LC-MS (liquid chromatography-mass spectrometry) detection method of alpha-hydroxybutyric acid

Abstract

The invention discloses an LC-MS (liquid chromatography-mass spectrometry) detection method of alpha-hydroxybutyric acid, and belongs to the technical field of quantitative detection of alpha-hydroxybutyric acid. The detection method comprises the following steps: step 1, preparing a standard working solution; 2, preparing an internal standard working solution; step 3, pre-treating a sample; step 4, putting the sample treated in the step 3 into EP tubes, adding an internal standard working solution into each EP tube, slightly and uniformly mixing, continuously carrying out vortex oscillation on the mixture at the room temperature at the medium speed, then carrying out centrifugal treatment, and transferring supernatant liquid in a centrifugal tube into a special bottle for liquid chromatography; and 5, placing the special bottle for liquid chromatography on a sample introduction disc of a high performance liquid chromatography-tandem triple quadrupole mass spectrometer, carrying out data acquisition and quantitative analysis, recording a chromatogram and a mass spectrum, and carrying out peak area calculation by adopting an internal standard method to obtain a detection result. The 2-HB can be accurately detected and quantified, and the metabolic state of a patient can be more accurately evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of quantitative detection of alpha-hydroxybutyric acid, and specifically relates to an LC-MS detection method of alpha-hydroxybutyric acid. Background Art

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology has been developing rapidly at home and abroad in recent years. It is a technology that combines the separation ability of high-performance liquid chromatography with the detection advantages of mass spectrometry. The compounds in the mixture are first separated by chromatography, and then these compounds are identified and quantified by mass spectrometry.

[0003] Since the 21st century, mass spectrometry has made great progress in various fields such as biopharmaceuticals, food safety, environmental monitoring, chemical energy, life sciences, and forensic toxicology. In particular, the development of liquid chromatography-mass spectrometry has greatly broadened the application field of mass spectrometry technology and has become the most mainstream mass spectrometry working mode. With the development of precision medicine, liquid chromatography-mass spectrometry technology has begun to be applied in the field of clinical testing.

[0004] Gamma-hydroxybutyric acid (GHB) is an endogenous molecule that can also be used as a therapeutic drug. In recent years, the abuse of GHB, a psychoactive drug, has attracted public attention at home and abroad. Therefore, the use of effective analytical methods is of great significance in forensic toxicology research.

[0005] Due to the different positions of hydroxyl substitution, γ-hydroxybutyric acid has two other isomers, β-hydroxybutyric acid (Beta-hydroxybutyric acid, BHB) and α-hydroxybutyric acid (α-hydroxybutyric acid, 2-HB). The specific chemical formula structure is shown in Figure 1 . 2-HB is an endogenous ketone body that provides analytical evidence for ketoacidosis. As ketoacidosis occurs, 2-HB levels will increase. High concentrations of 2-HB can be used as analytical evidence for ketoacidosis, thereby indicating a pathological diagnosis or a possible cause of death. Gall WE et al. discovered that α-hydroxybutyric acid (2-HB) is a serum marker that can directly determine insulin resistance (IR). Insulin resistance refers to the phenomenon that fat cells, muscle cells, and liver cells do not respond adequately to normal concentrations of insulin.

[0006] Sandra Rinne Dahl et al. found that GHB and its precursors 1,4-butane-diol (1,4-BD) and gamma-butyrolactone (GBL) are increasingly abused drugs in Norway and other Scandinavian countries. 1,4-BD and GBL are rapidly converted to GHB in the body. GHB and its precursor 1,4-BD induce euphoria, relaxation, and anxiolysis, with a mechanism of action similar to that of ethanol and benzodiazepines. As a precursor and metabolite of the endogenous neurotransmitter gamma-aminobutyric acid (GABA), GHB is present in small amounts in the body. Andresen et al. reviewed the pharmacodynamics, pharmacokinetics, toxic effects, addiction, analytical methods, and interpretation of results.

[0007] 2-HB is an organic acid derived from α-ketobutyric acid (α-KB). The research findings of Peddinti G, Varvel SA, Tricò D et al. show that the increase of 2-HB in serum is caused by at least the following two aspects: (1) Oxidative stress leads to increased hepatic glutathione synthesis, which in turn leads to an increase in α-ketobutyric acid products in the metabolic pathway; (2) Lipid oxidation causes an increase in the ratio of reduced nicotinamide adenine dinucleotide to oxidized nicotinamide adenine dinucleotide, resulting in an increase in the conversion of α-ketobutyric acid to 2-HB under the action of lactate dehydrogenase and α-hydroxyacid dehydrogenase. For people who have already developed IR, timely improvement of the body condition by optimizing lifestyle in the early stage can effectively prevent the malignant development of the disease.

