Method for detecting aceclodine in biological sample and application of aceclodine in pharmacokinetics

Abstract

The invention discloses a method for detecting aceclodine in a biological sample and application of the aceclodine in pharmacokinetics. The detection method comprises the following steps: taking a biological sample, adding 0.1-10 times of an aqueous solution containing a stabilizer into the biological sample, and uniformly mixing to obtain a biological sample solution; quantitatively transferring the biological sample solution, sequentially adding the internal standard substance working solution and the organic solvent, uniformly mixing in a vortex manner, and centrifuging; quantitatively transferring the obtained supernatant, adding pure water, uniformly mixing by vortex, centrifuging, and taking the supernatant to obtain a biological sample test solution; preparing a series of vinegar clinidine standard test solutions for the standard curve; and performing test analysis on the biological sample test solution and a series of vinegar clinidine standard test solutions for the standard curve by liquid chromatography-tandem mass spectrometry (LC-MS / MS). According to the invention, the method for detecting the aceclodine in the biological sample is applied to the pharmacokinetic research of the aceclodine in the living body, so that the technical blank in the aspect of aceclodine detection at present is filled up.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis and detection, and specifically relates to a liquid chromatography tandem mass spectrometry (LC-MS / MS) detection method for aceclidinium concentration in a biological sample and an application thereof. Background Art

[0002] Acetocline is a parasympathomimetic drug that can be used to treat diseases such as presbyopia. Its chemical structure is: The molecular weight is 169.2 and the chemical formula is C9H 15 NO2. Due to the small molecular weight and high polarity of aceclidin, serious matrix interference, ester bonds in the molecular structure, unstable chemical properties, and easy hydrolysis in biological matrices, these characteristics pose great challenges to the analysis of aceclidin in vivo concentration. Currently, there are no reports on methods for quantitative detection of aceclidin concentration in biological samples. However, detection methods with high specificity and good sensitivity are very important for non-clinical and clinical research on aceclidin. Summary of the invention

[0003] In order to solve the above-mentioned technical problems, one of the objectives of the present invention is to establish a method for detecting aceclidinium in biological samples with high sensitivity, strong specificity, fast analysis speed, small matrix effect, simple operation and good repeatability.

[0004] The second purpose of the present invention is to apply the detection method of aceclidin in biological samples to the study of aceclidin pharmacokinetics in vivo, so as to fill the current technical gap in aceclidin detection.

[0005] To achieve the above object, the technical solution adopted by the present invention is: a method for detecting aceclidin in a biological sample, using liquid chromatography tandem mass spectrometry LC-MS / MS method, the detection method comprises the following steps:

[0006] 1) Biological sample pretreatment: taking a biological sample, adding 0.1-10 times the amount of an aqueous solution containing a stabilizer thereto, and mixing well to obtain a biological sample solution;

[0007] 2) Preparation of biological sample test solution: pipette the biological sample solution, add the internal standard working solution and the organic solvent in sequence, vortex mix, and centrifuge; pipette the obtained supernatant, add pure water, vortex mix, centrifuge, take the supernatant, and obtain the biological sample test solution;

[0008] 3) Preparation of aceclidin series standard test solution for standard curve: pipette the aceclidin series standard working solution diluted with blank biological sample solution, add internal standard working solution and organic solvent in sequence, vortex mix, centrifuge; pipette the obtained supernatant, add pure water, vortex mix, centrifuge, take the supernatant, and obtain the aceclidin series standard test solution for standard curve;

[0009] 4) The biological sample test solution and the standard curve are tested and analyzed using aceclidinium series standard test solution and liquid chromatography tandem mass spectrometry LC-MS / MS.

[0010] Furthermore, in the above-mentioned method for detecting aceclidin in biological samples, the biological samples include biological samples from animals administered with aceclidin and biological samples from animals not administered with aceclidin, and the biological samples are plasma samples or tissue samples.

[0011] Furthermore, in the above-mentioned method for detecting aceclidin in biological samples, the blank biological sample solution is prepared by pre-treating biological samples of animals that have not been given aceclidin.

[0012] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the stabilizer is formic acid.

[0013] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the final concentration of formic acid in the biological sample solution is 0.5-2.5% by volume.

[0014] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the final concentration of formic acid in the biological sample solution is 0.6% by volume.

[0015] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the internal standard is atropine-d5.

[0016] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the organic solvent is acetonitrile.

[0017] Furthermore, in the above-mentioned method for detecting aceclidinium in biological samples, the test and analysis are performed using liquid chromatography tandem mass spectrometry LC-MS / MS, and the liquid chromatography conditions include: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A: aqueous solution containing 0.1% formic acid by volume; mobile phase B: acetonitrile-methanol solution containing 0.1% formic acid by volume.

[0018] Furthermore, in the above-mentioned method for detecting aceclidinium in a biological sample, the acetonitrile-methanol solution has a volume ratio of acetonitrile:methanol=1:1.

[0019] Furthermore, in the above-mentioned method for detecting aceclidin in biological samples, the liquid chromatography conditions also include: a gradient elution procedure.

[0020] Furthermore, in the above-mentioned method for detecting aceclidin in biological samples, the gradient elution procedure is as follows:

[0021]

[0022] Furthermore, in the above-mentioned method for detecting aceclidinium in biological samples, the mass spectrometry conditions include: ion source: electrospray ion source (ESI), positive ion multiple reaction monitoring mode (MRM), curtain gas (CUR): 30 psi; ion source temperature (TEM): 550°C; nebulizer gas (GS1): 55 psi; auxiliary heating gas (GS2): 55 psi; ion source voltage (IS): 5500 V; declustering voltage (DP): 109 eV; collision energy (CE): 24 eV.

