Detection method of gamma-aminobutyric acid and gamma-hydroxybutyric acid

Abstract

The invention discloses a method for detecting gamma-aminobutyric acid and gamma-hydroxybutyric acid, liquid chromatography tandem mass spectrometry is adopted for detection, ammonium bicarbonate is added in a mobile phase A and a mobile phase B, and ammonia water is also added in the mobile phase A; a chromatographic column with an amphoteric ionic bonded phase is adopted, and a hydrophilic interaction chromatographic mode is adopted. The detection method provided by the invention is high in detection sensitivity, the quantitative lower limit of GABA and GHB is increased to 20nM, the retention and peak patterns of GABA and GHB in a spectrogram are obviously enhanced, the analysis speed is obviously increased, and a PBS buffer solution is adopted as an alternative matrix, so that the application range of a matrix for in-vitro drug screening can be expanded.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and in particular to a method for detecting gamma-aminobutyric acid and gamma-hydroxybutyric acid. Background Art

[0002] γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays an extremely important role in the treatment of various neurological diseases, such as epilepsy, anxiety, Alzheimer's disease, intellectual degeneration, primary insomnia and biological treatment of cancer. γ-Hydroxybutyric acid (GHB) is a short-chain fatty acid, which is also an inhibitory neurotransmitter like GABA. It is widely present in various tissues of mammals and is mainly used to treat anesthesia, insomnia, clinical depression, etc. However, it is regulated by the State Food and Drug Administration because it can cause excitement, euphoria, anxiety and hypnosis at different dosages. However, the two can be converted into each other in the body, and GHB is both a metabolite and a precursor of GABA. Therefore, in the drug development stage, establishing a method for simultaneous detection of GHB and GABA can effectively determine the content of GABA and GHB converted into such related prodrugs in the body, and more efficiently evaluate the effectiveness, safety and toxicity of candidate compounds.

[0003] Currently, chromatography combined with mass spectrometry is mainly used to detect GABA and GHB to obtain sufficient detection sensitivity and selectivity. However, due to their small molecular weight and high polarity, GABA and GHB are not easily retained in traditional reversed-phase chromatography. In the open literature, derivatization is often used to increase the hydrophobicity of the compounds to enhance the retention of the compounds.

[0004] In general, in the prior art, the analysis technology of GABA and GHB mainly has the following problems:

[0005] 1. Limited detection limit: For GHB, although reverse phase chromatography can also be retained, its detection limit is relatively high. The quantitative limit of GHB reported in the literature is generally at the μM level. When the content of GHB generated in biological samples is low, it may not be accurately detected due to sensitivity issues, thereby affecting the metabolic stability of the compound and the accurate assessment of its content. In addition, the sample processing methods also mostly use complex and cumbersome SPE and LLE.

[0006] 2. Insufficient retention and long analysis time: GABA and GHB have small molecular weight and high polarity. The current liquid phase detection method for the simultaneous determination of the two mainly adopts reversed-phase chromatography. Compared with GHB, GABA has stronger polarity. Both are not retained in traditional reversed-phase C18 chromatographic columns. This is mainly achieved by reducing the flow rate and prolonging the liquid phase time. The currently reported analysis time is more than 10 minutes, and effective retention, separation and elution cannot be achieved on the chromatographic column, which is easy to cause certain interference and produce matrix effects.

[0007] 3. Sample processing is time-consuming and throughput is limited: Due to the problems caused by the structural characteristics of GABA and GHB, such as high polarity, difficulty in retention, and large matrix interference, derivatization is required during sample processing to enhance retention and ionization capabilities, or SPE and other methods are used to purify samples to reduce interference and improve sensitivity. This results in long sample processing time, complex steps, and low daily sample processing throughput. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for detecting gamma-aminobutyric acid and gamma-hydroxybutyric acid, which has a low limit of quantification, strong retention and separation capabilities, can reduce interference and matrix effects, and is time-saving.

[0009] In order to solve the above technical problems, the present invention provides a method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid, which adopts liquid chromatography tandem mass spectrometry for detection, wherein ammonium bicarbonate is added to both mobile phase A and mobile phase B, and the pH value of mobile phase A is 10.3±0.1;

[0010] The chromatographic column adopts a chromatographic column with a zwitterionic bonded phase and adopts a hydrophilic interaction chromatography mode.

