopropanol by volume ratio of 1.5 to 2.5:1 (preferably 2:1);

[0132] The flow rate is 0.5–0.6 mL / min, preferably 0.5 mL / min;

[0133] Gradient elution conditions:

[0134] 0.00–1.50 min, phase A uses concentration A1, and phase B uses concentration B1;

[0135] 1.50–2.00 min, phase A changes from concentration A1 to concentration A2 at a constant rate, and phase B changes from concentration B1 to concentration B2 at a constant rate.

[0136] 2.00–6.00 min, phase A uses concentration A2, and phase B uses concentration B2;

[0137] 6.01–7.50 min, phase A uses concentration A1, and phase B uses concentration B1;

[0138] Concentration A1 is selected from 15% to 20%, concentration B1 is selected from 80% to 85%, and concentration A1 + concentration B1 = 100%.

[0139] Concentration A2 is selected from 0% to 5%, concentration B2 is selected from 95% to 100%, and concentration A2 + concentration B2 = 100%.

[0140] The mass spectrometry analysis conditions were as follows: electrospray ionization (ESI) source, positive ion mode acquisition, and multiple reaction monitoring (MRM). Ion source parameters are shown in Table 7, and ion pair parameters are shown in Table 8.

[0141] Table 7

[0142]

[0143] Table 8

[0144]

[0145] Example 1

[0146] This embodiment illustrates a liquid chromatography-mass spectrometry (LC-MS) method for detecting the levels of 21 disease-related endogenous compounds in blood, including the following steps:

[0147] (I) Solution Preparation

[0148] Preparation of standard working solutions: Accurately weigh each compound standard, use the standard solution to a certain concentration, and store the solution at -20℃. Mix and dilute each compound standard stock solution to the concentrations shown in the table below to obtain standard working solutions for 21 endogenous compounds. The diluent used is 70% methanol-water. The prepared standard working solutions are shown in Tables 3 to 5.

[0149] Preparation of internal standard working solutions: Accurately weigh the internal standard for each compound, and use the internal standard solution to a certain concentration, storing it as an internal standard stock solution at -20℃. Take the stock solutions of each compound's internal standard and mix them, then dilute to the concentrations shown in the table below to obtain the internal standard working solutions. The diluent used is 70% methanol-water. The prepared internal standard working solutions are shown in Table 2.

[0150] (II) Standardization of Standard Solutions

[0151] Take 10 μL of the mixed hormone internal standard working solution and 10 μL of the mixed hormone standard working solution and place them in a 1.5 mL centrifuge tube 1, and blow them dry with nitrogen.

[0152] Take 10 μL of the mixed amino acid internal standard working solution, 10 μL of the mixed amino acid standard curve working solution, and 180 μL of methanol and place them in a 1.5 mL centrifuge tube 2. Vortex at 2500 rpm for 30 s. Then, take 20 μL of the mixture and add 180 μL of water. Vortex at 2500 rpm for 30 s. Finally, take 50 μL of the mixture and place it in a dried 1.5 mL centrifuge tube 1. Mix to prepare 8 standard solutions of different concentrations.

[0153] Take 10 μL of mixed ceramide internal standard working solution, 10 μL of mixed ceramide standard working solution and 300 μL of protein precipitant B (methanol and isopropanol volume ratio of 2:1) in a 1.5 mL centrifuge tube 3 and mix them to prepare 9 standard solutions of different concentrations II.

[0154] Standard solution I was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of standard solutions and internal standards at different concentrations. The peak areas of standard solution I and internal standards were obtained from the chromatograms. The ratio of the peak area of ​​standard solution I to the peak area of ​​internal standards at different concentrations was used as the ordinate y1 of the standard curve equation, and the ratio of the concentration of the standard working solution to the concentration of the internal standard working solution was used as the abscissa x1 of the standard curve equation. Linear regression was performed on the data of different concentrations obtained above to fit the standard curve equation y1=a×x1+b, and the linear equation coefficients a and b were obtained.

[0155] Standard solution II was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of standard solutions and internal standards at different concentrations. The peak areas of standard solution II and internal standards were obtained from these chromatograms. The ratio of the peak area of ​​standard solution II to the peak area of ​​internal standards at different concentrations was used as the ordinate (y1) of the standard curve equation, and the ratio of the concentration of the standard working solution to the concentration of the internal standard working solution was used as the abscissa (x1) of the standard curve equation. Linear regression was performed on the data of different concentrations obtained above to fit the standard curve equation y1 = c × x1 + d, and the linear equation coefficients c and d were obtained.