[0023] Furthermore, in the above-mentioned method for detecting aceclidin in biological samples, the mass spectrometry conditions also include: for aceclidin, the ion pair used for quantification is: m / z 171.4→111.0; for atropine-d5, the ion pair used for quantification is: m / z 295.4→124.1.

[0024] The invention provides an application of the method for detecting aceclidin in biological samples in the pharmacokinetics study of aceclidin.

[0025] The beneficial effects of the present invention are:

[0026] 1. Since aceclidin has a small molecular weight, high polarity, serious matrix interference, and contains an ester bond in its molecular structure, its chemical properties are unstable, and it is easily hydrolyzed in a biological matrix, so there is no literature report on methods for in vivo and in vitro detection of aceclidin in the prior art. The LC-MS / MS detection method of aceclidin in biological samples provided by the present invention stabilizes the sample by adding a stabilizer to the biological sample, which meets the requirements for biological sample detection.

[0027] 2. The method for detecting aceclidin in biological samples provided by the present invention screens formic acid aqueous solution as the biological sample stabilizer of the present invention by comparing different stabilizers. Formic acid aqueous solution, as an esterase inhibitor, minimizes the hydrolysis of aceclidin, is more suitable for the detection of aceclidin, and does not introduce more matrix noise.

[0028] 3. The method for detecting aceclidin in biological samples provided by the present invention screens out a final concentration of formic acid in the biological sample solution of 0.5-2.5% by investigating the stability of aceclidin. Within this range, aceclidin is stable in the matrix.

[0029] 4. Since aceclidin has a large polarity, logP is only 0.9 and pka is 9.17, a suitable internal standard is extremely important for accurate quantification. The LC-MS / MS detection method of aceclidin in biological samples provided by the present invention screens atropine-d5 as an internal standard. Under chromatographic analysis conditions, the internal standard atropine-d5 can be fully separated from each component in the sample, and the reproducibility and accuracy are good.

[0030] 5. Since the matrix effect of aceclidinium is large during analysis and there are many interfering substances near the peak position, a single organic phase cannot achieve complete separation. The LC-MS / MS detection method of aceclidinium in biological samples provided by the present invention uses an acetonitrile-methanol mixed mobile phase as the organic phase, which improves the separation selectivity of the compound and improves the peak shape. The present invention adopts a gradient elution method for analysis when determining aceclidinium, and the analysis time is only 5 minutes, which is short.

[0031] 6. The LC-MS / MS detection method for aceclidin in biological samples provided by the present invention, for mass spectrometry, when screening ion pairs, selects dynamic screening, and finds a group of parent ions and fragment ions with strong signals: m / z 171.4→111.0 (aceclidin); m / z 295.4→124.1 (atropine-d5, internal standard), which is easy to detect. The detection method of the present invention allows the quantitative lower limit of aceclidin in plasma to reach 10.00 ng / mL, and the quantitative lower limit of aceclidin in brain tissue to reach 50.00 ng / g, with high sensitivity, and is suitable for the detection of aceclidin in vivo.

[0032] In conclusion, the present invention establishes for the first time an LC-MS / MS method for determining the concentration of aceclidinium in biological samples, which has the characteristics of high sensitivity, fast analysis speed, small matrix effect, and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1-1 Chromatograms of aceclidinium (A) and internal standard (B) in plasma blank biological samples.

[0034] Figure 1-2 Chromatograms of aceclidinium (A) and internal standard (B) in blank biological samples of brain tissue.

[0035] Figure 2-1 Chromatograms of aceclidinium (A) and internal standard (B) in plasma biospecimen at zero concentration (QC0).

[0036] Figure 2-2 Chromatograms of aceclidinium (A) and internal standard (B) in brain tissue biological sample at zero concentration (QC0).

[0037] Figure 3-1 Chromatograms of aceclidinium (A) and internal standard (B) in plasma biospecimen at the lower limit of quantification (LLOQ).

[0038] Figure 3-2 Chromatograms of aceclidinium (A) and internal standard (B) in brain tissue biological samples at the lower limit of quantification (LLOQ).

[0039] Figure 4-1Chromatograms of aceclidin (A) and internal standard (B) in plasma biological samples 10 minutes after administration.

[0040] Figure 4-2 Chromatograms of aceclidin (A) and internal standard (B) in brain tissue biological samples 10 minutes after administration. DETAILED DESCRIPTION

[0041] Preparation of working solution:

[0042] 1. Preparation of aceclidinium stock solution and working solution

[0043] Preparation of aceclidinium stock solution: Accurately weigh the aceclidinium reference substance, place it in a 10 mL volumetric flask, dissolve it in an appropriate amount of 50% by volume methanol aqueous solution and make up to volume to prepare a 1.0 mg / mL aceclidinium stock solution.

[0044] Preparation of aceclidinium series standard working solutions: dilute the aceclidinium stock solution with 50% methanol aqueous solution to standard concentrations of 20.0 ng / mL, 40.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, 400.0 ng / mL, 1000.0 ng / mL, and 2000.0 ng / mL of aceclidinium series standard working solutions, respectively.

[0045] Preparation of aceclidin series quality control working solutions: dilute the aceclidin stock solution with 50% methanol aqueous solution to concentrations of 20.0 ng / mL, 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL of aceclidin series quality control working solutions, respectively.

[0046] 2. Preparation of internal standard stock solution and working solution

[0047] Preparation of internal standard stock solution: Accurately weigh an appropriate amount of atropine-d5 standard and place it in a volumetric flask, dissolve it in methanol and make up to volume to prepare an atropine-d5 stock solution with a concentration of 1.0 mg / mL.

[0048] Preparation of internal standard working solution: dilute the atropine-d5 stock solution with methanol to prepare a 5.000 ng / mL atropine-d5 working solution.