[0011] In the present invention, liquid chromatography tandem mass spectrometry (LC-MS / MS) is used for detection, and ammonium bicarbonate is added to the mobile phase to increase the retention of GHB and eliminate the nonspecific adsorption of GHB at low concentrations; ammonia water is added to the mobile phase A to adjust the pH thereof to 10.3±0.1, which effectively improves the retention and peak shape of GHB, and the signal of GHB is also increased by about 10 times; at the same time, in order to further enhance the retention and separation of GABA and GHB, a hydrophilic interaction chromatography mode is adopted, and a chromatographic column with a zwitterionic bonded phase is adopted.

[0012] In one specific embodiment, mobile phase A is 5% acetonitrile in water, 2 mM ammonium bicarbonate, and the pH is adjusted with aqueous ammonia;

[0013] The mobile phase B was 95% acetonitrile in water; the chromatographic column was Atlantis Premier BEH Z-HILIC.

[0014] In a specific embodiment, the chromatographic conditions also include a gradient elution program:

[0015] 0.01-0.2min: mobile phase B maintained at 100%;

[0016] 0.2-2.2min: mobile phase A: 0→40%, mobile phase B: 100%→60%;

[0017] 2.2-2.8min: Mobile phase A maintained at 40%, mobile phase B maintained at 60%;

[0018] 2.8-2.81min: mobile phase A: 40%→0%, mobile phase B: 60%→100%;

[0019] 2.81-3.0min: mobile phase B maintained at 100%;

[0020] The flow rate was 0.6 mL / min.

[0021] In a specific embodiment, the chromatographic conditions further include: the liquid phase is: Shimadzu LC 30-AD, and the injection volume is 3 μL.

[0022] In a specific embodiment, a standard stock solution is prepared: γ-hydroxybutyric acid sodium salt, γ-aminobutyric acid, sodium γ-hydroxybutyrate-d6 and γ-amino acid-d6 are weighed separately and dissolved in a solvent to form a corresponding standard stock solution; the preferred solvent is dimethyl sulfoxide, and the concentration of the standard stock solution is preferably 5-30mM, for example 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, preferably an integer for easy dilution.

[0023] In a specific embodiment, a standard working solution is prepared: a standard stock solution of γ-hydroxybutyric acid and a standard stock solution of γ-aminobutyric acid are mixed, and a solvent is added to dilute the mixture to form a standard working solution containing γ-hydroxybutyric acid and γ-aminobutyric acid; in the standard working solution, the concentrations of γ-hydroxybutyric acid and γ-aminobutyric acid are preferably the same, for example, they can be 0.5 mM, 1 mM, 1.5 mM, 2.0 mM, etc., to facilitate subsequent dilution. It should be noted that the solvent used to prepare the standard working solution is consistent with that used to prepare the standard stock solution, for example, preferably dimethyl sulfoxide.

[0024] In a specific embodiment, a stop solution containing an internal standard is prepared: a standard stock solution of sodium γ-hydroxybutyrate-d6 and a standard stock solution of γ-amino acid-d6 are mixed, diluted with a solvent, and then methanol is added to dilute to a final concentration to obtain a stop solution containing an internal standard; during the preparation process, the standard stock solution is preferably first diluted with a dimethyl sulfoxide solvent to a certain concentration, for example, to 0.5-2mM, preferably 0.5mM, 1mM, 1.5mM, 2.0mM, to facilitate subsequent dilution, and then diluted with a methanol solution to a desired concentration, for example Sodium γ-hydroxybutyrate-d6 is diluted to 0.5-2μM, preferably 0.5μM, 1μM, 1.5μM, 2.0μM, for subsequent use, and γ-amino acid-d6 is diluted to 250-1000nM, preferably 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1000nM for subsequent use.

[0025] In a specific embodiment, the main function of the PBS buffer is to be used as a substitute matrix. Since GHB and GABA are endogenous substances, they are widely present in biological samples, and the background content of biological samples from different sources varies greatly. Therefore, it is necessary to select a suitable substitute matrix for accurate quantification. The present invention preferably uses a PBS buffer with a pH of 7.4±0.1, which does not contain background response and is close to biological matrices such as plasma, and can reduce matrix differences for accurate quantification and corresponding promotion.

[0026] In a specific embodiment, the calibration curve sample is a mixed solution of a standard working solution and a PBS buffer solution. The preparation method is, for example, taking a standard working solution, mixing it thoroughly with a PBS buffer solution, and diluting the concentration to 20, 50, 100, 300, 800, 2500, 5000 and 20000 nM to obtain a calibration curve sample with a concentration gradient.

