Method for analyzing aceclodine hydrochloride in biological matrix

Abstract

The invention discloses a method for analyzing aceclodine hydrochloride in a biological matrix, and belongs to the technical field of drug analysis and detection, and the method comprises the following steps: adding the biological matrix into a precipitant, uniformly mixing by vortex, centrifuging for 8-12 minutes at the speed of 3800-4500 r / min under the condition of 4 DEG C, sucking supernatant, adding into deionized water, and uniformly mixing to obtain a sample to be detected; and performing liquid chromatography-tandem mass spectrometry analysis on the sample to be detected, and obtaining the content of the aceclodine hydrochloride in the biological matrix according to the standard curve of the aceclodine hydrochloride. Test consumables and instruments are easy to obtain, operation is easy and safe, batch operation can be achieved, and the requirement for large-batch detection is met.

Description

An analytical method for acetic acid ketidine hydrochloride in biological matrices Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, specifically relating to an analytical method for acetylcholine hydrochloride in a biological matrix. Background Technology

[0002] Aceclidine hydrochloride is an antihistamine with the molecular formula: (C9H) 16 ClNO2), with the structural formula as follows: Monitoring the concentration of acetylcholine hydrochloride in biological matrices (such as blood, urine, or tissue) is crucial for assessing efficacy and safety. Existing analytical methods largely rely on high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS), which suffer from insufficient sensitivity, long analysis times, or complex sample pretreatment. For example, HPLC methods are susceptible to matrix interference at low concentrations, while GC-MS requires a derivatization step, increasing operational complexity. Although liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology offers high specificity, parameter optimization for acetylcholine hydrochloride (such as ion source selection and collision energy settings) has not been fully explored, leading to poor reproducibility or unsatisfactory detection limits in practical applications. This invention aims to overcome these limitations by providing a rapid, stable, and applicable analytical method for acetylcholine hydrochloride in complex biological matrices. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an analytical method for acetylcholine hydrochloride in biological matrices. The method provided by this invention, by optimizing chromatographic separation and mass spectrometry detection parameters, can realize the analysis and detection of acetylcholine hydrochloride in biological matrices. The method of this invention is simple, fast and easy to operate.

[0004] The present invention provides the following technical solution: an analytical method for acetylcholine hydrochloride in a biological matrix, comprising the following steps: pretreating the biological matrix based on protein precipitation to obtain a sample to be tested; performing liquid chromatography-tandem mass spectrometry analysis on the sample to be tested, and obtaining the content of acetylcholine hydrochloride in the biological matrix according to the standard curve of acetylcholine hydrochloride.

[0005] Furthermore, the pretreatment includes the following steps: adding the biological matrix to a precipitant containing an internal standard solution, vortexing and mixing thoroughly, centrifuging at 3800~4500 r / min for 8~12 minutes at 4℃, taking the supernatant and adding it to deionized water, mixing thoroughly, and obtaining the sample to be tested.

[0006] Furthermore, the volume ratio of the biological matrix to the precipitant is 1:9, and the volume ratio of the supernatant to the deionized water is 1:1.

[0007] Furthermore, the precipitant is acetonitrile, and the internal standard solution is tolbutamide solution.

[0008] Furthermore, the biological matrix is ​​any one of plasma, serum, whole blood, excrement, or tissue homogenate.

[0009] Furthermore, the sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry using an AB Sciex 7500 Triple Quad instrument and an ACQUITY UPLC® BEH C18 1.7 μm 2.1 mm × 50 mm column.

[0010] Furthermore, the sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry, with the first mobile phase being an ultrapure aqueous solution containing 0.1% formic acid and the second mobile phase being an acetonitrile solution containing 0.1% formic acid.

[0011] Furthermore, the sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry. The chromatographic conditions used included: column temperature of 40℃; injection plate temperature of 4℃; injection volume of 5 µL; run time of 5 min; and gradient elution at a flow rate of 0.4 mL / min.

[0012] Furthermore, the gradient elution procedure includes: from 0.0 min to 0.5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%; from 0.5 min to 3.5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%; from 3.5 min to 4.2 min, the volume percentage of the first mobile phase is 5% and the volume percentage of the first mobile phase is 95%; from 4.2 min to 4.3 min, the volume percentage of the first mobile phase is 5% and the volume percentage of the first mobile phase is 95%; and from 4.3 min to 5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%.