[0049] The working solutions prepared were used in the following examples.

[0050] Example 1 Method for detecting aceclidin in biological samples - selection of aceclidin stabilizer

[0051] (I) The method comprises the following steps

[0052] 1. Pretreatment of simulated plasma biological samples

[0053] Take 25 μL of each of the 40.0 ng / mL and 1600.0 ng / mL aceclidin series quality control working solutions in a centrifuge tube, blow dry with nitrogen flow for later use. Take whole blood samples from mice that have not been given aceclidin, place fresh whole blood samples in heparin sodium blood collection tubes, centrifuge for 15 min (3500 rpm, 10°C), quantitatively draw 50 μL of upper plasma and add them to the above-mentioned dried centrifuge tubes, vortex to re-dissolve, and prepare simulated plasma biological samples with low (LQC) and high (HQC) concentrations of aceclidin.

[0054] Take simulated plasma biological samples with low (LQC) and high (HQC) concentrations of aceclidinium, respectively, add 0.125 times the volume of the stabilizer aqueous solution shown in Table 1-1, vortex mix, and obtain simulated plasma biological sample solutions.

[0055] The simulated plasma biological sample solution was placed at room temperature for 0 h, at room temperature for 6 h, and at 4°C for 6 h, and 3 replicates were prepared for each condition and concentration. After the different placement conditions were completed, the test was immediately started.

[0056] 2. Preparation of simulated plasma biological sample test solution

[0057] Quantitatively transfer 50 μL of simulated plasma biological sample solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain simulated plasma biological sample test solution.

[0058] 3. Test and analysis using LC-MS / MS

[0059] The simulated plasma biological sample test fluids placed under different storage conditions were tested and analyzed using liquid chromatography tandem mass spectrometry LC-MS / MS, and the chromatographic conditions and mass spectrometry conditions were the same as step 4 of Example 2.

[0060] (II) Results

[0061] Table 1-1 Degradation rate of aceclidinium in simulated plasma samples under different stabilizers

[0062]

[0063] Note: Degradation rate = (1-peak area of ​​biological samples after placement under various conditions / peak area of ​​biological samples at 0 h) × 100%.

[0064] As can be seen from Table 1-1, through the investigation of the stability of aceclidin, it was found that aceclidin is unstable in the plasma matrix. After being placed at room temperature or 4°C for 6 hours, the concentration dropped by more than 20%. In order to improve the stability, after comparing different stabilizers, the present invention found that the stability was greatly improved after adding an aqueous formic acid solution with an initial concentration of 5%-20%, and the aqueous formic acid solution was used as a stabilizer. This reagent is common and easy to obtain, low cost, simple operation, and low toxicity. When the initial concentration of formic acid is ≥5%, aceclidin is stable in the plasma matrix, but when the initial concentration of formic acid is too high, the plasma matrix partially denatures and becomes colloidal. Therefore, the present invention selects an aqueous formic acid solution with a final formic acid concentration of 0.5%-2.5% in the biological sample solution as a stabilizer. Preferably, the present invention chooses to add 0.125 times the amount of a 5% formic acid aqueous solution so that the final concentration of formic acid in the biological sample solution is 0.6%.

[0065] Example 2 Detection Method of Acetaminophen in Biological Samples - Selective Methodological Investigation

[0066] Six mouse plasma samples and brain tissue samples from different sources were used for selectivity investigation, with aceclidinium in plasma samples and brain tissue samples as the target analyte and atropine-d5 as the internal standard. The aceclidinium concentration in the samples was determined by LC-MS / MS method.

[0067] (I) The method comprises the following steps

[0068] Step 1: Biological sample pretreatment

[0069] 1. Preparation of plasma blank biological sample solution

[0070] A whole blood sample of a mouse not given aceclidinium was taken, placed in a sodium heparin blood collection tube, centrifuged for 15 min (3500 rpm, 10° C.), the upper plasma was quantitatively drawn, 0.125 times the volume of a 5% formic acid aqueous solution was added thereto, and the mixture was vortexed to obtain a plasma blank biological sample solution.

[0071] 2. Preparation of brain tissue blank biological sample solution

[0072] A brain tissue sample of a mouse not given the aceclidinium drug was taken, and a 0.6% formic acid aqueous solution with a volume percentage of 10 times the mass was added thereto, and the mixture was vortexed to obtain a blank biological sample solution of the brain tissue.

[0073] Step 2: Preparation of biological sample test fluid

[0074] 1. Preparation of plasma blank biological sample test solution

[0075] Take 50 μL of plasma blank biological sample solution, add 25 μL of methanol, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain plasma blank biological sample test solution.

[0076] 2. Preparation of brain tissue blank biological sample test solution

[0077] Take 50 μL of brain tissue blank biological sample solution, add 25 μL of methanol, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain brain tissue blank biological sample test solution.

[0078] Step 3: Preparation of aceclidinium series standard test solutions for standard curve

[0079] 1. Preparation of aceclidinium series standard test solutions for plasma standard curve

[0080] Accurately pipette a series of aceclidinium standard working solutions with standard concentrations of 20.0 ng / mL, 40.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, 400.0 ng / mL, 1000.0 ng / mL, and 2000.0 ng / mL, and dilute them with the plasma blank biological sample solution to prepare a series of standard curve plasma samples containing aceclidinium standard concentrations of 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 ng / mL, respectively.

[0081] Take 50 μL of each plasma sample for the series standard curve, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of the supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of the supernatant, and obtain the aceclidinium series standard test solution for the plasma standard curve.