[0027] In a specific embodiment, the quality control standard sample is a mixed solution of a standard working solution and a PBS buffer solution. The preparation method is, for example, taking a standard working solution, mixing it thoroughly with a PBS buffer solution, and preparing a quality control standard sample with a concentration gradient of 20, 80, 1000 and 16000 nM, wherein the concentrations of γ-aminobutyric acid and γ-hydroxybutyric acid are both gradient concentration values.

[0028] In a specific embodiment, the plasma spiked quality control sample is a mixture of γ-aminobutyric acid and γ-hydroxybutyric acid and a plasma matrix, and the preparation method is, for example, to take a standard working solution, fully mix it with the plasma matrix, and dilute the concentration of γ-aminobutyric acid and γ-hydroxybutyric acid in the standard working solution to 500, 2000 and 10000 nM to obtain a plasma spiked quality control sample. It should be noted that the plasma matrix itself contains GABA and GHB, and the aforementioned concentrations of 500, 2000 and 10000 nM refer to the concentrations after the dilution of the standard working solution, rather than the actual total concentrations of GABA and GHB in the plasma spiked quality control sample, because the original concentrations of GABA and GHB in the plasma matrix are unknown and need to be measured.

[0029] Since the present invention adopts PBS buffer as a substitute matrix, there are two types of quality control samples, namely quality control standard samples and plasma spiked quality control samples. The former can be used to examine the accuracy of PBS buffer as a substitute matrix under this method, and the latter is used to evaluate the accuracy of this method in real plasma samples.

[0030] In a specific embodiment, the single blank sample is a mixed solution formed by a PBS buffer and a stop solution containing an internal standard. The preparation method is, for example, taking a PBS buffer, adding a stop solution containing an internal standard and mixing them thoroughly. The two can be mixed, for example, at a volume ratio of 1:2-8. Preferably, the PBS buffer is 100 μL and the stop solution containing the internal standard is 400 μL.

[0031] In a specific embodiment, the double blank sample is a mixed solution of PBS buffer and methanol solution. The preparation method is, for example, taking PBS buffer, adding methanol solution and mixing thoroughly. The two can be mixed, for example, at a volume ratio of 1:2-8. Preferably, PBS buffer is 100 μL and methanol solution is 400 μL.

[0032] In a specific embodiment, the sample to be tested is a mixed solution formed by a plasma matrix and a stop solution containing an internal standard. The preparation method is, for example, taking a plasma matrix, adding a stop solution containing an internal standard and mixing them thoroughly. The two can be mixed, for example, at a volume ratio of 1:2-8. Preferably, the plasma matrix is ​​100 μL and the stop solution containing the internal standard is 400 μL.

[0033] In a specific embodiment, the sample processing method before the machine is: take the calibration curve sample, the quality control standard sample and the single blank sample, and add 2-5 times the volume of the stop solution containing the internal standard; take the double blank sample, add 2-5 times the volume of the methanol solution thereto, shake for 5-20 minutes, centrifuge, and take the supernatant for liquid chromatography tandem mass spectrometry analysis. After the sample is processed, the signal of GABA can be increased by at least 20 times.

[0034] The beneficial effects of the present invention include at least:

[0035] 1. Improve detection sensitivity: Increase the lower limit of quantification of GABA and GHB to 20nM, expanding the detection concentration range;

[0036] 2. During the analysis, the retention and peak shape of GABA and GHB were significantly enhanced, which was beneficial for the analysis;

[0037] 3. Improve the analysis speed. First, the protein precipitation time is greatly shortened in the sample processing stage compared with SEP. The protein precipitation time is about 30 minutes, while SEP requires at least 1.5 hours. Second, the analysis time is greatly shortened compared with the existing literature reports. The total time of the present invention is 3 minutes, while the time reported in the existing literature is at least 10 minutes. Therefore, the sample processing capacity and detection throughput can be greatly improved.