[0156] (III) Sample processing

[0157] 1. Pretreatment methods for serum hormones and amino acids:

[0158] Take 10 μL of mixed amino acid internal standard working solution and 10 μL of sample a to be tested, add 180 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 20 μL of supernatant I, add 180 μL of water and mix well to obtain solution I.

[0159] Take 10 μL of the mixed hormone internal standard working solution and 200 μL of the test sample b, add 450 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 450 μL of the supernatant and mix with 450 μL of water, then add it to an activated and equilibrated SPE plate (Oasis PRiME), and wash successively with water, 30% (v / v) methanol aqueous solution, and 10% (v / v) acetonitrile aqueous solution. Finally, elute with 50 μL of acetonitrile, collect the eluent, and dry it under nitrogen. Reconstitute with solution I. After centrifugation at 4000 rpm for 5 min, the supernatant is used as injection sample I for injection analysis.

[0160] 2. Pretreatment method for serum ceramides:

[0161] Take 10 μL of mixed ceramide internal standard working solution and 10 μL of the sample to be tested, add 300 μL of precipitant B (methanol:isopropanol volume ratio of 2:1), mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 5 min, and take the supernatant as injection sample II for injection analysis.

[0162] (iv) Detection of the sample to be tested

[0163] The injected sample I was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of the target analyte and the corresponding internal standard. The peak areas of the target analyte and the corresponding internal standard were obtained from the chromatograms. The ratio y1 of the peak area of ​​the target analyte and the corresponding internal standard was substituted into the standard curve equation y1 = a*x1 + b from step (I) above. The relative concentration x1 of the target analyte and the corresponding internal standard in the sample was calculated. The concentration of the internal standard working solution was known. Thus, the concentration of the 18 analytes in the body fluid sample was calculated.

[0164] The injected sample II was detected using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to obtain chromatograms of the target analyte and the corresponding internal standard. The peak areas of the target analyte and the corresponding internal standard were obtained from the chromatograms. The ratio y1 of the peak area of ​​the target analyte and the corresponding internal standard was substituted into the standard curve equation y1 = c*x1 + d in step (I) above. The relative concentration x1 of the target analyte and the corresponding internal standard in the sample was calculated. The concentration of the internal standard working solution was known. Thus, the concentrations of the three analytes in the body fluid sample were calculated.

[0165] 1. Detection conditions for sample I:

[0166] The chromatographic column used for the chromatographic analysis was a Kinetex F5 (2.6 μm, 3.0 × 50 mm).

[0167] The mobile phases were: Phase A: water containing 0.4 mmol / L ammonium fluoride; Phase B: methanol.

[0168] The flow rate was 0.5 mL / min.

[0169] The gradient elution conditions are shown in Table 9:

[0170] Table 9

[0171] time A％ B％ 0.00 95.00 5.00 1.00 95.00 5.00 1.01 85.00 15.00 3.00 85.00 15.00 3.50 65.00 35.00 12.00 20.00 80.00 12.50 0.00 100.00 13.00 0.00 100.00 13.01 95.00 1.00 15.00 95.00 1.00

[0172] The mass spectrometry conditions used were electrospray ionization (ESI) with simultaneous acquisition in both positive and negative ion modes and multiple reaction monitoring (MRM). The ion source parameters are shown in Table 5, and the ion pair parameters are shown in Table 6.

[0173] The obtained chromatogram is as follows Figure 1 , Figure 3 As shown. Figure 1 This is a chromatogram of hormones and amino acids in the standard solution in an embodiment of the present invention. Figure 3 This is a chromatogram of hormones and amino acids in serum in an embodiment of the present invention.

[0174] 2. Detection conditions for sample II:

[0175] The chromatographic column used for the chromatographic analysis was a Kinetex F5 (2.6 μm, 3.0 × 50 mm).

[0176] The mobile phase was an aqueous solution containing 0.1% formic acid and 2 mmol / L ammonium acetate by volume; Phase B was a mixed solvent of methanol and isopropanol in a volume ratio of 2:1.

