A method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid

Abstract

This invention discloses a method for detecting γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB) using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Ammonium bicarbonate is added to both mobile phase A and mobile phase B, and ammonia is also added to mobile phase A. The chromatographic column is a zwitterionic column, and hydrophilic interaction chromatography mode is used. The detection method provided by this invention has high sensitivity, raising the limits of quantification for GABA and GHB to 20 nM. The retention and peak shape of GABA and GHB in the chromatogram are significantly enhanced, and the analysis speed is significantly improved. Furthermore, the use of PBS buffer as an alternative matrix expands the range of matrix applications for in vitro drug screening.

Description

Technical Field

[0001] This invention relates to the field of drug detection, and in particular to a method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays a crucial role in the treatment of various neurological diseases, such as epilepsy, anxiety disorders, Alzheimer's disease, intellectual decline, primary insomnia, and cancer biotherapy. Gamma-hydroxybutyric acid (GHB) is a short-chain fatty acid, also an inhibitory neurotransmitter like GABA, widely found in various tissues of mammals. It is primarily used to treat anesthesia, insomnia, and clinical depression, but due to its potential to cause excitatory, euphoric, anxiety-inducing, and hypnotic effects depending on dosage, it is regulated by the National Medical Products Administration. However, GHB and GABA can interconvert in the body; GHB is both a metabolite and a precursor of GABA. Therefore, establishing methods for simultaneously detecting GHB and GABA during drug development can effectively determine the conversion of related prodrugs into GABA and GHB in the body, allowing for more efficient evaluation of the efficacy, 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 the small molecular weight and high polarity of GABA and GHB, they are not easily retained in traditional reversed-phase chromatography. Public literature often uses derivatization to increase the hydrophobicity of the compounds in order to enhance their retention.

[0004] In summary, existing techniques for analyzing GABA and GHB mainly suffer from the following problems:

[0005] 1. Limited detection limit: Although reversed-phase chromatography can preserve GHB, its detection limit is relatively high. The reported lower limit of quantification for GHB 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, thus affecting the accurate assessment of the compound's metabolic stability and content. In addition, the sample processing methods often use complex and cumbersome SPE and LLE.

[0006] 2. Insufficient retention and long analysis time: GABA and GHB have small molecular weights and high polarity. Current liquid chromatography methods for the simultaneous determination of both mainly use reversed-phase chromatography. Compared with GHB, GABA has stronger polarity. Both are not retained on traditional reversed-phase C18 columns. This is mainly achieved by reducing the flow rate and extending the liquid chromatography time. The reported analysis time is over 10 minutes, and effective retention, separation and elution cannot be achieved on the chromatographic column, which can easily lead to interference and matrix effects.

[0007] 3. Sample processing is time-consuming and has limited throughput: Due to the structural characteristics of GABA and GHB, such as high polarity, difficulty in retention, and large matrix interference, derivatization is required in sample processing to enhance retention and ionization capabilities, or SPE and other methods are used to purify the sample 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 γ-aminobutyric acid and γ-hydroxybutyric acid, which has a low limit of quantitation, strong retention and separation ability, can reduce interference and matrix effects, and has a short detection time.

[0009] To solve the above-mentioned technical problems, the present invention provides a method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid, which uses 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 used is a zwitterionic bonded phase, and the hydrophilic interaction chromatography mode is employed.

[0011] In this invention, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for detection. Adding ammonium bicarbonate to the mobile phase can increase the retention of GHB and eliminate the non-specific adsorption of GHB at low concentrations. Adding ammonia to mobile phase A to adjust its pH to 10.3±0.1 can effectively improve the retention and peak shape of GHB, and the GHB signal 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 zwitterionic bonding phase is used.

[0012] In one specific scheme, the mobile phase A is a 5% aqueous solution of acetonitrile, containing 2 mM ammonium bicarbonate, and the pH is adjusted with ammonia.

[0013] Mobile phase B was a 95% aqueous solution of acetonitrile; the chromatographic column was an Atlantis Premier BEH Z-HILIC.

[0014] In one specific implementation, the chromatographic conditions further include a gradient elution program as follows:

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

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

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

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

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

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

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

[0022] In one specific embodiment, standard stock solutions are prepared by weighing sodium γ-hydroxybutyrate, γ-aminobutyric acid, sodium γ-hydroxybutyrate-d6, and γ-amino acid-d6 respectively and dissolving them in a solvent to form the corresponding standard stock solutions; preferably, dimethyl sulfoxide is used as the solvent, and the concentration of the standard stock solution is preferably 5-30 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, and 30 mM, preferably an integer to facilitate dilution.