[0038] 4. Using PBS buffer as a substitute matrix can expand the application scope of the matrix for in vitro drug screening: The biological samples involved in the in vitro pharmacokinetic model are extensive and the background content varies greatly. The phosphate buffer screened in this method is close to the content of each in vitro biological matrix, which can eliminate the influence of matrix differences to the greatest extent. The plasma matrix mentioned in this method is quite different, and this method can be directly extended to more and closer in vitro evaluation models, such as liver microsome stability experiments, gastrointestinal fluid stability experiments and various permeability evaluation models. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 is the spectrum of experimental group 1 of embodiment 1 of the present invention;

[0041] Figure 2 is the spectrum of experimental group 2 of Example 1 of the present invention;

[0042] Figure 3 is the spectrum of experimental group 3 of Example 1 of the present invention;

[0043] Figure 4 is the spectrum of experimental group 4 of Example 1 of the present invention;

[0044] Figure 5 is the spectrum of experimental group 1 of Example 2 of the present invention;

[0045] Figure 6 is the spectrum of experimental group 2 of Example 2 of the present invention;

[0046] Figure 7 is the spectrum of embodiment 3 of the present invention;

[0047] Figure 8 is the standard curve of GABA in Example 4 of the present invention in the concentration range of 20-20000 nM (r>0.99);

[0048] Fig. 9 It is a spectrum of a single blank sample and a GABA quantitative lower limit sample in the GABA linear range and quantitative lower limit experiment of Example 4 of the present invention;

[0049] Fig.10 is the standard curve of GHB in the concentration range of 20-20000 nM (r>0.99) of Example 4 of the present invention;

[0050] Fig.11 It is a spectrum of a single blank sample and a GHB lower limit of quantitation sample in the GHB linear range and lower limit of quantitation experiment of Example 4 of the present invention;

[0051] Fig.12 This is the spectrum of the blood matrix (test sample) of Example 4 of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Experimental reagents: methanol, acetonitrile, isopropanol, and ammonia water were of chromatographic grade, and ammonium bicarbonate was of analytical grade.

[0054] Standard products: γ-hydroxybutyric acid sodium salt (purity ≥ 99%, purchased from Shanghai Yuansi Biaowu Technology Co., Ltd.), γ-aminobutyric acid (purity 98%, purchased from Beijing Liuhe Ningyuan Pharmaceutical Technology Co., Ltd.), γ-hydroxybutyric acid sodium-d6 (purity 98.2%, purchased from Shanghai Yuansi Biaowu Technology Co., Ltd.), γ-aminobutyric acid-d6 (purity 100%, purchased from MedChemExpress).

[0055] Consumables: Low-adsorption pipette tips with specifications of 1 mL, 300 μL, and 10 μL, 1.5 mL centrifuge tubes, and 2 mL 96-well round-bottom low-adsorption plates were all purchased from Eppendorf.

[0056] Instruments: Tecan D300e picoliter-up ultra-micropipette, purchased from Tecan (Shanghai) Trading Co., Ltd.; Centrifuge5810R high-speed centrifuge, purchased from Eppendorf; High-performance liquid chromatography tandem mass spectrometry (LC-MS / MS): liquid phase Shimadzu LC 30-AD, purchased from Shimadzu (Shanghai) Laboratory Equipment Co., Ltd.; automatic sample injector CTCPAL3-RSI, purchased from Guangzhou Zhida Laboratory Technology Co., Ltd.; mass spectrometer SCIEX Triple Quad 6500+, purchased from SCIEX.

[0057] γ-hydroxybutyric acid standard stock solution: Weigh γ-hydroxybutyric acid sodium salt and dissolve it in dimethyl sulfoxide to prepare a 10 mM γ-hydroxybutyric acid standard stock solution, and keep it at -80°C for use.

[0058] γ-hydroxybutyrate-d6 standard stock solution: Weigh γ-hydroxybutyrate sodium salt-d6 and dissolve it in dimethyl sulfoxide to prepare a 10mM γ-hydroxybutyrate-d6 standard stock solution, and keep it at -80°C for use.

[0059] γ-aminobutyric acid standard stock solution: Weigh γ-aminobutyric acid and dissolve it in dimethyl sulfoxide to prepare a 10 mM γ-aminobutyric acid standard stock solution, and keep it at -80°C for use.

[0060] γ-aminobutyric acid-d6 standard stock solution: Weigh γ-aminobutyric acid-d6 and dissolve it in dimethyl sulfoxide to prepare a 10 mM γ-aminobutyric acid-d6 standard stock solution, and keep it at -80°C for use.

[0061] Standard working solution: Take equal volumes of γ-hydroxybutyric acid standard stock solution and γ-aminobutyric acid standard stock solution, mix them, add dimethyl sulfoxide to dilute, and the concentrations of γ-hydroxybutyric acid and γ-aminobutyric acid after dilution are both 1 mM.