[0008] Foreign studies have found that 2-HB can better distinguish IR subjects from insulin-sensitive people. IR is the common pathophysiological basis for diseases such as type 2 diabetes. Existing IR evaluation methods are all indirect methods, including dynamic assessment methods and static assessment methods. The dynamic assessment methods mainly include the hyperinsulinemic-euglycemic clamp test (HI-clamp), 72h dynamic blood glucose test and oral glucose tolerance test (OGTT). The static assessment method that is currently widely used and easy to implement is the homeostasis model assessment of insulin resistance (HOMA-IR).

[0009] Based on the above research, this study attempted to establish an LC-MS detection method for detecting 2-HB in order to quantitatively monitor changes in 2-HB content in the blood. Summary of the invention

[0010] In view of the deficiencies in the prior art, the present invention proposes an LC-MS detection method for α-hydroxybutyrate, which can accurately detect and quantify 2-HB by using a specific mass spectrometry technique, and can accurately measure it even in complex biological samples.

[0011] The technical solution of the present invention is as follows:

[0012] In a first aspect, the present invention provides an LC-MS detection method for α-hydroxybutyric acid, the detection method comprising the following steps:

[0013] Step 1: Prepare standard working solution;

[0014] Step 2: prepare internal standard working solution;

[0015] Step 3: Sample pretreatment;

[0016] Step 4: Place the sample after the treatment in step 3 in an EP tube, add the internal standard working solution to each EP tube, gently mix, continue to vortex the mixture at room temperature at a medium speed, then centrifuge, and transfer the supernatant in the centrifuge tube to a special bottle for liquid chromatography;

[0017] Step 5: Place the liquid chromatography bottle on the injection plate of the high performance liquid chromatography-tandem triple quadrupole mass spectrometer, perform data acquisition and quantitative analysis, record the chromatogram and mass spectrum, and use the internal standard method to calculate the peak area to obtain the test result.

[0018] In some embodiments of the present invention, the specific process of step 1 includes:

[0019] Weigh pure sodium α-hydroxybutyrate and transfer it to a centrifuge tube, add methanol solution to prepare a primary standard working solution, vortex at room temperature to dissolve the solid powder fully, seal with a sealing film and place in a -20°C refrigerator for later use;

[0020] When in use, transfer the methanol solution to another centrifuge tube and add the primary standard working solution to dilute it. Prepare the standard working solution, mix it by vortexing thoroughly and then divide it into centrifuge tubes. Seal them with sealing film and freeze them in a -20℃ refrigerator for later use.

[0021] In some embodiments of the present invention, sodium α-hydroxybutyrate and methanol solution are mixed in a volume ratio of 1:1 to prepare a 10 mM primary standard working solution.

[0022] In some embodiments of the present invention, when used, methanol solution is added to the primary standard working solution to dilute it 100 times to prepare a 100 μM standard working solution.

[0023] In some embodiments of the present invention, the specific process of step 2 includes:

[0024] Dissolve the 2-HB-d3 isotope marker in methanol solution to prepare an isotope marker stock solution, seal it with a sealing film and place it in a -20°C refrigerator for later use;

[0025] When in use, transfer the methanol solution to a new centrifuge tube, add the isotope marker stock solution to dilute it, prepare the isotope internal standard working solution, seal it with a sealing film and place it in a -20°C refrigerator for later use.

[0026] In some embodiments of the present invention, the concentration of the isotope label stock solution is 500 μM.

[0027] In some embodiments of the present invention, when used, the isotope marker stock solution is diluted 100 times by adding methanol solution to prepare a 5 μM isotope internal standard working solution.

[0028] In some embodiments of the present invention, the sample pretreatment operation in step 3 includes: centrifuging the blood collection tube at 3000 rpm for 15 minutes using a low-speed centrifuge, and when obvious stratification occurs, carefully aspirating the supernatant and transferring it to a 1.5 mL cryotube and placing it in a -80°C refrigerator for freezing and standby use.

[0029] In some embodiments of the present invention, in step 4, the sample is mixed with the isotope internal standard working solution in a volume ratio of 1:4.