[0082] 2. Preparation of aceclidinium series standard test solutions for brain tissue standard curve

[0083] Accurately pipette aceclidinium series standard working solutions with standard concentrations of 20.0 ng / mL, 40.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, 400.0 ng / mL, 1000.0 ng / mL, and 2000.0 ng / mL, and dilute them with brain tissue blank biological sample solutions to prepare a series of standard curve brain tissue samples containing aceclidinium standard concentrations of 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 ng / mL, respectively.

[0084] Take 50 μL of each brain tissue sample for the series standard curve, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of the supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of the supernatant, and obtain the aceclidinium series standard test solution for the brain tissue standard curve.

[0085] Step 4: Test and analyze using LC-MS / MS

[0086] The plasma blank biological sample test solution and the brain tissue blank biological sample test solution obtained in step 2, and the plasma standard curve aceclidin series standard test solution and the brain tissue standard curve aceclidin series standard test solution obtained in step 3 are tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS.

[0087] 1. Chromatographic conditions

[0088] Acquity UPLC ultra-high performance liquid chromatograph from WATERS, USA; ABQtrap5500 mass spectrometer from AB Sciex, USA; data acquisition and processing software: Analyst 1.7.2.

[0089] Chromatographic column: Waters, Waters, USA C 18 (4.6×100mm,5μm);

[0090] Mobile phase A: aqueous solution containing 0.1% formic acid by volume;

[0091] Mobile phase B: acetonitrile-methanol solution containing 0.1% formic acid by volume, wherein the acetonitrile-methanol solution has a volume ratio of acetonitrile:methanol=1:1;

[0092] Column temperature: 40°C;

[0093] Flow rate: 0.5 mL min -1 ;

[0094] Injection volume: 10 μL;

[0095] Analysis time: 5min;

[0096] The gradient elution program is as follows:

[0097]

[0098] 2. Mass spectrometry conditions

[0099] Ion source: electrospray ionization (ESI), positive ion multiple reaction monitoring (MRM);

[0100] Curtain air (CUR): 30psi;

[0101] Ion source temperature (TEM): 550°C;

[0102] Atomizing gas (GS1): 55psi;

[0103] Auxiliary heating gas (GS2): 55psi;

[0104] Ion source voltage (IS): 5500 V;

[0105] Declustering potential (DP): 109 eV;

[0106] Collision energy (CE): 24 eV;

[0107] The ion pairs used for quantification were: m / z 171.4→111.0 (aceclidinium); m / z 295.4→124.1 (atropine-d5, internal standard).

[0108] (II) Results

[0109] Table 2-1 Results of plasma selectivity investigation

[0110]

[0111]

[0112] Table 2-2 Results of selective investigation of brain tissue

[0113]

[0114] Figure 1-1 Chromatograms of aceclidinium (A) and internal standard (B) in plasma blank biological samples. Figure 1-2 The chromatograms of aceclidin (A) and internal standard (B) in blank biological samples of brain tissue were shown in Figure 1. No endogenous substances in plasma and brain tissue were found to interfere with the analyte aceclidin and internal standard atropine-d5.

[0115] As can be seen from Table 2-1 and Table 2-2, endogenous substances in plasma and brain tissue do not interfere with the determination of the analyte aceclidin and the internal standard atropine-d5. The results show that the detection method provided by the present invention has good selectivity and meets the requirements for biological sample detection. Example 3 Detection method of aceclidin in biological samples - linear methodology investigation

[0116] (I) The method is as follows

[0117] 1. Preparation of zero concentration biological sample (QC0) test solution

[0118] Preparation of plasma blank biological sample solution: same as step 1 in Example 2.

[0119] Preparation of blank brain tissue biological sample solution: same as step 1 in Example 2.

[0120] Preparation of zero-concentration plasma biological sample test solution: take 50 μL of plasma blank biological sample solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain zero-concentration plasma biological sample test solution.

[0121] Preparation of zero-concentration brain tissue biological sample test solution: take 50 μL of brain tissue blank biological sample solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain zero-concentration brain tissue biological sample test solution.

[0122] 2. Preparation of aceclidinium series standard test solutions for standard curve

[0123] Preparation of aceclidinium series standard test solutions for plasma standard curve: The method is the same as step 3 of Example 2.

[0124] Preparation of aceclidinium series standard test solutions for brain tissue standard curve: The method is the same as step 3 of Example 2.

[0125] 3. Test and analysis using LC-MS / MS

[0126] The obtained zero-concentration plasma biological sample test solution and the zero-concentration brain tissue biological sample test solution, and the obtained plasma standard curve aceclidin series standard test solution or the obtained brain tissue standard curve aceclidin series standard test solution are tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS, and the method is the same as step 4 of Example 2.

[0127] (II) Linear Investigation

[0128] The internal standard method uses the peak area ratio (x) of the test component aceclidinium to the internal standard atropine-d5 and the concentration ratio (y) of the test component aceclidinium to the internal standard atropine-d5 for weighted linear regression. The linear range is 10-1000 ng / mL, and the minimum limit of quantification is 10 ng / mL. The standard curve regression equation for plasma biological samples is: y = 0.0324x + 0.000137, r = 0.9999. The standard curve regression equation for brain tissue biological samples is: y = 0.0204x + 0.001449, r = 0.9998. The typical chromatogram of the standard curve is shown in Figure 2-1 , Figure 2-2 , Figure 3-1 and Figure 3-2 The results show that the method of the present invention has high sensitivity and good linearity, and meets the needs of biological sample detection.

[0129] Example 4 Detection Method of Acetaminophen in Biological Samples - Accuracy and Precision Methodological Investigation

[0130] (I) The method is as follows

[0131] Step 1: Biological sample pretreatment

[0132] 1. Preparation of plasma blank biological sample solution: the method is the same as step 1 in Example 2.