[0062] Stop solution containing internal standard: Measure the γ-hydroxybutyric acid-d6 standard stock solution and the γ-aminobutyric acid-d6 standard stock solution, dilute them to 1 mM with DMSO respectively, mix them in a volume ratio of 2:1, and then continue to dilute them with methanol solution to 1 μM for γ-hydroxybutyric acid-d6 and 500 nM for γ-aminobutyric acid-d6, and store them at 4°C for use.

[0063] Preparation of PBS buffer: Dissolve phosphate buffer powder (purchased from Thermo Fisher Scientific) in 500 mL of ultrapure water to prepare a 100 mM PBS solution with a pH of 7.2, then add 150 mM sodium chloride solution, adjust the pH with 1% phosphoric acid or 1 M sodium hydroxide solution to a pH of 7.4 ± 0.1, and store in a 4 °C refrigerator.

[0064] Calibration curve samples: The standard working solution was mixed with PBS buffer to obtain calibration curve samples with concentration gradients of 20, 50, 100, 300, 800, 2500, 5000 and 20000 nM for both γ-hydroxybutyric acid and γ-aminobutyric acid.

[0065] Quality control standard samples: Take the standard working solution and mix it with PBS buffer to prepare quality control standard samples with a concentration gradient of 20, 80, 1000 and 16000 nM;

[0066] Plasma spiked quality control samples: Take the standard working solution and mix it with the plasma matrix (purchased from BIOIVT) to prepare plasma spiked quality control samples with concentrations of 500, 2000 and 10000 nM;

[0067] Single blank sample: Take 100 μL of PBS buffer and mix it with 400 μL of stop solution containing internal standard to obtain a single blank sample;

[0068] Double blank sample: Take 100 μL PBS buffer and mix it with 400 μL methanol solution to obtain a double blank sample;

[0069] Sample to be tested: Take 100 μL of plasma matrix and mix it with 400 μL of stop solution containing internal standard to obtain the sample to be tested.

[0070] Liquid chromatography tandem mass spectrometry detection methods include:

[0071] The chromatographic and mass spectrometric conditions are shown in Table 1 below:

[0072] Table 1

[0073]

[0074]

[0075] Pre-treatment of calibration curve samples, quality control standard samples, plasma spiked quality control samples and single blank samples: 100 μL of the sample to be loaded on the machine was mixed with 400 μL of the stop solution containing the internal standard, the protein was precipitated, shaken for 10 min, centrifuged at 4000 rpm for 30 min, 100 μL of the supernatant was transferred to a 96-well plate, and loaded on the machine for testing.

[0076] Double blank sample pre-treatment: Take 100 μL of the sample to be loaded and mix it with 400 μL of methanol solution to precipitate protein, shake for 10 minutes, centrifuge at 4000 rpm for 30 minutes, take 100 μL of supernatant to 96-well plate, and load it for testing.

[0077] Example 1 Selection of mobile phase and chromatographic column

[0078] Experimental Group 1:

[0079] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium acetate added;

[0080] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium acetate added;

[0081] Column: ACQUITY UPLC BEH Amide 1.7 μm;

[0082] The gradient elution program was:

[0083] 0.01-0.2min: mobile phase A maintains 5%, mobile phase B maintains 95%;

[0084] 0.2-1.0min: mobile phase A: 5%→50%, mobile phase B: 95%→50%;

[0085] 1.0-1.8min: Mobile phase A maintains 50%, mobile phase B maintains 50%;

[0086] 1.80-1.81: Mobile phase A is 50%→5%, mobile phase B is 50%→95%;

[0087] 1.81-2.0: Mobile phase A is maintained at 5%, mobile phase B is maintained at 95%;

[0088] The flow rate was 0.6 mL / min.

[0089] Mass spectrometry conditions are the same as Table 1;

[0090] Sample preparation: dilute the γ-hydroxybutyric acid standard stock solution with dimethyl sulfoxide to a concentration of 1 μM; dilute the γ-aminobutyric acid standard stock solution with dimethyl sulfoxide to a concentration of 1 μM.

[0091] The spectrum results are as follows Figure 1 As shown, it can be seen that GABA and GHB are not retained.

[0092] Experimental Group 2:

[0093] The mobile phase, gradient elution procedure and samples were the same as those in Experimental Group 1. The chromatographic column was replaced with Atlantis Premier BEHZ-HILIC 1.7 μm. The mass spectrometry conditions were the same as those in Table 1. The results are shown in Figure 2 As shown in the figure, it can be seen that the retention of GABA is significantly improved, but GHB is not retained. This shows that the chromatographic column Atlantis Premier BEH Z-HILIC 1.7μm has significantly improved the retention of GABA.