[0030] In some embodiments of the present invention, the mixture of the sample and the isotope internal standard working solution in step 4 is vortexed twice at room temperature at a medium speed for 15 seconds with an interval of 3 seconds in between.

[0031] In some embodiments of the present invention, the chromatographic conditions for data acquisition in step 5 are: using a Phenomenex Kinetex EVO C18 chromatographic column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, flow rate is 0.3 mL / min, autosampler temperature is 4 ° C, chromatographic column temperature is set to 40 ° C, injection volume is 5.00 μL, the starting mobile phase is pre-equilibrated with the chromatographic column for 15 minutes before injection, gradient elution is used, elution time is 3.5 minutes, each needle is balanced for 2 minutes before injection, and the elution program is:

[0032]

[0033] In some embodiments of the present invention, the mass spectrometry conditions for data acquisition in step 5 are as follows: ion source: electrospray ion source negative ion mode, multiple reaction monitoring mode analysis; air curtain pressure: 30.0 psi; collision pressure: 7 psi; spray pressure: 19.0 psi; atomization auxiliary heating pressure: 0 psi; spray voltage: -4500 V; inlet voltage: -10 V; outlet voltage: -9 V; dwell time: 450.0 ms; source temperature: 550 ° C; MRM ion parameters of 2-HB and its internal standard 2-HB-d3 are shown in the following table:

[0034]

[0035] In a second aspect, the present invention also provides an application of an LC-MS detection method of α-hydroxybutyrate in evaluating the progress of the therapeutic effect of diabetic ketoacidosis.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. High sensitivity and specificity: Mass spectrometry technology can provide highly sensitive detection, so that even trace amounts of 2-HB can be accurately detected.

[0038] 2. Accurate quantification: Mass spectrometry technology can accurately quantify the content of β-HB, which helps to more accurately assess the patient's metabolic status.

[0039] 3. Rapid detection: The present invention can complete the determination of β-HB in a shorter time.

[0040] 4. Reliable diagnosis and monitoring: Mass spectrometry detection of 2-HB shows higher sensitivity and reliability than traditional methods in the diagnosis and treatment monitoring of diabetic ketoacidosis.

[0041] 5. Early diagnosis: The determination of 2-HB is of great significance for the early diagnosis of diabetes, which helps in timely detection and intervention.

[0042] 6. Comprehensiveness: Mass spectrometry technology can not only detect 2-HB, but also simultaneously detect other related metabolites, providing a more comprehensive metabolic analysis.

[0043] 7. Improve the evaluation of treatment effects: The determination of 2-HB can be used as an important indicator to measure the efficacy of diabetic ketoacidosis, which helps doctors evaluate the treatment effect and adjust the treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 1 is the structural formula of γ-hydroxybutyric acid, β-hydroxybutyric acid and α-hydroxybutyric acid; from left to right they are α-hydroxybutyric acid, β-hydroxybutyric acid and γ-hydroxybutyric acid.

[0045] Figure 2This is the detection peak diagram of α-hydroxybutyric acid and internal standard.

[0046] Figure 3 is the standard curve of 2-HB. DETAILED DESCRIPTION

[0047] The present invention is further described below with reference to specific examples.

[0048] This embodiment specifically provides an LC-MS detection method for α-hydroxybutyric acid, which is as follows:

[0049] 1. Experimental Materials and Methods

[0050] 1.1 Reagents

[0051] Sodium α-hydroxybutyrate (DL-2-Hydroxybutyrate) was purchased from CFW Laboratories, USA, with a purity ≥96.0%; d3-α-hydroxybutyrate [Sodium DL-2-Hydroxybutyrate-2,3,3-d3, 2-HB-d3] isotope marker was purchased from CFW Laboratories, USA, with an isotope abundance of 98.8%; methanol (LC / MS grade), acetonitrile (LC / MS grade), and formic acid (LC / MS grade) were all purchased from Fisher Scientific, USA; the ultrapure water used in the experiment was 4.5L distilled drinking water purchased from Guangzhou Watsons; the plasma samples used in this experiment were all from the plasma of 32 random stroke patients who visited Beijing Tiantan Hospital Affiliated to Capital Medical University from October 2021 to February 2024.