[0177] Flow rate: 0.5 mL / min;

[0178] The gradient elution conditions are shown in Table 10:

[0179] Table 10

[0180] Time A％ B％ 0.00 15.00 85.00 1.50 15.00 85.00 2.00 5.00 95.00 6.00 5.00 95.00 6.01 15.00 85.00 7.50 15.00 85.00

[0181] The mass spectrometry analysis conditions were as follows: electrospray ionization (ESI) source, positive ion mode acquisition, and multiple reaction monitoring (MRM). Ion source parameters are shown in Table 7, and ion pair parameters are shown in Table 8.

[0182] The obtained chromatogram is as follows Figure 2 , Figure 4 As shown. Figure 2 This is a chromatogram of ceramide in the standard solution in an embodiment of the present invention. Figure 4 This is a chromatogram of ceramide in serum in an embodiment of the present invention.

[0183] Example 2

[0184] The technical method in Example 1 is demonstrated as follows:

[0185] I. Linearity of the Method

[0186] According to the determination conditions in Example 1, the concentrations were measured from low to high. Standard curves were obtained by plotting the ratio of the peak area of ​​the 21 analytes to the peak area of ​​the internal standard versus the ratio of the concentration of the 21 analytes to the concentration of the internal standard. The results showed that the concentrations of the 21 analytes were within the linear range, indicating good linearity and a correlation coefficient R0. 2 >0.99. The concentrations of the 21 analytes within the linear range are shown in Table 11:

[0187] Table 11

[0188]

[0189] According to the data shown in Table 11, the detection method of the present invention has a wide linear range, exhibits good linearity within the aforementioned linear range, and has a correlation coefficient R. 2 >0.99.

[0190] II. Recovery and Precision of the Method

[0191] The standard working solution was prepared into three concentrations (low, medium, and high) for spiked recovery and precision experiments, and the results were determined according to the method in Example 1. The recovery and precision are shown in Tables 12 to 14.

[0192] Table 12

[0193]

[0194] Table 13

[0195]

[0196]

[0197] Table 14

[0198]

[0199] III. Limit of Quantitation and Limit of Detection of this Method

[0200] Low-concentration serum samples were taken separately, and the limits of quantitation and detection were tested. The limit of detection was three times the signal-to-noise ratio, and the limit of quantitation was 10 times the signal-to-noise ratio and the coefficient of variation of the quantitative results of 6 consecutive tests was not less than 20%. The determination was carried out according to the method of Example 1. The limits of quantitation and detection are shown in Tables 15 to 17.

[0201] Table 15

[0202] Unit: ng / mL C16 C18 C24 LOQ 10.00 10.00 10.00 LOD 3.00 3.00 3.00

[0203] Table 16

[0204] Unit: ng / mL ILE ASP GLU MET PHE TRY KYN LOQ 7.26 7.01 9.32 7.95 6.95 7.47 0.50 LOD 2.18 2.10 2.80 2.38 2.09 2.24 0.15

[0205] Table 17

[0206] Unit: ng / mL DHEA DHP AP IP 3α-diol P PE T DHT E3 E2 LOQ 0.958 0.906 0.935 0.938 2.500 0.017 0.95 0.019 0.198 0.193 0.200 LOD 0.287 0.272 0.281 0.281 0.788 0.005 0.285 0.006 0.059 0.058 0.06

[0207] Based on the above verification experiments, the recovery rate, precision, and sensitivity of the detection method in Example 1 all meet the requirements. The method in Example 1 can detect the content of 21 endogenous compounds in body fluids with good precision and high spike recovery rate, thus improving the accuracy and sensitivity of the detection results.

[0208] Example 3

[0209] This embodiment illustrates a method for detecting 21 endogenous substances in cerebrospinal fluid:

[0210] (a) Solution preparation, same as in Example 1;

[0211] (ii) Standardization of the standard solution, same as in Example 1;

[0212] (III) Sample processing

[0213] 1. Pretreatment methods for cerebrospinal fluid hormones and amino acids:

[0214] Take 10 μL of the mixed amino acid internal standard working solution and 20 μL of the sample to be tested (a), add 170 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 20 μL of supernatant I, add 180 μL of water and mix well. This yields a mixed solution 2 containing amino acids.

[0215] Take 10 μL of the mixed hormone internal standard working solution and 500 μL of the test sample b, mix at 2500 rpm for 1 min, and add to the activated and equilibrated SPE plate (Oasis PRiME). Elute sequentially with water, 30% methanol aqueous solution, and 10% acetonitrile aqueous solution. Finally, elute with 50 μL of acetonitrile, collect the eluent, and dry it under nitrogen. Reconstitute with mixed solution 1 containing amino acids. Centrifuge at 14000 rpm for 10 min, and use the supernatant as injection sample I for analysis.