[0094] Experimental group three:

[0095] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0096] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0097] The gradient elution procedure was the same as that of experimental group 1, and the chromatographic column was the same as that of experimental group 2;

[0098] The mass spectrometry conditions are the same as those in Table 1, and the samples are the same as those in experimental group 1. The results are as follows Figure 3 As shown, it can be seen that the retention of GHB has been significantly improved compared with experimental groups 1 and 2. This shows that adding ammonium bicarbonate to the mobile phase is beneficial to improving the retention of GHB.

[0099] Experimental Group 4:

[0100] Mobile phase A: 5% acetonitrile aqueous solution, to which 2 mM ammonium bicarbonate and ammonia were added, and the pH value was adjusted to 10.3 ± 0.1;

[0101] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0102] The gradient elution procedure was the same as that of experimental group 1, and the chromatographic column was the same as that of experimental group 2;

[0103] The mass spectrometry conditions are the same as those in Table 1, and the samples are the same as those in experimental group 1. The results are as follows Figure 4 As shown, it can be seen that the retention and peak shape of GHB and GABA are significantly improved, indicating that adjusting the pH value of mobile phase A has a favorable effect on the retention and peak shape of GHB and GABA.

[0104] In this embodiment, since GABA and GHB have relatively large polarity, two bonded phase chromatographic columns in hydrophilic interaction chromatography mode, BEH Amide and BEH Z-HILIC, were selected for trial. After the BEH Z-HILIC chromatographic column with better retention effect was determined, ammonium bicarbonate was added to mobile phases A and B, and ammonia water was added to mobile phase A, which significantly improved the retention and peak shape of GABA and GHB.

[0105] Example 2 Optimization of nonspecific adsorption

[0106] Experimental Group 1:

[0107] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium fluoride added;

[0108] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium fluoride added;

[0109] Chromatographic column: Atlantis Premier BEH Z-HILIC 1.7μm

[0110] Sample preparation: Take the GHB standard stock solution and dilute it with methanol solution to obtain two samples with concentrations of 10nM and 20nM;

[0111] The gradient elution procedure was the same as in Example 1, and the mass spectrometry conditions were the same as in Table 1. The results were as follows: Figure 5 As shown, it can be seen that there is no signal response at a concentration of 10 nM GHB.

[0112] Experimental Group 2:

[0113] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0114] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0115] The chromatographic columns were: Atlantis Premier BEH Z-HILIC 1.7 μm;

[0116] Sample: Take the GHB standard stock solution and dilute it step by step with dimethyl sulfoxide to obtain two samples with concentrations of 10 nM and 20 nM.

[0117] The gradient elution procedure was the same as in Example 1, and the mass spectrometry conditions were the same as in Table 1. The results were as follows: Figure 6 As shown, it can be seen that both 10 nM and 20 nM concentrations of GHB have obvious signal responses, and the relationship is about 1.7 times.

[0118] In this embodiment, since GHB contains a carboxyl group, it can undergo a certain chelation reaction with the metal on the LC-MS / MS instrument system to produce a nonspecific adsorption problem, resulting in a detection concentration lower than the actual concentration; by adding ammonium bicarbonate to the mobile phase, the chelation reaction can be effectively inhibited, thereby obtaining a more accurate detection result; similarly, there are hydroxyl groups in GBAB, and adding ammonium bicarbonate to the mobile phase can also effectively inhibit its chelation reaction with metals.

[0119] Example 3 Selection of solvent in stop solution

[0120] Experimental Group 1:

[0121] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0122] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0123] The chromatographic columns were: Atlantis Premier BEH Z-HILIC 1.7 μm;

[0124] Sample preparation: Take the GABA standard stock solution, dilute it to 100 μM with dimethyl sulfoxide, and then dilute it to 1 μM with acetonitrile;

[0125] The gradient elution procedure was the same as in Example 1, and the mass spectrometry conditions were the same as in Table 1. The results were as follows: Figure 7 shown.

[0126] Experimental Group 2:

[0127] Mobile phase A: 5% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0128] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0129] The chromatographic columns were: Atlantis Premier BEH Z-HILIC 1.7 μm;

[0130] Sample preparation: Take the GABA standard stock solution, dilute it to 100 μM with dimethyl sulfoxide, and then dilute it to 1 μM with methanol;

[0131] The gradient elution procedure was the same as in Example 1, and the mass spectrometry conditions were the same as in Table 1. The results were as follows: Figure 7 shown.