[0133] 2. Preparation of blank brain tissue biological sample solution: The method is the same as step 1 in Example 2.

[0134] 3. Preparation of Acetoclide Series Quality Control Plasma Biological Sample Solutions

[0135] Take a series of aceclidinium quality control working solutions with concentrations of 20.0 ng / mL, 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL, and dilute them with plasma blank biological sample solutions to prepare a series of aceclidinium quality control plasma biological sample solutions with aceclidinium concentrations of 10.0 ng / mL (LLOQ), 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC).

[0136] 4. Preparation of Acetoclin Series Quality Control Brain Tissue Biological Sample Solutions

[0137] Take a series of aceclidin quality control working solutions with concentrations of 20.0 ng / mL, 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL, and dilute them with brain tissue blank biological sample solutions, respectively, to prepare a series of aceclidin quality control brain tissue biological sample solutions containing aceclidin with concentrations of 10.0 ng / mL (LLOQ), 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC).

[0138] Step 2: Preparation of biological sample test fluid

[0139] 1. Preparation of Acetoclide Series Quality Control Plasma Biological Sample Test Solution

[0140] Take 50 μL of each of the aceclidinium series quality control plasma biological sample solutions of 10.0 ng / mL (LLOQ), 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC), add 25 μL of 5.000 ng / mL atropine-d5 working solution, add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of the supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of the supernatant, and obtain the aceclidinium series quality control plasma biological sample test solutions.

[0141] 2. Preparation of Acetoclin Series Quality Control Brain Tissue Biological Sample Test Solution

[0142] Take 50 μL of 10.0 ng / mL (LLOQ), 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC) aceclidinium series quality control brain tissue biological sample solutions, respectively, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant, and obtain aceclidinium series quality control brain tissue biological sample test solutions, respectively.

[0143] Step 3: Preparation of aceclidinium series standard test solutions for standard curve

[0144] 1. Preparation of aceclidinium series standard test solutions for plasma standard curve: The method is the same as step 3 of Example 2.

[0145] 2. Preparation of aceclidinium series standard test solutions for brain tissue standard curve: The method is the same as step 3 of Example 2.

[0146] Step 4: Test and analyze using LC-MS / MS

[0147] The obtained aceclidin series quality control plasma biological sample test solution and aceclidin series quality control brain tissue biological sample test solution, and the obtained plasma standard curve aceclidin series standard test solution or brain tissue standard curve aceclidin series standard test solution were tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS, and the method was the same as step 4 of Example 2.

[0148] (II) Results

[0149] The precision and accuracy within and between batches were investigated; the actual concentration was calculated according to the linear regression equation, and the measured mean value, deviation and precision (%CV) of each concentration were calculated. The results are shown in Table 3-1 and Table 3-2.

[0150] As shown in Table 3-1 and Table 3-2, the results show that the intra-batch and inter-batch precision of aceclidinium are less than 15%, and the relative deviation is less than 15%. The results show that this method is accurate, precise and reproducible. It is suitable for biological sample analysis.

[0151] Table 3-1 Results of plasma accuracy and precision investigation

[0152]

[0153]

[0154] Note: SD is standard deviation; %CV is precision.

[0155] Table 3-2 Results of the accuracy and precision study of brain tissue

[0156]

[0157]

[0158] Note: SD is standard deviation; %CV is precision.

[0159] Example 5 Detection Method of Acetaminophen in Biological Samples - Matrix Effect and Extraction Recovery Methodology Investigation (I) The method is as follows

[0160] Step 1: Biological sample pretreatment

[0161] 1. Preparation of plasma blank biological sample solution: the method is the same as step 1 in Example 2.

[0162] 2. Preparation of blank brain tissue biological sample solution: The method is the same as step 1 in Example 2.

[0163] 3. Preparation of Acetoclide Series Quality Control Plasma Biological Sample Solutions

[0164] Take aceclidinium series quality control working solutions with concentrations of 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL, and dilute them with plasma blank biological sample solutions, respectively, to prepare aceclidinium series quality control plasma biological sample solutions with aceclidinium concentrations of 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC), respectively.

[0165] 4. Preparation of Acetoclin Series Quality Control Brain Tissue Biological Sample Solutions

[0166] Take aceclidinium series quality control working solutions with concentrations of 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL, and dilute them with brain tissue blank biological sample solutions, respectively, to prepare aceclidinium series quality control brain tissue biological sample solutions with aceclidinium concentrations of 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC), respectively.

[0167] Step 2: Preparation of biological sample test fluid

[0168] 1. Preparation of extracted aceclidin series quality control plasma or brain tissue biological sample test solution

[0169] Take 50 μL of aceclidin series quality control plasma or brain tissue biological sample solutions containing aceclidin concentrations of 20.0 ng / mL (LQC), 400.0 ng / mL (MQC), and 800.0 ng / mL (HQC), add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant, and obtain the extracted aceclidin series quality control plasma or brain tissue biological sample test solution, respectively.

[0170] 2. Preparation of unextracted aceclidinium series quality control plasma or brain tissue biological sample test solution

[0171] Take 75 μL of plasma blank biological sample solution or brain tissue blank biological sample solution, add 37.5 μL of 50% methanol-water, then add 262.5 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 200 μL of supernatant, add 25 μL of aceclidin series quality control working solution with concentrations of 40.0 ng / mL, 800.0 ng / mL, and 1600.0 ng / mL, and 25 μL of 5.000 ng / mL atropine-d5 working solution, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant, and obtain unextracted aceclidin series quality control plasma or brain tissue biological sample test solution, respectively.