[0216] 2. Pretreatment methods for cerebrospinal fluid ceramides:

[0217] Take 10 μL of mixed ceramide internal standard working solution and 100 μL of the sample to be tested, add 300 μL of precipitant (methanol:isopropanol volume ratio = 2:1), mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 5 min, and take 100 μL of supernatant as injection sample II for injection analysis.

[0218] (iv) Testing of the sample to be tested, same as in Example 1.

[0219] The obtained chromatogram is as follows Figure 1 , Figure 2 , Figure 5 , Figure 6 As shown. Figure 1 This is a chromatogram of hormones and amino acids in standard working solution L4 in an embodiment of the present invention; Figure 2 This is a chromatogram of ceramide in standard working solution L4 in an embodiment of the present invention; Figure 5 This is a chromatogram of hormones and amino acids in cerebrospinal fluid in an embodiment of the present invention; Figure 6 This is a chromatogram of ceramides in cerebrospinal fluid in an embodiment of the present invention.

[0220] Example 4

[0221] The technical method in this embodiment 3 is demonstrated as follows:

[0222] I. Linearity of the Method

[0223] According to the determination conditions in Example 3, the concentrations were measured from low to high. A standard curve was obtained by plotting the ratio of the peak area of ​​the 21 analytes to the peak area of ​​the internal standard versus the ratio of the concentration of the 21 analytes to the concentration of the internal standard. The results showed that the concentrations of the 21 analytes were within the linear range, indicating good linearity and a correlation coefficient R0. 2 >0.99. The concentrations of the 21 analytes within the linear range are shown in Table 18:

[0224] Table 18

[0225]

[0226] According to the data shown in Table 18, the detection method of the present invention has a wide linear range, exhibits good linearity within the aforementioned linear range, and has a correlation coefficient R. 2 >0.99.

[0227] II. Recovery and Precision of the Method

[0228] The standard working solution was prepared into three concentrations (low, medium, and high) for spike recovery and precision experiments, and the results were determined according to the method in Example 3. The recovery and precision are shown in Tables 19 to 21.

[0229] Table 19

[0230]

[0231] Table 20

[0232]

[0233] Table 21

[0234]

[0235] III. Limit of Quantitation and Limit of Detection of this Method

[0236] Low-concentration cerebrospinal fluid samples were taken and subjected to limit of quantitation and limit of detection experiments. The limit of detection was three times the signal-to-noise ratio, and the limit of quantitation was 10 times the signal-to-noise ratio, with a coefficient of variation of not less than 20% for six consecutive quantitative results. The determination was carried out according to the method in Example 3. The limits of quantitation and limits of detection are shown in Tables 22 to 24.

[0237] Table 22

[0238] Unit: ng / mL C16 C18 C24 LOQ 1.00 1.00 1.00 LOD 0.30 0.30 0.30

[0239] Table 23

[0240] Unit: ng / mL ILE ASP GLU MET PHE TRY KYN LOQ 3.63 3.50 4.66 3.97 3.48 3.74 0.25 LOD 1.09 1.05 1.40 1.19 1.04 1.12 0.08

[0241] Table 24

[0242] Unit: ng / mL DHEA DHP AP IP DHA LOQ 0.479 0.453 0.468 0.469 1.250 LOD 0.144 0.136 0.140 0.141 0.394 PE T DHT E3 E2 P 0.475 0.01 0.099 0.097 0.100 0.009 0.143 0.003 0.030 0.029 0.03 0.003

[0243] Based on the above verification experiments, the recovery rate, precision, and sensitivity of the method in Example 3 all meet the requirements. The method can detect the content of 21 endogenous compounds in body fluids with good precision and high spike recovery rate, which improves the accuracy and sensitivity of the detection results.

[0244] Example 5

[0245] I. Comparison of the impact of different gradient conditions on detection results:

[0246] Serum samples were pretreated according to the conditions in Example 1, and hormones and amino acids were detected. The difference was that the flow rate was 0.4 mL / min; the gradient elution conditions are shown in Table 25 below:

[0247] Table 25

[0248] Time A％ B％ 0.00 90.00 10.00 1.00 90.00 10.00 1.01 75.00 25.00 3.00 75.00 25.00 3.50 60.00 40.00 12.00 10.00 90.00 12.50 0.00 100.00 13.00 0.00 100.00 13.01 90.00 10.00 15.00 90.00 10.00

[0249] Experimental results are as follows Figure 7 As shown in the figure. The experimental results show that the expected results can be obtained under these elution conditions.