[0132] In this embodiment, since the ionization efficiency of organic solvents for compounds is different, good ionization efficiency can greatly improve the detection sensitivity of the target compound. Although acetonitrile and methanol are both commonly used organic solvents as protein precipitation stop solutions, there is no obvious difference in the signal response between the two for GHB, but for GABA, the ionization efficiency in methanol is significantly better than that in acetonitrile. At the same concentration, the signal enhancement can be more than 20 times. Therefore, the present invention preferably uses methanol as the solvent for the stop solution.

[0133] In Example 2 and Example 3, the pH of mobile phase A was not adjusted to 10.3±0.1, but this had no effect on nonspecific adsorption and the selection of the stop solution.

[0134] Example 4 Methodological Verification

[0135] 1-Linear range and lower limit of quantitation

[0136] Mobile phase A: 5% acetonitrile in water, to which 2 mM ammonium bicarbonate was added, and the pH was adjusted to 10.3 ± 0.1 with aqueous ammonia;

[0137] Mobile phase B: 95% acetonitrile in water with 2 mM ammonium bicarbonate added;

[0138] The chromatographic columns were: Atlantis Premier BEH Z-HILIC 1.7 μm;

[0139] The gradient elution program was:

[0140] 0.01-0.2min: mobile phase B maintained at 100%;

[0141] 0.2-2.2min: mobile phase A: 0→40%, mobile phase B: 100%→60%;

[0142] 2.2-2.8min: Mobile phase A maintained at 40%, mobile phase B maintained at 60%;

[0143] 2.8-2.81min: mobile phase A: 40%→0%, mobile phase B: 60%→100%;

[0144] 2.81-3.0min: mobile phase B maintained at 100%;

[0145] The flow rate was 0.6 mL / min.

[0146] The gradient system program was optimized to 3 min, and the extension of the liquid phase time had little effect on the results. It was mainly to further increase the separation of the target analytes in the matrix.

[0147] Mass spectrometry conditions are the same as Table 1;

[0148] Samples: calibration curve samples and single blank samples;

[0149] GABA test results are as follows Figure 8 As shown, the typical regression equation after fitting is y = 4.14979e -5 X+6.44252e -4 (r>0.99), with good linear relationship in the range of 20-20000nM, and the lower limit of quantification is at least 20nM. Fig. 9 Spectra of single blank sample and GABA quantitative lower limit sample (calibration curve sample of 20 nM);

[0150] GHB test results are as follows Fig.10 As shown, the typical regression equation after fitting is y = 6.22606e -4 X+0.00599 (r>0.99), good linear relationship within 20-20000nM, and the lower limit of quantification is at least 20nM, Fig.11 The graphs are for a single blank sample and a GHB lower limit of quantitation sample (calibration curve sample of 20 nM).

[0151] 2-Precision and Accuracy

[0152] The chromatographic conditions and mass spectrometry conditions were the same as those in the linear range and lower limit of quantitation experiments;

[0153] Samples: quality control standard samples, plasma spiked quality control samples, double blank samples and samples to be tested;

[0154] Among them, the precision and accuracy of the quality control samples of GABA and GHB in the surrogate matrix are shown in Table 2, and the precision and accuracy of the quality control samples of GABA and GHB in spiked plasma are shown in Table 3.

[0155] After determination (chromatographic conditions and mass spectrometry conditions are the same as the linear range and quantitative limit), the concentration of GABA in the blood matrix of the test sample was 107.2nM and the concentration of GHB was 211.5nM. The spectrum is as follows Fig.12 shown.

[0156] Table 2

[0157]

[0158] Table 3

[0159]

[0160] As can be seen from Table 2, the average accuracy of GABA in the quality control standard samples was between 95.2% and 104% of the theoretical value, and the precision was less than 16.4%, among which the average accuracy of the quality control standard samples of LLOQ (20nM) was 104%, the intra-batch precision was 16.4%, and the signal-to-noise ratio was greater than 5; the average accuracy of GBH was between 91.7% and 109% of the theoretical value, and the precision was less than 11.3%, among which the average accuracy of the quality control standard samples of LLOQ (20nM) was 99.3%, the intra-batch precision was 11.3%, and the signal-to-noise ratio was greater than 5; all met the methodological requirements.