[0023] In one specific embodiment, a standard working solution is prepared by mixing γ-hydroxybutyric acid (GHB) standard stock solution and γ-aminobutyric acid (GABA) standard stock solution, and then diluting with a solvent to form a standard working solution containing GHB and GABA. Preferably, the concentrations of GHB and GABA in the standard working solution are the same, for example, both can be 0.5 mM, 1 mM, 1.5 mM, 2.0 mM, etc., to facilitate further 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, dimethyl sulfoxide (DMSO) is preferred.

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

[0025] In one specific implementation, the main function of PBS buffer is to be used as an alternative 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 alternative matrix for accurate quantification. The present invention preferably uses 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. It can reduce matrix differences for accurate quantification and promote its application.

[0026] In one specific embodiment, the calibration curve sample is a mixture of a standard working solution and a PBS buffer solution. The preparation method is, for example, to take the standard working solution, mix it thoroughly with the PBS buffer solution, and dilute 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 one specific implementation, the quality control standard sample is a mixed solution of standard working solution and PBS buffer. The preparation method is, for example, to take the standard working solution, mix it thoroughly with PBS buffer, and prepare quality control standard samples with concentration gradients of 20, 80, 1000 and 16000 nM, wherein the concentrations of γ-aminobutyric acid and γ-hydroxybutyric acid are gradient concentration values.

[0028] In one specific embodiment, the spiked plasma control sample is a mixture of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB) with the plasma matrix. The preparation method, for example, involves taking a standard working solution, thoroughly mixing it with the plasma matrix, and then gradient-diluting the GABA and GHB concentrations of the standard working solution to 500, 2000, and 10000 nM to obtain the spiked plasma control sample. It should be noted that the plasma matrix itself contains GABA and GHB. The aforementioned concentrations of 500, 2000, and 10000 nM refer to the concentrations after dilution of the standard working solution, not the actual total concentrations of GABA and GHB in the spiked plasma control sample, because the original concentrations of GABA and GHB in the plasma matrix are unknown and need to be measured.

[0029] Since this invention uses PBS buffer as an alternative matrix, there are two types of quality control samples: quality control standard samples and plasma spiked quality control samples. The former can be used to examine the accuracy of this method with PBS buffer as an alternative matrix, while the latter is used to evaluate the accuracy of this method in real plasma samples.

[0030] In one specific embodiment, the blank sample is a mixed solution formed by PBS buffer and a stop solution containing an internal standard. The preparation method is, for example, to take PBS buffer and add the stop solution containing an internal standard and mix thoroughly. The two can be mixed at a volume ratio of 1:2-8, preferably 100 μL of PBS buffer and 400 μL of stop solution containing an internal standard.

[0031] In one specific embodiment, the double blank sample is a mixed solution formed by PBS buffer and methanol solution. The preparation method is, for example, to take PBS buffer and add methanol solution and mix thoroughly. The two can be mixed at a volume ratio of 1:2-8, preferably 100 μL of PBS buffer and 400 μL of methanol solution.

[0032] In one specific embodiment, the sample to be tested is a mixed solution formed by plasma matrix and a stop solution containing internal standard. The preparation method is, for example, to take plasma matrix and add stop solution containing internal standard and mix thoroughly. The two can be mixed at a volume ratio of 1:2-8, preferably 100 μL of plasma matrix and 400 μL of stop solution containing internal standard.

[0033] In one specific implementation, the sample preparation method before analysis is as follows: Take the calibration curve sample, quality control standard sample, and single blank sample respectively, and add 2-5 times the volume of stop solution containing internal standard to each; take the double blank sample, add 2-5 times the volume of methanol solution to each, shake for 5-20 minutes, centrifuge, and take the supernatant for liquid chromatography-tandem mass spectrometry analysis. After sample preparation, the GABA signal can be enhanced by at least 20 times.