[0250] II. Comparison of the effects of different flow rates on detection performance:

[0251] Serum samples were pretreated according to the conditions in Example 1, and hormones and amino acids were detected. The difference was that the mobile phase was: Phase A: 0.5 mmol / L ammonium fluoride in water; Phase B: methanol.

[0252] Experimental results are as follows Figure 8As shown in the figure. The experimental results show that the expected results can be obtained under the given mobile phase conditions.

[0253] Example 6

[0254] I. Comparison of the impact of different gradient conditions on detection results:

[0255] Serum samples were pretreated according to the conditions in Example 1, and then ceramide was detected, except that the flow rate was 0.6 mL / min; the gradient elution conditions are shown in Table 26 below:

[0256] Table 26

[0257] Time A％ B％ 0.00 20.00 80.00 1.50 20.00 80.00 2.00 0.00 100.00 6.00 0.00 100.00 6.01 20.00 80.00 7.50 20.00 80.00

[0258] Experimental results are as follows Figure 9 As shown in the figure. The experimental results show that the expected results can be obtained under these elution conditions.

[0259] II. Comparison of the effects of different flow rates on detection performance:

[0260] Serum samples were pretreated according to the conditions in Example 1 and then ceramide was detected. The difference was that the dynamic phase was: Phase A: 0.2% formic acid and 2 mmol / L ammonium acetate in water.

[0261] Experimental results are as follows Figure 10 As shown in the figure. The experimental results show that the expected results can be obtained under the given mobile phase conditions.

[0262] Comparative Example 1

[0263] The method of Example 1 is used, except that the following conditions are changed:

[0264] 1. Pretreatment conditions for detecting amino acids and hormones in serum samples:

[0265] Take 10 μL of the mixed amino acid hormone internal standard working solution and 200 μL of serum sample to be tested, add 600 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 20 μL of supernatant, add 180 μL of water and mix well. The resulting sample solution is then analyzed. The experimental results show that all hormone channels have no response values. Even increasing the sample volume by 5 times still fails to meet the limit of quantitation, indicating that a single protein precipitation pretreatment of the same sample cannot detect hormones.

[0266] 2. Pretreatment conditions for serum sample detection of ceramides:

[0267] Take 10 μL of mixed ceramide internal standard working solution and 10 μL of serum sample to be tested, add 300 μL of precipitant B (methanol), mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 5 min, and inject the supernatant for analysis. The obtained chromatogram is shown below. Figure 11 As shown.

[0268] The results showed that the peak shape of ceramide under these pretreatment conditions was poor and could not meet the detection requirements.

[0269] Comparative Example 2

[0270] The methods of Examples 1 and 3 are used, with the only difference being the change of the following conditions:

[0271] When processing serum samples, the solid-phase extraction step in the pretreatment process involved sequential rinsing with water and then with a 10% (v / v) acetonitrile aqueous solution. Finally, 50 μL of acetonitrile was used to collect the supernatant. After collection, the supernatant was dried under nitrogen and reconstituted with the initial mobile phase for detection. The experimental results are shown in Table 27.

[0272] Table 27

[0273]

[0274] The results showed that even with only two rinsing steps (water and 10% acetonitrile water), the amino acids to be tested were still detected. This indicates that the solid-phase extraction rinsing was incomplete, which led to higher amino acid test results than the true values, resulting in inaccurate test results.

[0275] Comparative Example 3

[0276] The method of Example 3 is used, except that the following conditions are changed:

[0277] Pretreatment steps: Take 20 μL of mixed amino acid hormone internal standard working solution and 10 μL of cerebrospinal fluid sample to be tested, add 180 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 20 μL of supernatant, add 180 μL of water and mix well. Obtain injection solution A.

[0278] Take 10 μL of the mixed amino acid hormone internal standard working solution, add 500 μL of cerebrospinal fluid sample to be tested, add 600 μL of methanol, mix at 2500 rpm for 5 min, centrifuge at 14000 rpm for 10 min, take 20 μL of supernatant, add 180 μL of water and mix well. This yields injection solution B. Perform instrumental analysis, and the experimental results are shown in Table 28.