[0161] As can be seen from Table 3, in the plasma spiked quality control samples, the average accuracy of GABA is between 101% and 109% of the theoretical value, and the precision is less than 2.79%; the average accuracy of GBH is between 84.4% and 98.8% of the theoretical value, and the precision is less than 4.24%, which meets the methodological requirements. It should be noted that in the methodological validation of endogenous substance detection, the amount of the analyte added needs to be more than three times the background concentration or statistically sufficient to show a higher concentration than the endogenous substance. Since the measured concentration of GABA in the blood matrix is ​​107.2nM and the concentration of GHB is 211.5nM, the minimum concentration of the standard working solution after dilution in the plasma spiked quality control samples is preferably 500nM, which can well distinguish the background content in plasma.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid, characterized in that: Liquid chromatography tandem mass spectrometry was used for detection, wherein ammonium bicarbonate was added to both mobile phase A and mobile phase B, and the pH value of mobile phase A was 10.3±0.1; The chromatographic column adopts a chromatographic column with a zwitterionic bonded phase and adopts a hydrophilic interaction chromatography mode.

2. The detection method according to claim 1, characterized in that Mobile phase A was 5% acetonitrile in water containing 2 mM ammonium bicarbonate, and the pH was adjusted with aqueous ammonia; Mobile phase B was 95% acetonitrile in water; The chromatographic column was Atlantis Premier BEH Z-HILIC.

3. The detection method according to claim 1, characterized in that Chromatographic conditions also include: Liquid phase: Shimadzu LC 30-AD The injection volume was 3 μL.

4. The detection method according to claim 1, characterized in that Prepare standard stock solutions: weigh γ-hydroxybutyric acid sodium salt, γ-aminobutyric acid, γ-hydroxybutyric acid sodium-d6 and γ-amino acid-d6 respectively and dissolve them in solvents to form corresponding standard stock solutions; Prepare standard working solution: mix the γ-hydroxybutyric acid standard stock solution and the γ-aminobutyric acid standard stock solution, add solvent to dilute, and form a standard working solution containing γ-hydroxybutyric acid and γ-aminobutyric acid; Prepare a stop solution containing an internal standard: mix the γ-sodium hydroxybutyrate-d6 standard stock solution and the γ-amino acid-d6 standard stock solution, dilute with a solvent, and then add methanol to dilute to the final concentration to obtain a stop solution containing an internal standard; PBS buffer: pH 7.4 ± 0.

1.

5. The detection method according to claim 4, characterized in that: The solvent of the standard stock solution is dimethyl sulfoxide, and the concentration of the standard stock solution is 5-30mM; the concentration of the γ-hydroxybutyric acid standard working solution and the γ-aminobutyric acid standard working solution are both 0.5-2mM; When preparing the stop solution containing the internal standard, the stock solutions of γ-hydroxybutyrate-d6 and γ-amino acid-d6 were diluted to 0.5-2 mM before adding methanol. After further dilution with methanol, the concentration of γ-hydroxybutyrate-d6 was 0.5-2 μM, and the concentration of γ-amino acid-d6 was 250-1000 nM.

6. The detection method according to claim 4, characterized in that: Preparation of calibration curve samples: Take the standard working solution, mix it with PBS buffer, and prepare calibration curve samples with concentrations of 20, 50, 100, 300, 800, 2500, 5000, and 20000 nM; Preparation of quality control standard samples: Take the standard working solution, mix it with PBS buffer, and prepare quality control standard samples with a concentration gradient of 20, 80, 1000 and 16000 nM; Preparation of plasma spiked quality control samples: Take the standard working solution, mix it with the plasma matrix, and prepare plasma spiked quality control samples with concentrations of 500, 2000, and 10000 nM. Single blank sample preparation: Take PBS buffer and mix it with the stop solution containing the internal standard to obtain a single blank sample; Double blank sample preparation: Take PBS buffer and mix it with methanol solution to obtain a double blank sample; Preparation of test samples: Take plasma matrix and mix it with the stop solution containing internal standard to obtain the test sample.

7. The detection method according to claim 4, characterized in that: The sample processing method before the machine is as follows: take the calibration curve sample, quality control standard sample and double blank sample respectively, and add 2-5 times the volume of the stop solution containing the internal standard respectively, take the double blank sample, add 2-5 times the volume of the methanol solution thereto, shake for 5-20 minutes, centrifuge, and take the supernatant for liquid chromatography tandem mass spectrometry analysis.

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