[0013] Furthermore, the sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry. The mass spectrometry conditions used included: electrospray ionization source; positive ion multiple reaction monitoring mode; positive ion mode; curtain gas: 40 psi; collision gas: 10 psi; ion voltage: 5500 V; ion source temperature: 550℃; nebulizing gas: 35 psi; auxiliary heating gas: 70 psi; inlet voltage: 10 V; collision cell outlet voltage: 13 V.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention extracts acetylcholine hydrochloride from biological matrix based on protein precipitation method, which is suitable for rapid sample processing. The lower limit of quantification is 250 mg / mL, which basically meets the requirements of quantitative detection. It provides a method for analyzing acetylcholine hydrochloride in biological matrix using LC-MS / MS. By optimizing chromatographic separation and mass spectrometry detection parameters, the detection sensitivity and accuracy are significantly improved. It is suitable for drug metabolism research, clinical monitoring and toxicological analysis, etc., and can meet the research needs of pharmacokinetic and toxicokinetic studies of acetylcholine hydrochloride in preclinical / clinical trials, and meet the requirements of the guidelines; (2) The method provided by the present invention has high sensitivity and specificity. It adopts multiple reaction monitoring (MRM) mode and can detect concentrations from pg / ml to ng / ml. It can effectively distinguish between target analytes and matrix interference, verify a large linear range (e.g., 0.25-50 ng / ml) and high precision (RSD<10%); The test consumables and instruments of the present invention are easy to obtain, simple and safe to operate, and can be operated in batches to meet the needs of large-scale detection. Attached Figure Description

[0015] Figure 1 is the standard curve of acetic acid ketidine hydrochloride in Example 1 of the present invention; Figure 2 is the chromatogram of the sample with the lower limit of quantitation in Example 1 of the present invention. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0017] In the description of this invention, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more. The terms "comprising," "including," "having," "containing," etc., as used herein, are open-ended terms, meaning that they include but are not limited to.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] The reagents and instruments used in this invention are all commercially available and can be purchased from the market.

[0020] A method for analyzing acetylcholine hydrochloride in a biological matrix is ​​provided, comprising the following steps: S1. Pretreatment of the biological matrix based on protein precipitation method: the biological matrix is ​​added to a precipitant containing an internal standard solution, vortexed and mixed evenly, centrifuged at 3800~4500 r / min for 8~12 minutes at 4℃, the supernatant is collected and added to deionized water, mixed evenly, and the sample to be tested is obtained; S2. LC-MS / MS analysis of the sample to be tested is performed, and the content of acetylcholine hydrochloride in the biological matrix is ​​obtained according to the standard curve of acetylcholine hydrochloride.

[0021] In some possible embodiments, pretreatment analysis is performed before testing by adding 270 µL of acetonitrile to 30 µL of blank biological matrix, vortexing and mixing, centrifuging at 4000 r / min for 10 min at 4°C, taking 100 µL of the supernatant, adding it to 100 µL of deionized water, mixing, and taking 5 µL for LC-MS / MS analysis to eliminate the influence of contamination and interference.

[0022] In some possible embodiments, protein precipitation can be used to process samples quickly, but it should be noted that phospholipid residues may affect ionization efficiency.

[0023] In the analytical method of the present invention, the internal standard method is used to correct for ion suppression / enhancement, which can ensure quantitative accuracy. In some possible embodiments, the internal standard used in the internal standard method is tolbutamide.

[0024] In some possible embodiments, the volume ratio of biological matrix to precipitant is 1:9, the volume ratio of supernatant to deionized water is 1:1, and the precipitant is acetonitrile.

[0025] In some possible embodiments, the biological matrix includes any one of plasma, serum, whole blood, excrement, or tissue homogenate.