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

[0035] 1. Improved detection sensitivity: The lower limit of quantitation for GABA and GHB was raised to 20 nM, expanding the range of detection concentrations;

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

[0037] 3. Improved analysis speed: First, in the sample processing stage, protein precipitation time is greatly reduced compared to SEP, with protein precipitation time being approximately 30 minutes, while SEP requires at least 1.5 hours; second, the analysis time is greatly reduced compared to existing literature reports, with the total time of this invention being 3 minutes, while the time reported in existing literature is at least 10 minutes; therefore, it can greatly improve sample processing capacity and detection throughput.

[0038] 4. Using PBS buffer as an alternative matrix expands the range of matrix applications for in vitro drug screening: In vitro pharmacokinetic models involve a wide variety of biological samples with significant differences in background concentrations. The phosphate buffer used in this method has a concentration close to that of various in vitro biological matrices, minimizing the impact of matrix differences. The plasma matrix mentioned in this method exhibits considerable variation; therefore, this method can be directly extended to more closely related in vitro evaluation models, such as liver microsomal stability assays, gastrointestinal fluid stability assays, and various permeability assessment models. Attached Figure Description

[0039] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 This is the spectrum of Experimental Group 1 in Embodiment 1 of the present invention;

[0041] Figure 2 This is the spectrum of experimental group two in Embodiment 1 of the present invention;

[0042] Figure 3 This is the spectrum of experimental group three in Embodiment 1 of the present invention;

[0043] Figure 4 This is the spectrum of experimental group four in Embodiment 1 of the present invention;

[0044] Figure 5 This is the spectrum of experimental group one in Embodiment 2 of the present invention;

[0045] Figure 6 This is the spectrum of experimental group two in Embodiment 2 of the present invention;

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

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

[0048] Figure 9 These are the spectra of a single blank sample and a sample with the lower limit of quantitation of GABA in the GABA linear range and quantitation limit experiment of Embodiment 4 of the present invention.

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

[0050] Figure 11 These are the spectra of a single blank sample and a sample with the lower limit of quantitation of GHB in the GHB linear range and lower limit of quantitation experiment of Embodiment 4 of the present invention.

[0051] Figure 12 This is a spectrum of the blood matrix (sample to be tested) in Example 4 of the present invention. Detailed Implementation

[0052] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

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

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

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

[0056] Instruments: Tecan D300e Pipe-Sized Micropipettes, purchased from Tecan (Shanghai) Trading Co., Ltd.; Centrifuge 5810R High-Speed ​​Centrifuge, purchased from Eppendorf; High-Performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS): Shimadzu LC 30-AD, purchased from Shimadzu (Shanghai) Laboratory Equipment Co., Ltd.; CTCPAL3-RSI Autosampler, purchased from Guangzhou Zhida Laboratory Technology Co., Ltd.; SCIEX Triple Quad 6500+ Mass Spectrometer, purchased from SCIEX.

[0057] γ-Hydroxybutyric acid standard stock solution: Weigh out sodium γ-hydroxybutyrate and dissolve it in dimethyl sulfoxide to prepare a 10 mM γ-hydroxybutyric acid standard stock solution, and keep it at -80℃ for later use.

[0058] γ-Hydroxybutyric acid-d6 standard stock solution: Weigh out sodium γ-hydroxybutyrate-d6 and dissolve it in dimethyl sulfoxide to prepare a 10 mM γ-hydroxybutyric acid-d6 standard stock solution, and keep it at -80℃ for later 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℃ for later 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℃ for later use.

[0061] Standard working solution: Mix equal volumes of γ-hydroxybutyric acid standard stock solution and γ-aminobutyric acid standard stock solution, and dilute with dimethyl sulfoxide. The concentrations of γ-hydroxybutyric acid and γ-aminobutyric acid after dilution are both 1 mM.

[0062] Termination solution containing internal standard: Measure γ-hydroxybutyric acid-d6 standard stock solution and γ-aminobutyric acid-d6 standard stock solution, dilute them to 1 mM with DMSO, mix them at a volume ratio of 2:1, and then further dilute them with methanol solution to 1 μM γ-hydroxybutyric acid-d6 and 500 nM γ-aminobutyric acid-d6. Store at 4℃ for later use.

[0063] PBS buffer preparation: 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 and adjust the pH with 1% phosphoric acid or 1 M sodium hydroxide solution to a pH range of 7.4 ± 0.1. Store at 4°C.

[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 γ-hydroxybutyric acid and γ-aminobutyric acid.