[0172] 3. Preparation of Acetoclide Series Quality Control Sample Test Solution for Solution Samples

[0173] Take the aceclidin series quality control working solutions with concentrations of 40.0ng / mL, 800.0ng / mL, and 1600.0ng / mL, and dilute them with pure water to prepare a series of solution samples containing aceclidin concentrations of 20.0ng / mL (LQC), 400.0ng / mL (MQC), and 800.0ng / mL (HQC). Take 50μL of the series solution samples, add 25μL of 5.000ng / mL atropine-d5 working solution, and then add 175μL of acetonitrile, vortex mix, centrifuge for 5min (14500rpm, 10℃), take 10μL of the supernatant, and obtain the aceclidin series quality control sample test solutions of the solution samples.

[0174] Step 3: Preparation of aceclidinium series standard test solutions for standard curve

[0175] 1. Preparation of aceclidinium series standard test solutions for plasma standard curve: The method is the same as step 3 of Example 2.

[0176] 2. Preparation of aceclidinium series standard test solutions for brain tissue standard curve: The method is the same as step 3 of Example 2.

[0177] Step 4: Test and analyze using LC-MS / MS

[0178] The obtained extracted aceclidin series quality control plasma or brain tissue biological sample test solution, the unextracted aceclidin series quality control plasma or brain tissue biological sample test solution, the aceclidin series quality control sample test solution of the solution sample, and the obtained aceclidin series standard test solution for the plasma standard curve and the aceclidin series standard test solution for the brain tissue standard curve were tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS, and the method was the same as step 4 of Example 2.

[0179] (II) Results

[0180] The peak areas of the aceclidine series quality control sample test solution and the internal standard atropine-d5 of the solution sample, as well as the peak areas of the unextracted aceclidine series quality control plasma or brain tissue biological sample test solution and the internal standard atropine-d5 were used to calculate the internal standard normalized matrix factor. The extraction recovery rates of aceclidine and internal standard atropine-d5 were calculated using the peak areas of the unextracted aceclidine series quality control plasma or brain tissue biological sample test solution and the internal standard, as well as the peak areas of the extracted aceclidine series quality control plasma or brain tissue biological sample test solution and the internal standard. The results showed that the normalized matrix effect factors were all less than ±15%, and the extraction recoveries were all between 90% and 110%. The matrix effects of aceclidine are shown in Tables 4-1 and 4-2, the extraction recoveries of aceclidine are shown in Tables 5-1 and 5-2, and the extraction recoveries of internal standard atropine-d5 are shown in Tables 5-3 and 5-4. The results showed that this method had no matrix effect, the extraction recovery rate met the requirements, and met the requirements for biological sample analysis.

[0181] Table 4-1 Matrix effect investigation (plasma)

[0182]

[0183] Note: SD is standard deviation; %CV is precision.

[0184] Table 4-2 Matrix effect investigation (brain tissue)

[0185]

[0186]

[0187] Note: SD is standard deviation; %CV is precision.

[0188] Table 5-1 Investigation of the recovery rate of the test substance extraction (plasma)

[0189]

[0190] Note: SD is standard deviation; %CV is precision.

[0191] Table 5-2 Investigation of the recovery rate of the analytes (brain tissue)

[0192]

[0193]

[0194] Note: SD is standard deviation; %CV is precision.

[0195] Table 5-3 Internal standard extraction recovery rate investigation (plasma)

[0196]

[0197] Note: SD is standard deviation; %CV is precision.

[0198] Table 5-4 Investigation of internal standard extraction recovery rate (brain tissue)

[0199]

[0200] Note: SD is standard deviation; %CV is precision.

[0201] Example 6 Detection Method of Acetaminophen in Biological Samples - Stability Methodology Investigation

[0202] Mouse plasma samples and brain tissue samples were used for method validation, with aceclidin in plasma samples and brain tissue samples as the target analyte and atropine-d5 as the internal standard. The aceclidin concentration in the samples was determined by LC-MS / MS.

[0203] (I) The method comprises the following steps

[0204] Step 1: Biological sample pretreatment

[0205] 1. Pretreatment of simulated plasma biological samples

[0206] Preparation of plasma blank biological sample solution: same as step 1 in Example 2.

[0207] Take 25 μL of 40.0 ng / mL and 1600.0 ng / mL aceclidinium series quality control working solutions, blow dry under nitrogen flow at room temperature, add 50 μL of plasma blank biological sample solution to reconstitute, and obtain simulated plasma biological sample stability solutions.

[0208] 2. Simulate the pretreatment of brain tissue biological samples

[0209] Preparation of blank brain tissue biological sample solution: same as step 1 in Example 2.

[0210] Take 25 μL of 40.0 ng / mL and 1600.0 ng / mL aceclidinium series quality control working solutions, blow dry under nitrogen flow at room temperature, add 50 μL of brain tissue blank biological sample solution to reconstitute, and obtain simulated brain tissue biological sample stability solutions.

[0211] The simulated plasma biological sample stability solution or simulated brain tissue biological sample stability solution was placed at room temperature for 24 hours, at 4°C for 24 hours, freeze-thawed for 3 times, and stored at -80°C for 30 days. Three copies were prepared in parallel for each condition and concentration. After the different storage conditions were completed, the test was immediately started.

[0212] Step 2: Preparation of biological sample test fluid

[0213] 1. Preparation of plasma biological sample stability test solution

[0214] Quantitatively remove 50 μL of the plasma biological sample stability solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of the supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of the supernatant to obtain the plasma biological sample stability test solution.

[0215] 2. Preparation of brain tissue biological sample stability test solution

[0216] Quantitatively remove 50 μL of the brain tissue biological sample stability solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of the supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of the supernatant to obtain the brain tissue biological sample stability test solution.