[0052] 1.2 Instruments

[0053] Kinetex EVO C18 (100 mm × 2.1 mm, 2.6 μm) chromatographic column was purchased from Phenomenex, USA, and the high performance liquid chromatography-tandem triple quadrupole mass spectrometer was used. TM4500MD (equipped with electrospray ESI ion source) was purchased from AB SCIEX, USA, Heraeus Fresco 21 refrigerated high-speed micro-tabletop centrifuge was purchased from ThermoFisher Scientific, USA, BY-320A medical low-speed centrifuge was purchased from Beijing Baiyang Company, METTLER TOLEDO balance (maximum weighing capacity 220 g, precision division value 0.1 mg) was purchased from METTLER, Germany, VORTEX Genie 2T vortex oscillator was purchased from Scientific Industries, USA, 1.5 mL micro-transparent centrifuge tubes were purchased from AXYGEN, USA, and 5.0 mL transparent centrifuge tubes were purchased from AXYGEN, USA.

[0054] 1.3 Method development

[0055] 1.3.1 Solution preparation

[0056] 1.3.1.1 Preparation of standard working solution

[0057] Accurately weigh 2.3 mg of pure sodium α-hydroxybutyrate and transfer it to a 5.0 mL centrifuge tube, accurately add 1.824 mL of methanol solution to prepare a 10 mM primary standard working solution, vortex at room temperature to fully dissolve the solid powder, seal with a sealing film and place in a -20 ° C refrigerator for later use.

[0058] When in use, transfer 1980 μL of methanol solution to another 5.0 mL centrifuge tube, and add 20 μL of primary standard working solution to dilute it 100 times to prepare a 100 μM standard working solution. After sufficient vortexing and mixing, divide it into 1.5 mL centrifuge tubes, seal them with sealing film and place them in a -20°C refrigerator for later use.

[0059] 1.3.1.2 Preparation of internal standard working solution

[0060] Use methanol solution to dissolve 2-HB-d3 isotope marker to prepare 500μM isotope marker stock solution, seal with parafilm and place in -20℃ refrigerator for freezing. When used, take 9.8mL of methanol solution and transfer to a new 15mL centrifuge tube, add 200μL of 500μM isotope marker stock solution to dilute it 100 times, prepare 5μM isotope internal standard working solution, seal with parafilm and place in -20℃ refrigerator for freezing.

[0061] 1.3.2 Sample pretreatment

[0062] The matrix of biological samples is complex, and the targets to be detected are mostly endogenous compounds. Therefore, in order to reduce matrix interference, extend the service life of the chromatographic column, prevent system blockage and damage, and improve detection sensitivity and specificity, it is often necessary to pre-treat the samples by precipitation, extraction, purification, enrichment, etc.

[0063] The 32 blood collection tubes were centrifuged at 3000 rpm for 15 min using a low-speed centrifuge. When obvious stratification occurred, the supernatant was carefully aspirated and transferred to a 1.5 mL cryotube and placed in a -80°C refrigerator for later use.

[0064] Take 20 μL of supernatant from each sample and place it in a 1.5 mL EP tube. Add 80 μL of 5 μM isotope internal standard working solution (volume ratio 1:4) to each EP tube. Mix gently and continue to vortex the mixture twice at room temperature at medium speed for 15 seconds, with a 3-second interval. Use a refrigerated high-speed centrifuge to centrifuge at 20.0 × g for 25 minutes at 4 ° C, and transfer the supernatant in the centrifuge tube to a special bottle for liquid chromatography for analysis.

[0065] 1.3.3 Instrument conditions

[0066] 1.3.3.1 Liquid chromatography conditions

[0067] Chromatographic conditions include instruments, chromatographic columns, mobile phases, injection volumes and column temperatures, among which the most important are the selection of chromatographic columns and mobile phases.

[0068] A Phenomenex Kinetex EVO C18 (100 mm × 2.1 mm, 2.6 μm) chromatographic column was used, mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, the flow rate was 0.3 mL / min, the autosampler temperature was 4 °C, the column temperature was set to 40 °C, the injection volume was 5.00 μL, the starting mobile phase was pre-equilibrated with the column for 15 min before injection, gradient elution was used, the elution time was 3.5 min, the elution program was shown in Table 1, and each injection was equilibrated for 2 min before injection.

[0069] Table 1 Liquid chromatography mobile phase gradient elution program

[0070]

[0071] 1.3.3.2 Mass spectrometry conditions

[0072] Mass spectrometry conditions, including the selected mass spectrometry type, ionization mode, ion channel and other related mass spectrometry parameters.