[0065] Quality control standard samples: Take the standard working solution, mix it with PBS buffer, and prepare quality control standard samples with concentration gradients 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 of PBS buffer and mix it with 400 μL of methanol solution to obtain a double blank sample;

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

[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] Pretreatment of calibration curve samples, quality control standard samples, plasma spiked quality control samples and single blank samples before testing: Mix 100 μL of the sample to be tested with 400 μL of stop solution containing internal standard, precipitate the protein, shake for 10 min, centrifuge at 4000 rpm for 30 min, take 100 μL of supernatant into a 96-well plate and test.

[0076] Pretreatment of double blank samples before testing: Mix 100 μL of the sample to be tested with 400 μL of methanol solution to precipitate the protein, shake for 10 min, centrifuge at 4000 rpm for 30 min, take 100 μL of supernatant into a 96-well plate and test.

[0077] Example 1: Selection of Mobile Phase and Chromatographic Column

[0078] Experimental Group 1:

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

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

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

[0082] The gradient elution procedure is as follows:

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

[0084] 0.2-1.0 min: Mobile phase A changes from 5% to 50%, mobile phase B changes from 95% to 50%;

[0085] 1.0-1.8 min: Mobile phase A maintained at 50%, mobile phase B maintained at 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%, and 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: Take γ-hydroxybutyric acid standard stock solution and dilute it with dimethyl sulfoxide to a concentration of 1 μM; take γ-aminobutyric acid standard stock solution and dilute it with dimethyl sulfoxide to a concentration of 1 μM.

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

[0092] Experimental Group 2:

[0093] The mobile phase, gradient elution program, and samples were the same as in Experimental Group 1. The chromatographic column was changed to an Atlantis Premier BEHZ-HILIC 1.7 μm, and the mass spectrometry conditions were the same as in Table 1. The results are as follows: Figure 2 As shown, GABA retention is significantly improved, but GHB is not retained. This indicates that the Atlantis Premier BEH Z-HILIC 1.7μm column significantly improves GABA retention.

[0094] Experimental Group 3:

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

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

[0097] The gradient elution program was the same as in Experiment 1, and the chromatographic column was the same as in Experiment 2.

[0098] The mass spectrometry conditions were the same as in Table 1, the samples were the same as in Experimental Group 1, and the results were as follows: Figure 3 As shown, the retention of GHB was significantly improved compared to experimental groups one and two. This indicates that adding ammonium bicarbonate to the mobile phase is beneficial for improving GHB retention.

[0099] Experimental Group 4:

[0100] Mobile phase A: 5% acetonitrile aqueous solution, with 2 mM ammonium bicarbonate added, and then ammonia added to adjust the pH to 10.3 ± 0.1;

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

[0102] The gradient elution program was the same as in Experiment 1, and the chromatographic column was the same as in Experiment 2.

[0103] The mass spectrometry conditions were the same as in Table 1, the samples were the same as in Experimental Group 1, and the results were as follows: Figure 4 As shown, the retention and peak shape of GHB and GABA are significantly improved, indicating that adjusting the pH of mobile phase A has a beneficial effect on the retention and peak shape of GHB and GABA.

[0104] In this embodiment, due to the high polarity of GABA and GHB, two bonded phase chromatographic columns using hydrophilic interaction chromatographic mode, BEH Amide and BEH Z-HILIC, were selected for testing. After determining that the BEH Z-HILIC column had better retention effect, ammonium bicarbonate was added to mobile phases A and B, and ammonia was added to mobile phase A, which significantly improved the retention and peak shape of GABA and GHB.

[0105] Example 2: Optimization of Non-specific Adsorption

[0106] Experimental Group 1:

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

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

[0109] The chromatographic column was: Atlantis Premier BEH Z-HILIC 1.7μm.

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

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

[0112] Experimental Group 2:

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

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

[0115] The chromatographic column was: Atlantis Premier BEH Z-HILIC 1.7μm;

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

[0117] The gradient elution procedure is the same as in Example 1, the mass spectrometry conditions are the same as in Table 1, and the results are as follows: Figure 6 As shown, both 10 nM and 20 nM GHB concentrations showed significant signal responses, with a relationship of approximately 1.7 times.