[0279] Table 28

[0280] sample Sample size to be tested Hormone response value amino acid response value Injection solution A 20μL No response All responses were within the instrument's detection range. Injection solution B 500μL No response <![CDATA[The responses are all greater than 6e 6 beyond the detection range of the instrument]]>

[0281] The results showed that a single protein precipitation pretreatment of the same sample could not simultaneously detect hormones and amino acids, and the hormone signal detected by protein precipitation was far below the target response. Therefore, the detection requirements could not be met.

[0282] Comparative Example 4

[0283] The methods of Examples 1 and 3 are used, with the only difference being the change of the following conditions:

[0284] The mobile phases are: Phase A: water; Phase B: methanol.

[0285] The serum samples were tested, and the experimental results are as follows: Figure 12 , Figure 13 As shown.

[0286] The results showed that the signal-to-noise ratio of hormones P and E2 could not meet the detection requirements under these mobile phase conditions.

[0287] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the combined detection of multiple amino acids, hormones, and ceramides by liquid chromatography-tandem mass spectrometry, characterized in that, The amino acids include isoleucine, aspartic acid, glutamic acid, methionine, phenylalanine, tryptophan, and kynurenine; the hormones include progesterone, pregnenolone, dihydroprogesterone, tetrahydroprogesterone, allogeneic progesterone, testosterone, dehydroepiandrosterone, androstenedione (3α,17β), dihydrotestosterone, estriol, and estradiol; the ceramides include ceramide C16 (d18:1 / 16:0), ceramide C18 (d18:1 / 18:0), and ceramide C24 (d18:1 / 24:0); The method includes at least the following steps: S1. Standard curve equation I and standard curve equation II are prepared respectively. Prepare standard solution I, and use high performance liquid chromatography-mass spectrometry to detect standard solution I to obtain standard curve equation I for calculating hormone and amino acid content; Prepare standard solution II, and use high performance liquid chromatography-mass spectrometry to detect standard solution II to obtain standard curve equation II for calculating ceramide content; S2. Pre-processing of the sample to be tested, including: The sample to be tested is divided into three parts: sample a, sample b, and sample c; the sample to be tested is a serum sample or a cerebrospinal fluid sample. Take the mixed amino acid internal standard working solution and the test sample a, add protein precipitant A, mix well and centrifuge, take the supernatant I, add water to dilute to obtain solution I; Mix the mixed hormone internal standard working solution with the test sample b, add protein precipitant A, mix well, centrifuge, take the supernatant II, redissolve in water, perform solid-phase extraction, collect the eluent containing hormone components, dry it and mix it with the solution I, the supernatant after centrifugation is used as injection sample I, for injection analysis; the solid-phase extraction uses an SPE plate loaded with Oasis PRiME pretreatment column; the eluent for the solid-phase extraction is acetonitrile, and before collecting the eluent, it is rinsed sequentially with water, a 20%~40% (v / v) methanol aqueous solution, and a 5%~15% (v / v) acetonitrile aqueous solution; Take the mixed ceramide internal standard working solution and the sample c to be tested, add protein precipitant B, mix well and centrifuge, and take the supernatant as the injection sample II; The protein precipitant A is selected from methanol; the protein precipitant B is selected from a mixed solvent of methanol and isopropanol in a volume ratio of 1.5 to 2.5:

1. S3. Detect the injected sample I and the injected sample II respectively, including: The injected sample I and the injected sample II were detected by high performance liquid chromatography-mass spectrometry. The detection results of the injected sample I were substituted into the standard curve equation I, and the detection results of the injected sample II were substituted into the standard curve equation II to obtain the content of various amino acids, hormones and ceramides in the sample to be tested. When using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), a pentafluorophenyl column was used. When using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to detect the standard solution I and the injected sample I, the chromatographic analysis conditions include: Mobile phase: Phase A: Water containing 0.4 ~ 0.5 mmol / L ammonium fluoride; Phase B: Methanol; Gradient elution conditions: 0.00 ~ 1.00 min, phase A uses concentration A1, and phase B uses concentration B1; 1.01 ~ 3.00 min, phase A uses concentration A2, and phase B uses concentration B2; 3.00 ~ 3.50 min, phase A changes from concentration A2 to concentration A3 at a constant rate, and phase B changes from concentration B2 to concentration B3 at a constant rate. 3.50~12.00 min, concentration A3 changes at a constant rate to concentration A4; Phase B: concentration B3 changes at a constant rate to concentration B4. From 12.01 to 12.50 min, the concentration of phase A4 was changed at a constant rate to 0; in phase B, the concentration of phase B4 was changed at a constant rate to 100%. 12.50 ~ 13.00 min, Phase A is 0%; Phase B is 100%; 13.01 ~ 15.00 min, phase A uses concentration A1, and phase B uses concentration B1; Concentration A1 is selected from 95% to 90%, concentration B1 is selected from 5% to 10%, and concentration A1 + concentration B1 = 100%. Concentration A2 is selected from 85% to 75%, concentration B2 is selected from 15% to 25%, and concentration A2 + concentration B2 = 100%; Concentration A3 is selected from 60% to 65%, concentration B3 is selected from 35% to 40%, and concentration A3 + concentration B3 = 100%; Concentration A4 is selected from 10% to 20%, concentration B4 is selected from 80% to 90%, and concentration A4 + concentration B4 = 100%; The chromatographic analysis conditions for detecting the standard solution II and the injected sample II using high performance liquid chromatography-mass spectrometry include: Mobile phase: Phase A: an aqueous solution containing 0.1% to 0.2% formic acid and 1 to 2 mmol / L ammonium acetate by volume; Phase B: a mixed solvent of methanol and isopropanol by volume ratio of 1.5 to 2.5:1; Gradient elution conditions: 0.00 ~ 1.50 min, phase A uses concentration A1, and phase B uses concentration B1; 1.50 ~ 2.00 min, phase A changes from concentration A1 to concentration A2 at a constant rate, and phase B changes from concentration B1 to concentration B2 at a constant rate. 2.00~6.00 min, phase A uses concentration A2, and phase B uses concentration B2; 6.01 ~ 7.50 min, phase A uses concentration A1, and phase B uses concentration B1; Concentration A1 is selected from 15% to 20%, concentration B1 is selected from 80% to 85%, and concentration A1 + concentration B1 = 100%; Concentration A2 is selected from 0~5%, concentration B2 is selected from 95%~100%, and concentration A2 + concentration B2 = 100%.

2. The method according to claim 1, characterized in that, S1 includes: Preparation of internal standard working solution, preparation of standard curve working solution, and preparation of standard solution. The preparation of the internal standard working solution includes: preparing the mixed hormone internal standard working solution, the mixed amino acid internal standard working solution, and the mixed ceramide internal standard working solution respectively; The preparation of the standard curve working solution includes: preparing a mixed hormone standard curve working solution, a mixed amino acid standard curve working solution, and a mixed ceramide standard curve working solution respectively.

3. The method according to claim 2, characterized in that, The preparation of the standard solution I includes: taking the mixed amino acid internal standard working solution and the mixed amino acid standard curve working solution of gradient concentration, adding the protein precipitant A and mixing well to obtain a mixed solution, adding water to the mixed solution and mixing well to obtain solution I'; taking the mixed hormone internal standard working solution and the mixed hormone standard curve working solution of gradient concentration, drying them and dissolving them in solution I'; thus preparing the standard solution I of gradient concentration. The preparation of the standard solution II includes: taking the mixed ceramide internal standard working solution, the mixed ceramide standard working solution of gradient concentrations, and the protein precipitant B to prepare the standard solution II of gradient concentrations; The standard solution I was detected by high performance liquid chromatography-mass spectrometry to obtain the standard curve equation I; The standard solution II was detected by high performance liquid chromatography-mass spectrometry to obtain the standard curve equation II.

4. The method according to claim 3, characterized in that, When preparing the standard solution I, the mixing conditions are: vortex mixing at 1000-2500 rpm for 30 seconds to 1 minute; and / or, The volume ratio of the mixed amino acid internal standard working solution to the mixed amino acid standard curve working solution is 1:1; the volume ratio of the mixed amino acid internal standard working solution to protein precipitant A is 1:15-20; the volume ratio of the mixture to water is 1:8-10; and / or, The volume ratio of the mixed hormone internal standard working solution to the mixed hormone standard working solution is 1:1; and / or, The volume ratio of the mixed ceramide internal standard working solution, the mixed ceramide standard working solution, and the protein precipitant B is 1:1:25~35.