[0073] Ion source: electrospray ionization (ESI) negative ion mode (ESI-), multiple reaction monitoring (MRM) mode analysis; curtain gas pressure (CUR): 30.0psi; collision gas pressure (CAD): 7psi; spray gas pressure (GS1): 19.0psi; nebulization auxiliary heating gas pressure (GS2): 0psi; spray voltage (ionspray voltage, IS): -4500V; entrance potential (EP): -10V; exit potential (CXP): -9V; dwell time: 450.0ms; source temperature: 550℃; MRM ion parameters of 2-HB and its internal standard 2-HB-d3 are shown in Table 2.

[0074] Table 2 MRM ion pairs and mass spectrometry acquisition parameters of 2-HB and its internal standard 2-HB-d3

[0075]

[0076] 1.3.4 Quantitative methods

[0077] The internal standard method is a commonly used method for quantitative analysis of biological samples using LC-MS technology. The internal standard is introduced to correct the errors caused by factors such as loss caused by sample pretreatment, injection volume differences, and the influence of matrix effects on the ionization process. The properties of a suitable internal standard should be similar to those of the component to be measured, and the internal standard peak should also be close to the peak of the component to be measured.

[0078] In this embodiment, the internal standard method is used to perform quantitative analysis and calculation for the determination of α-hydroxybutyric acid. All mixed working standard solutions are analyzed in order of concentration from small to large to obtain a working curve of quantitative ion peak area ratio and concentration ratio.

[0079] 1.4 Methodological Validation

[0080] 1.4.1 Method Specificity

[0081] Take 100μM standard working solution, add precooled (4℃) methanol solution to dilute 100 times to 10μM, take 20μL of 10μM standard working solution and place it in a 1.5mL EP tube, then add 80μL of 5μM isotope internal standard working solution (volume ratio 1:4) to the EP tube, mix gently and continue to vortex the mixture twice at room temperature at medium speed for 15s, with a 3s interval in between. Transfer all the liquid to a special bottle for liquid chromatography. Apply the instrument conditions of 1.3.3 to detect and analyze it, and repeat 3 times.

[0082] 1.4.2 Method accuracy

[0083] The accuracy experiment was conducted by using the spike recovery method. The 100μM standard solution was diluted with precooled (4℃) methanol solution to prepare samples with three molar concentrations: high (10μM), medium (5μM), and low (1μM). Each concentration was divided into three parts, each part was tested once, and the spike recovery rate was calculated. The spike recovery rate was acceptable between 80% and 120%.

[0084] 1.4.3 Method precision

[0085] The samples of low, medium and high molar concentrations prepared in 1.4.2 were selected. Each concentration sample was processed in 3 portions, and each sample was measured 3 times to obtain the average value. Three batches were measured continuously, with a total of 81 samples. The intra-batch precision of each concentration sample in each batch and the inter-batch precision of each concentration sample between batches were evaluated. In general, the imprecision within the range of ≤15% is acceptable.

[0086] 1.4.4 Standard curve and limit of quantitation

[0087] A 100 μM standard working solution was frozen in a -20°C refrigerator for later use, and diluted with pre-cooled (4°C) methanol solution to prepare a series of standard solutions of 0, 0.049, 0.098, 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM.

[0088] To take 20μL of standard solutions of different molar concentrations, first add 4 times the volume (80μL) of 5.0μM internal standard solution to 1.5mL EP, and then accurately add 20μL of standard solutions of different concentrations. After sufficient vortex mixing, transfer all of each mixed solution to the corresponding liquid chromatography bottle. Inject from low concentration to high concentration in sequence, and inject each mixed standard solution three times to take the average value. The X-axis is the ratio of the concentration of 2-HB to the internal standard 2-HB-d3, and the Y-axis is the ratio of the peak area of ​​2-HB to the internal standard 2-HB-d3. Use ORIGIN software for univariate linear regression to obtain the standard curve and correlation coefficient (r) of 2-HB.

[0089] According to CLSI C50-A, the lower limit of quantification (LLOQ) of 2-HB was investigated by stepwise dilution method using the lowest concentration mixed standard solution, and the standard concentration when the signal / noise (S / N) ratio was equal to 10 was taken as the LLOQ of 2-HB.

[0090] 1.4.5 Detection limit

[0091] The detection limit is the minimum concentration or amount of the target analyte that can be specifically detected from a sample using a specific analytical method within a given confidence level.