[0118] In this embodiment, because GHB contains carboxyl groups, it can chelate with metals in the LC-MS / MS instrument system, resulting in non-specific adsorption and causing the detected concentration to be lower than the actual concentration. By adding ammonium bicarbonate to the mobile phase, the chelation can be effectively suppressed, thereby obtaining more accurate detection results. Similarly, GBAB also contains hydroxyl groups, and adding ammonium bicarbonate to the mobile phase can also effectively suppress its chelation with metals.

[0119] Example 3: Selection of Solvent in Termination Solution

[0120] Experimental Group 1:

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

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

[0123] The chromatographic column was: Atlantis Premier BEH Z-HILIC 1.7μm;

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

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

[0126] Experimental Group 2:

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

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

[0129] The chromatographic column was: Atlantis Premier BEH Z-HILIC 1.7μm;

[0130] Sample preparation: Take 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 is the same as in Example 1, the mass spectrometry conditions are the same as in Table 1, and the results are as follows: Figure 7 As shown.

[0132] In this embodiment, since organic solvents have different ionization efficiencies for compounds, 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 termination solutions, for GHB, there is no significant difference in the signal response between the two. However, 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 greater. Therefore, the present invention prefers methanol as the solvent for the termination solution.

[0133] In Examples 2 and 3, the pH of the mobile phase A was not adjusted to 10.3 ± 0.1, but this did not affect the nonspecific adsorption or the selection of the termination solution.

[0134] Example 4 Methodological Validation

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

[0136] Mobile phase A: 5% aqueous acetonitrile solution with 2 mM ammonium bicarbonate added, and pH adjusted to 10.3 ± 0.1 with ammonia.

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

[0138] The chromatographic column was: Atlantis Premier BEH Z-HILIC 1.7μm;

[0139] The gradient elution procedure is as follows:

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

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

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

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

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

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

[0146] The gradient system program was optimized to 3 minutes. The extension of the liquid phase time had little impact on the results and was mainly to further increase the separation of the target analyte 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 fitted canonical regression equation is y = 4.14979e -5 X+6.44252e -4 (r>0.99), exhibiting good linearity in the range of 20-20000 nM, with a lower limit of quantitation of at least 20 nM. Figure 9 The spectra of a single blank sample and a GABA lower limit of quantification sample (calibration curve sample at 20 nM);

[0150] The test results of GHB are as follows Figure 10 As shown, the fitted canonical regression equation is y = 6.22606e -4 X + 0.00599 (r > 0.99) showed good linearity in the range of 20–20000 nM, with a lower limit of quantitation of at least 20 nM. Figure 11 The spectra are for a single blank sample and a GHB limit of quantitation sample (calibration curve sample at 20 nM).

[0151] 2-Precision and accuracy

[0152] Chromatographic and mass spectrometry conditions were the same as those used in experiments on linear range and lower limit of quantitation.

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

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

[0155] After determination (under the same chromatographic and mass spectrometric conditions as the linear range and lower limit of quantitation), the concentration of GABA in the blood matrix of the test sample was 107.2 nM, and the concentration of GHB was 211.5 nM. The chromatogram is shown below. Figure 12 As shown.

[0156] Table 2

[0157]

[0158] Table 3

[0159]

[0160] Table 2 shows that the average accuracy of GABA in the quality control standard samples is between 95.2% and 104% of the theoretical value, and the precision is less than 16.4%. Among them, the average accuracy of the LLOQ (20nM) quality control standard samples is 104%, the intra-batch precision is 16.4%, and the signal-to-noise ratio is greater than 5. The average accuracy of GBH is between 91.7% and 109% of the theoretical value, and the precision is less than 11.3%. Among them, the average accuracy of the LLOQ (20nM) quality control standard samples is 99.3%, the intra-batch precision is 11.3%, and the signal-to-noise ratio is greater than 5. All meet the methodological requirements.

[0161] Table 3 shows that in the plasma spiked control samples, the average accuracy of GABA was between 101% and 109% of the theoretical value, with a precision of less than 2.79%; the average accuracy of GBH was between 84.4% and 98.8% of the theoretical value, with a precision of less than 4.24%, meeting the methodological requirements. It should be noted that in the methodological validation of endogenous substance detection, the amount of 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 concentration of GABA in the measured blood matrix is ​​107.2 nM and the concentration of GHB is 211.5 nM, the minimum preferred concentration of the diluted standard working solution in the plasma spiked control samples is 500 nM, which can effectively distinguish the background content in the 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 within the scope of protection of the present invention.