[0217] Step 3: Preparation of aceclidinium series standard test solutions for standard curve

[0218] 1. Preparation of aceclidinium series standard test solutions for plasma standard curve: same as step 3 of Example 2.

[0219] 2. Preparation of aceclidinium series standard test solutions for brain tissue standard curve: Same as step 3 of Example 2.

[0220] Step 4: Test and analyze using LC-MS / MS

[0221] The obtained plasma biological sample stability test solution and brain tissue biological sample stability test solution, and the obtained plasma standard curve aceclidin series standard test solution or brain tissue standard curve aceclidin series standard test solution were tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS, and the method was the same as step 4 of Example 2.

[0222] (II) Results

[0223] The stability results of biological sample stability solution at room temperature for 24 hours are shown in Tables 6-1 and 6-2, 4℃ for 24 hours, 3 freeze-thaw cycles, 30-day storage, and -80℃ for stability. The results are shown in Tables 9-1 and 9-2. The experimental results show that the %RE (accuracy deviation) of the average value of all samples and the theoretical value does not exceed ±10%, which is significantly better than the test standard requirement of no more than ±15%, and the %CV (precision) of the stability sample measurement value at each concentration level is ≤±10%, which is significantly better than the test standard requirement of ≤15.0%. The results show that the stability of aceclidin in biological samples under all conditions is good and the stability meets the test requirements.

[0224] Table 6-1 Stability of biological samples placed at room temperature for 24 hours after preparation (plasma)

[0225]

[0226] Note: SD is standard deviation; %CV is precision.

[0227] Table 6-2 Stability of biological samples placed at room temperature for 24 hours after preparation (brain tissue)

[0228]

[0229]

[0230] Note: SD is standard deviation; %CV is precision.

[0231] Table 7-1 Stability of biological samples after preparation at 4°C for 24 hours (plasma)

[0232]

[0233] Note: SD is standard deviation; %CV is precision.

[0234] Table 7-2 Stability of biological samples after preparation at 4°C for 24 hours (brain tissue)

[0235]

[0236] Note: SD is standard deviation; %CV is precision.

[0237] Table 8-1 Stability of biological samples after freeze-thaw cycles for 3 times (plasma)

[0238]

[0239]

[0240] Note: SD is standard deviation; %CV is precision.

[0241] Table 8-2 Stability of biological samples after 3 freeze-thaw cycles (brain tissue)

[0242]

[0243] Note: SD is standard deviation; %CV is precision.

[0244] Table 9-1 Stability of biological samples after preparation at -80℃ for long term storage (30 days) (plasma)

[0245]

[0246] Note: SD is standard deviation; %CV is precision.

[0247] Table 9-2 Stability of samples at -80℃ long-term storage (30 days) (brain tissue)

[0248]

[0249]

[0250] Note: SD is standard deviation; %CV is precision.

[0251] Example 7 Application of the Detection Method of Acetidine in Biological Samples in the Pharmacokinetic Study of Acetidine

[0252] After 3% aceclidin eye drops were administered to both eyes of mice, plasma and brain tissue were collected 3min, 10min, 30min, 1h, 2h, and 4h after administration, and the aceclidin concentrations in plasma and brain tissue were determined by LC-MS / MS.

[0253] (I) The method comprises the following steps

[0254] Step 1: Biological sample pretreatment

[0255] 1. Preparation of blank plasma biological sample solution: same as step 1 in Example 2.

[0256] 2. Preparation of blank brain tissue biological sample solution: same as step 1 in Example 2.

[0257] 3. Preparation of plasma biological sample solution

[0258] Whole blood samples were obtained from mice given aceclidinium, and the fresh whole blood samples were placed in sodium heparin blood collection tubes and centrifuged for 15 min (3500 rpm, 10° C.). The upper plasma was quantitatively aspirated, and 0.125 times the volume of a 5% formic acid aqueous solution was added. The mixture was vortexed to obtain a plasma biological sample solution with a final formic acid concentration of 0.6%.

[0259] 4. Preparation of brain tissue biological sample solution

[0260] Take the brain tissue sample of the mouse administered with aceclidine, add 10 times the mass of a 0.6% formic acid aqueous solution by volume percentage, and vortex mix to obtain a brain tissue biological sample solution with a final formic acid concentration of 0.6%.

[0261] Step 2: Preparation of biological sample test fluid

[0262] 1. Preparation of plasma biological sample test solution

[0263] Take 50 μL of plasma biological sample solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain plasma biological sample test solution.

[0264] 2. Preparation of brain tissue biological sample test solution

[0265] Take 50 μL of brain tissue biological sample solution, add 25 μL of 5.000 ng / mL atropine-d5 working solution, then add 175 μL of acetonitrile, vortex mix, centrifuge for 10 min (14500 rpm, 10°C), take 50 μL of supernatant, add 450 μL of pure water, vortex mix, centrifuge for 5 min (14500 rpm, 10°C), take 10 μL of supernatant to obtain brain tissue biological sample test solution.

[0266] Step 3: Preparation of aceclidinium series standard test solutions for standard curve:

[0267] 1. Preparation of aceclidinium series standard test solutions for plasma standard curve: same as step 3 of Example 2.

[0268] 2. Preparation of aceclidinium series standard test solutions for brain tissue standard curve: Same as step 3 of Example 2.

[0269] Step 4: Test and analyze using LC-MS / MS method:

[0270] The plasma biological sample test solution and the brain tissue biological sample test solution obtained in step 2, and the plasma standard curve aceclidin series standard test solution and the brain tissue standard curve aceclidin series standard test solution obtained in step 3 were tested and analyzed by liquid chromatography tandem mass spectrometry LC-MS / MS. The chromatographic conditions and mass spectrometry conditions were the same as those in step 4 of Example 2.