[0092] The LC-MS / MS method was used for detection. 20 μL of pure water and 80 μL of 5.0 μM internal standard solution (volume ratio 1:4) were prepared in a 1.5 mL EP tube to prepare 10 blank groups. After sufficient vortexing and mixing, each mixed solution was transferred to the corresponding liquid chromatography bottle for analysis. Each sample was tested 3 times, and the total number of samples was 30.

[0093] According to the International Union of Pure and Applied Chemistry (IUPAC) regulations on the detection limit DL of analytical methods, repeated measurements without adding the measured component (i.e. blank test) are performed under the same conditions as the analysis of the actual sample, and the number of tests is at least 20. When the confidence level is 95%, the detection limit (DL) is the significant difference between the sample measurement value and the measurement value of the zero concentration sample.

[0094] DL=4.6σ

[0095] Where: σ-blank parallel determination (within batch) standard deviation (repeated determination more than 20 times).

[0096] 1.4.6 Matrix Effects

[0097] The purpose of the matrix effect experiment is to verify whether there is a difference in the mass spectrometric response of the target analyte in the biological sample and the pure solution, and whether it affects the accurate quantification of the target analyte.

[0098] The clinical plasma samples were pre-treated as described in 1.3.2. Each sample was tested once and the peak area CV value was calculated. If the difference was less than a certain ratio (CV ≤ 20%), it was proved that the matrix effect did not affect the accurate quantification of the target analyte.

[0099] 2. Results and Discussion

[0100] 2.1 Methodological evaluation results

[0101] 2.1.1 Method Specificity

[0102] Using the instrument conditions of 1.3.3, the retention time of 2-HB was 1.09 min and the retention time of the internal standard 2-HB-d3 was 1.18 min. The peak shape was good, and the separation and response were good. Figure 2 .

[0103] 2.1.2 Method accuracy

[0104] The accuracy results of samples with three different molar concentrations are shown in Table 3, indicating that the recovery rates were between 98.2% and 104%, and the relative standard deviations (RSDs) were between 1.62% and 2.71%, all within the acceptable range (±5%), demonstrating that the accuracy of the established LC-MS method meets the requirements and is suitable for the determination of serum specimens.

[0105] Table 3 Spike recovery test results

[0106]

[0107] 2.1.3 Method precision

[0108] The intra-batch precision of this method is ≤3.21%, and the inter-batch precision is ≤2.50%. The method has good precision and is suitable for the determination of serum specimens (see Table 4).

[0109] Table 4 Precision verification results

[0110]

[0111]

[0112] 2.1.4 Standard curve and limit of quantitation

[0113] The concentration ratio of 2-HB to the internal standard 2-HB-d3 was used as the X-axis, and the peak area ratio of 2-HB to the internal standard 2-HB-d3 was used as the Y-axis. A linear regression was performed using ORIGIN software, and the regression equation was Y = 0.05943X + 0.0071, r = 0.9991, see Figure 3 The concentration when the average signal-to-noise ratio (RSN) is RSN=10 is taken as the quantitative limit. The experimental results show that the quantitative limit of 2-HB detected by this method is 0.40μM. Therefore, 2-HB has a good linear relationship in the concentration range of 0.4 to 100.0μM, and the linear range and quantitative limit are both sufficient for the detection of human serum samples.

[0114] 2.1.5 Detection limit

[0115] The results of the blank parallel test group are shown in Table 5. The standard deviation was calculated and the detection limit was 0.050 μM according to formula (1).

[0116] Table 5 Results of blank parallel test group

[0117]

[0118]

[0119] 2.1.6 Matrix Effects

[0120] The results of 32 clinical serum matrix samples are shown in Table 6. The coefficient of variation CV ≤ 6.16%, which is within the acceptable range (± 20%), indicating that the matrix effect is small and can be ignored, and is suitable for the determination of serum samples.

[0121] Table 6 Serum matrix results

[0122]

[0123] 2.2 Discussion

[0124] 2.2.1 Advancedness of the method

[0125] The LC-MS method established in this example has simple sample pretreatment, an analysis time of only 3.5 min, is rapid and sensitive, has a small matrix effect, and is suitable for serum sample detection.