Claims

1. A method for detecting γ-aminobutyric acid and γ-hydroxybutyric acid, characterized by, The detection is performed by using liquid chromatography tandem mass spectrometry, wherein: the mobile phase A is 5% acetonitrile aqueous solution, the pH value of which is 10.3±0.1, and the mobile phase A contains 2mM ammonium bicarbonate; the mobile phase B is 95% acetonitrile aqueous solution, wherein the mobile phase B contains 2mM ammonium bicarbonate; the chromatographic column is Atlantis Premier BEH Z-HILIC, and a hydrophilic interaction chromatography mode is adopted; the mass spectrometry conditions are as follows: the mass spectrometer is SCIEX Triple Quad 6500+; the ion source is ESI; the scanning mode is MRM; in the positive ion mode, the Q1 Mass of γ-aminobutyric acid is 104.1m / z, the Q1 Mass is 69.1m / z, the DP is 70V, and the CE is 21V; in the positive ion mode, the Q1 Mass of the γ-aminobutyric acid internal standard is 110.1m / z, the Q1 Mass is 92.3m / z, the DP is 70V, and the CE is 15V; in the negative ion mode, the Q1 Mass of γ-hydroxybutyric acid is 103.1m / z, the Q1 Mass is 85.1m / z, the DP is-50V, and the CE is-11V; in the negative ion mode, the Q1 Mass of the γ-hydroxybutyric acid internal standard is 109.1m / z, the Q1 Mass is 90.1m / z, the DP is-50V, and the CE is-14V; the preparation of the sample to be detected: take the plasma matrix, add the internal standard-containing termination solution, and mix well; the volume ratio of the plasma matrix to the internal standard-containing termination solution is 1:2-8; the sample treatment method before machine operation is as follows: take the calibration curve sample, the quality control standard sample and the double blank sample respectively, and add 2-5 times the volume of the internal standard-containing termination solution to each of them; take the double blank sample, add 2-5 times the volume of the methanol solution to it, shake for 5-20 minutes, and then centrifuge and take the supernatant for liquid chromatography tandem mass spectrometry analysis; the termination solution is methanol.

2. The detection method of claim 1, wherein, The chromatographic conditions further include: the liquid phase is Shimadzu LC 30-AD the injection volume is 3μL.

3. The detection method as described in claim 1, characterized in that, preparing the standard stock solution: respectively weigh γ-hydroxybutyric acid sodium salt, γ-aminobutyric acid, γ-hydroxybutyric acid sodium-d6 and γ-amino acid-d6, and dissolve them in a solvent to form corresponding standard stock solutions; preparing the standard working solution: mix the γ-hydroxybutyric acid standard stock solution and the γ-aminobutyric acid standard stock solution, dilute with a solvent, and form a standard working solution containing γ-hydroxybutyric acid and γ-aminobutyric acid; preparing the internal standard-containing termination solution: mix the γ-hydroxybutyric acid sodium-d6 standard stock solution and the γ-amino acid-d6 standard stock solution, dilute with a solvent, and then add methanol to dilute to a final concentration to obtain the internal standard-containing termination solution; the PBS buffer has a pH value of 7.4±0.

1.

4. The detection method of claim 3, wherein, 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 is 0.5-2mM. When preparing the termination solution containing the internal standard, the stock solution of sodium γ-hydroxybutyric acid-d6 and γ-amino acid-d6 was diluted to 0.5-2 mM before adding methanol. After further dilution with methanol, the concentration of sodium γ-hydroxybutyric acid-d6 was 0.5-2 μM, and the concentration of γ-amino acid-d6 was 250-1000 nM.

5. The detection method as described in claim 3, characterized in that, Preparation of calibration curve samples: Take the standard working solution and mix it with PBS buffer to 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 and mix it with PBS buffer to prepare quality control standard samples with concentration gradients of 20, 80, 1000, and 16000 nM; Preparation of plasma spiked quality control samples: Take the standard working solution and mix it with plasma matrix to prepare plasma spiked quality control samples with concentrations of 500, 2000, and 10000 nM; Preparation of single blank samples: Take PBS buffer and mix it with the termination solution containing the internal standard to obtain single blank samples; Preparation of double blank samples: Take PBS buffer and mix it with methanol solution to obtain double blank samples.

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