5. The method according to claim 1, characterized in that, In S2, During the preparation of the injected sample I, the mixing conditions are 2000-2500 rpm for 3-5 min, and the centrifugation conditions are 12000-14000 rpm for 5-10 min; and / or, During the preparation of the sample II, the mixing conditions are 2000-2500 rpm for 3-5 min, and the centrifugation conditions are 12000-14000 rpm for 5-10 min.

6. The method according to any one of claims 1 to 5, characterized in that, In S2, when the sample to be tested is a serum sample: The volume ratio of the test sample a, the test sample b, and the test sample c is 1:20:1; and / or, The specific steps for the injection of sample I include: taking the mixed hormone internal standard working solution and the sample to be tested b, mixing them, adding the protein precipitant A, mixing, centrifuging, taking the supernatant II, adding water to redissolve, and performing solid-phase extraction. During the preparation of sample I, the volume ratio of the test sample a to the mixed amino acid internal standard working solution is 1:1, the volume ratio of the test sample a to the protein precipitant A is 1:15 to 20, and the volume ratio of the supernatant I to water is 1:8 to 10. The volume ratio of the test sample b to the mixed hormone internal standard working solution is 20:1; the volume ratio of the test sample b to protein precipitant A is 1:2 to 2.5; the volume ratio of supernatant II to water is 1:1 to 1.2; and / or, During the preparation of sample II, the volume ratio of the test sample c to the mixed ceramide internal standard working solution is 1:1, and the volume ratio of the test sample c to protein precipitant B is 1:25 to 35.

7. The method according to claim 6, characterized in that, When the sample to be tested is a serum sample, the volumes of sample a, sample b, and sample c are 10 ~ 20 μL, 200 ~ 400 μL, and 10 ~ 20 μL, respectively.

8. The method according to any one of claims 1 to 5, characterized in that, When the sample to be tested is a cerebrospinal fluid sample: The volume ratio of the test sample a, the test sample b, and the test sample c is 1:25:5; and / or, During the preparation of the injected sample I, the volume ratio of the test sample a to the mixed amino acid internal standard working solution is 2:1, the volume ratio of the test sample a to protein precipitant A is 2:15-20, and the volume ratio of supernatant I to water is 1:8-10; and / or, The volume ratio of the test sample b to the mixed hormone internal standard working solution is 50:1; and / or, During the preparation of the sample II, the volume ratio of the test sample c to the mixed ceramide internal standard working solution is 10:1, and the volume ratio of the test sample c to the protein precipitant B is 1:2.5 to 3.

5.

9. The method according to claim 8, characterized in that, The volumes of test sample a, test sample b, and test sample c are 20 ~ 30 μL, 500 ~ 750 μL, and 100 ~ 150 μL, respectively.

10. The method according to any one of claims 1 to 5, characterized in that, When using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS), a Kinetex F5 column with a packing particle size of 2.6 µm was used for analysis.

11. The method according to claim 10, characterized in that, When using high-performance liquid chromatography-mass spectrometry to detect the standard solution I and the injected sample I: The flow rate for chromatographic analysis is 0.4 ~ 0.5 mL / min.

12. The method according to claim 10, characterized in that, When using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to detect the standard solution I and the injected sample I, the mass spectrometry analysis conditions are as follows: electrospray ionization source, simultaneous acquisition in positive and negative ion modes, multiple reaction monitoring; nebulization temperature: 500℃~600℃; electrospray voltage: 5500 / -4000 V; curtain gas: 20~30 L / min. Collision gas: 6 ~ 10 L / min; Atomizing gas: 55 ~ 65 L / min; Auxiliary gas: 55 ~ 65 L / min.

13. The method according to any one of claims 1 to 5, characterized in that, The flow rate during chromatographic analysis of the standard solution II and the injected sample II using high performance liquid chromatography-mass spectrometry was 0.5 ~ 0.6 mL / min.

14. The method according to any one of claims 1 to 5, characterized in that, When using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to detect the standard solution II and the injected sample II, the mass spectrometry analysis conditions are as follows: electrospray ionization source, positive ion mode acquisition, multiple reaction monitoring; nebulization temperature: 500℃~600℃; electrospray voltage: 5500 V; curtain gas: 20~30 L / min. Collision gas: 6 ~ 10 L / min; Atomizing gas: 30 ~ 40 L / min; Auxiliary gas: 30 ~ 40 L / min.

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