[0126] The LC-MS detection method for serum 2-HB established in this example has laid a preliminary foundation for the clinical dynamic quantitative monitoring of changes in 2-HB content in the blood, and provides a new detection method for the prevention and treatment of IR. The research on apparently healthy and diseased populations can be further expanded in the future to provide a reference for the auxiliary diagnosis of IR in the Chinese population.

[0127] In summary, this example completes the development and verification of the LC-MS detection method for α-hydroxybutyric acid. The method is simple, rapid, sensitive and efficient, with accurate measurement results, high recovery rate and good precision, and is suitable for clinical routine and large-sample epidemiological surveys of α-hydroxybutyric acid content.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A LC-MS detection method for α-hydroxybutyric acid, characterized in that: The detection method comprises the following steps: Step 1: Prepare standard working solution; Step 2: Prepare internal standard working solution; Step 3: Sample pretreatment; Step 4: Place the sample after the treatment in step 3 in an EP tube, add the internal standard working solution to each EP tube, gently mix, continue to vortex the mixture at room temperature at a medium speed, then centrifuge, and transfer the supernatant in the centrifuge tube to a special bottle for liquid chromatography; Step 5: Place the liquid chromatography bottle on the injection plate of the high performance liquid chromatography-tandem triple quadrupole mass spectrometer, perform data acquisition and quantitative analysis, record the chromatogram and mass spectrum, and use the internal standard method to calculate the peak area to obtain the test result.

2. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: The specific process of step 1 includes: Weigh pure sodium α-hydroxybutyrate and transfer it to a centrifuge tube, add methanol solution to prepare a primary standard working solution, vortex at room temperature to dissolve the solid powder fully, seal with a sealing film and place in a -20°C refrigerator for later use; When in use, transfer the methanol solution to another centrifuge tube, add the primary standard working solution to dilute it, and prepare a 100 μM standard working solution. After sufficient vortexing and mixing, divide it into centrifuge tubes, seal it with sealing film, and place it in a -20°C refrigerator for later use.

3. The LC-MS detection method of α-hydroxybutyric acid according to claim 2, characterized in that: Sodium α-hydroxybutyrate and methanol solution were mixed in a volume ratio of 1:1 to prepare a 10 mM primary standard working solution.

4. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: The specific process of step 2 includes: Dissolve the 2-HB-d3 isotope marker in methanol solution to prepare an isotope marker stock solution, seal it with a sealing film and place it in a -20°C refrigerator for later use; Before use, transfer the methanol solution to a new centrifuge tube, add the isotope marker stock solution to dilute it, and prepare a 5 μM isotope internal standard working solution. Seal with a sealing film and place in a -20°C refrigerator for later use.

5. The LC-MS detection method of α-hydroxybutyric acid according to claim 4, characterized in that: The concentration of the isotope label stock solution was 500 μM.

6. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: The sample pretreatment operations in step 3 include: centrifuging the blood collection tube at 3000 rpm for 15 minutes using a low-speed centrifuge, and when obvious stratification occurs, carefully aspirate the supernatant and transfer it to a 1.5 mL cryotube and place it in a -80°C refrigerator for freezing.

7. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: In step 4, the sample and the isotope internal standard working solution are mixed in a volume ratio of 1:

4.

8. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: The chromatographic conditions for data collection in step 5 were as follows: a Phenomenex Kinetex EVO C18 column was used, mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, flow rate was 0.3 mL / min, autosampler temperature was 4°C, column temperature was set to 40°C, injection volume was 5.00 μL, the starting mobile phase was pre-equilibrated with the column for 15 min before injection, gradient elution was used, elution time was 3.5 min, and each injection was equilibrated for 2 min before injection. The elution program was as follows:

9. The LC-MS detection method of α-hydroxybutyric acid according to claim 1, characterized in that: Mass spectrometry conditions for data acquisition in step 5: ion source: electrospray ion source negative ion mode, analysis using multiple reaction monitoring mode; Air curtain pressure: 30.0psi; Collision air pressure: 7psi; Spray air pressure: 19.0psi; Atomization auxiliary heating air pressure: 0psi; Spray voltage: -4500V; Inlet voltage: -10V; Exit voltage: -9 V; dwell time: 450.0 ms; source temperature: 550°C; MRM ion parameters of 2-HB and its internal standard 2-HB-d3 are shown in the following table:

10. Use of the LC-MS detection method of α-hydroxybutyrate according to any one of claims 1 to 9 in evaluating the progress of therapeutic efficacy of diabetic ketoacidosis.

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