[0271] (II) Drawing of standard curve

[0272] The standard curve obtained in step 3 is tested and analyzed using a series of aceclidinium standard test solutions and liquid chromatography tandem mass spectrometry LC-MS / MS to draw a standard curve, as shown in Table 10-1.

[0273] Table 10-1 Parameters of plasma and brain aceclidinium standard curves

[0274] Matrix type Linear equations Slope intercept r plasma y=0.796x+0.000691 0.796 0.000691 0.9947 brain y=0.864x+0.00109 0.864 0.00109 0.9973

[0275] (III) Determination of series of quality control test solutions

[0276] The method is the same as Example 4.

[0277] Table 10-2 Test results of aceclidinium plasma series quality control solutions

[0278]

[0279] Table 10-3 Test results of aceclidinium series quality control solutions

[0280]

[0281]

[0282] (IV) Determination of aceclidin concentration in plasma and brain tissue of mice after administration of 3% aceclidin eye drops in both eyes

[0283] The standard curve method was used to deduce the concentration values ​​of aceclidin in biological samples at different administration time points (within the range of the standard curve).

[0284] Table 10-4 Acetocline plasma sample determination results (quantitative limit: 10 ng / mL)

[0285]

[0286] Note: SD is standard deviation; BLQ means the sample concentration is lower than the lower limit of quantification; NA means no valid value.

[0287] Table 10-5 Acetoclin brain tissue sample determination results (quantitative limit: 50ng / g)

[0288]

[0289] Note: SD is standard deviation; BLQ means the sample concentration is lower than the lower limit of quantification; NA means no valid value.

[0290] From Table 10-1 to Table 10-5, Figure 4-1 and Figure 4-2It can be seen that the results show that the linear range of aceclidin in plasma is 10.00~1000ng / mL, and the linear range of aceclidin in brain tissue is 50.00~5000ng / g, with good linearity (r≥0.99), intra-batch and inter-batch precision are within ±15%, and the accuracy is within 85-115%. After aceclidin eye drops were given to both eyes of mice, the concentration of aceclidin in plasma reached a peak at 3min, then quickly eliminated, and was below the quantitative limit at 30min; no aceclidin was detected in the brain at each time point.

[0291] Conclusion: This method is rapid, sensitive and specific, and is suitable for accurate determination of aceclidinium concentration in mouse plasma and brain and for pharmacokinetic studies.

Claims

1. A method for detecting aceclidin in a biological sample, characterized in that: The liquid chromatography tandem mass spectrometry LC-MS / MS method is used, and the detection method includes the following steps: 1) Biological sample pretreatment: taking a biological sample, adding 0.1-10 times the amount of an aqueous solution containing a stabilizer thereto, and mixing well to obtain a biological sample solution; 2) Preparation of biological sample test solution: pipette the biological sample solution, add the internal standard working solution and the organic solvent in sequence, vortex mix, and centrifuge; pipette the resulting supernatant, add pure water, vortex mix, centrifuge, and take the supernatant. obtaining a biological sample test fluid; 3) Preparation of aceclidin series standard test solution for standard curve: pipette the aceclidin series standard working solution diluted with blank biological sample solution, add internal standard working solution and organic solvent in sequence, vortex mix, centrifuge; pipette the obtained supernatant, add pure water, vortex mix, centrifuge, take the supernatant, and obtain the aceclidin series standard test solution for standard curve; 4) The biological sample test solution and the standard curve are tested and analyzed using aceclidinium series standard test solution and liquid chromatography tandem mass spectrometry LC-MS / MS.

2. The method for detecting aceclidin in a biological sample according to claim 1, characterized in that: The biological sample is a plasma sample or a tissue sample.

3. The method for detecting aceclidin in a biological sample according to claim 1, characterized in that: The stabilizer is formic acid; preferably, in the biological sample solution, the final concentration of formic acid is 0.5-2.5% by volume; more preferably, in the biological sample solution, the final concentration of formic acid is 0.6% by volume.

4. The method for detecting aceclidin in a biological sample according to claim 1, characterized in that: The internal standard is atropine-d5; the organic solvent is acetonitrile.

5. The method for detecting aceclidin in a biological sample according to claim 1, characterized in that: The test and analysis are performed by liquid chromatography tandem mass spectrometry LC-MS / MS, and the liquid chromatography conditions include: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase A: aqueous solution containing 0.1% formic acid by volume; mobile phase B: acetonitrile-methanol solution containing 0.1% formic acid by volume; preferably, the acetonitrile-methanol solution has a volume ratio of acetonitrile:methanol = 1:

1.

6. The method for detecting aceclidin in a biological sample according to claim 5, characterized in that: Liquid chromatography conditions also include: gradient elution program.

7. The method for detecting aceclidin in a biological sample according to claim 6, characterized in that: The gradient elution procedure is as follows:

8. The method for detecting aceclidin in a biological sample according to claim 5, 6 or 7, characterized in that: The mass spectrometry conditions included: ion source: electrospray ion source (ESI), positive ion multiple reaction monitoring (MRM), curtain gas (CUR): 30 psi; ion source temperature (TEM): 550°C; nebulizer gas (GS1): 55 psi; auxiliary heating gas (GS2): 55 psi; ion source voltage (IS): 5500 V; declustering potential (DP): 109 eV; collision energy (CE): 24 eV.

9. The method for detecting aceclidin in a biological sample according to claim 8, characterized in that: The mass spectrometry conditions also include: for acecliidine, the ion pair used for quantification is: m / z 171.4→111.0; for atropine-d5, the ion pair used for quantification is: m / z 295.4→124.

1.

10. Use of the method for detecting aceclidine in a biological sample according to any one of claims 1 to 9 in a study on the pharmacokinetics of aceclidine.

Images