[0026] In some possible embodiments, the test sample was analyzed by LC-MS / MS using an ABSciex 7500 Triple Quad instrument and an ACQUITY UPLC® BEH C18 1.7 μm 2.1 mm × 50 mm (Waters) column. The first mobile phase was an ultrapure aqueous solution containing 0.1% formic acid, and the second mobile phase was an acetonitrile solution containing 0.1% formic acid. The chromatographic conditions included: column temperature of 40 °C; injection plate temperature of 4 °C; injection volume of 5 µL; run time of 5 min; and gradient elution at a flow rate of 0.4 mL / min, as shown in Table 1.

[0027] Table 1 Gradient elution program

[0028] In some possible embodiments, the sample to be tested is analyzed by LC-MS / MS, and the mass spectrometry conditions are shown in Table 2.

[0029] Table 2 Mass Spectrometry Conditions

[0030] Example 1

[0031] This embodiment provides the preparation and analysis of a standard curve for acetylcholine hydrochloride in a biological matrix based on the analytical method for acetylcholine hydrochloride provided by the present invention.

[0032] Before testing, pretreatment analysis was performed by adding 270 µL of acetonitrile to 30 µL of blank biological matrix, vortexing and mixing, centrifuging at 4000 r / min for 10 min at 4℃, taking 100 µL of the supernatant and adding it to 100 µL of deionized water, mixing, and taking 5 µL for LC-MS / MS analysis to eliminate the influence of contamination and interference.

[0033] In some possible embodiments, according to the requirements of the General Rules of Pharmacopoeia 9012, a standard curve sample of acetylcholine hydrochloride was prepared and diluted with a blank protein matrix to form standard solutions of different concentration gradients, with concentrations of 0.25 ng / mL, 0.5 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 12.5 ng / mL, 25 ng / mL, 40 ng / mL, and 50 ng / mL, respectively. 30 µL of the standard solution to be tested was taken and added to 270 µL of acetonitrile containing 10 ng / mL tolbutamide. After vortexing and mixing thoroughly, the mixture was centrifuged at 4000 r / min for 10 minutes at 4°C. 100 µL of the supernatant was then added to 100 µL of deionized water and mixed thoroughly to obtain the test sample. The test sample containing the acetylcholine hydrochloride standard sample was analyzed by LC-MS / MS. The precision and accuracy data of the standard curve are shown in Figure 1.

[0034] As shown in Figure 1, the standard curve equation for acetic acid ketidine hydrochloride is: y = 0.01584x + 7.02845 × 10 -4 (r=0.99814, r) 2 =0.99629). The linear range of the standard curve is 0.25~50 ng / mL, which validates a large linear range and can detect concentrations from pg / ml to ng / ml, effectively distinguishing target analytes from matrix interference.

[0035] Example 2

[0036] This embodiment is based on the requirements of the General Rules of Pharmacopoeia 9012. Quality control samples of acetylcholine hydrochloride were prepared according to these rules. Acetylcholine hydrochloride was diluted with a blank protein matrix to prepare quality control samples of different concentration gradients, with concentrations of 0.25 ng / mL, 0.75 ng / mL, 8.00 ng / mL, and 35.00 ng / mL, respectively. This embodiment uses the same processing method as in Example 1. 30 µL of quality control samples of different concentrations of acetylcholine hydrochloride were added to 270 µL of acetonitrile containing 10 ng / mL tolbutamide. After vortexing and mixing thoroughly, the mixture was centrifuged at 4000 r / min for 10 minutes at 4°C. 100 µL of the supernatant was added to 100 µL of deionized water and mixed thoroughly to obtain the test samples. The prepared quality control sample solutions of different concentrations of acetylcholine hydrochloride were analyzed by LC-MS / MS. The detection results are shown in Table 3.

[0037] Table 3. Accuracy Results of Quality Control Samples

[0038] As shown in Table 3, based on the standard curve obtained from the test in the example, and using the analytical method provided by this invention, the concentrations at the four test points are basically close to the theoretical concentrations, indicating high precision (RSD<10%).

[0039] Figure 2 is the chromatogram of the sample with a lower limit of quantification of 0.25 ng / ml in the standard curve. As can be seen from Figure 2, according to the standard curve of acetic acid hydrochloride, the method of the present invention is used to analyze acetic acid hydrochloride in biological matrix with a high signal-to-noise ratio and no impact. This shows that the method provided by the present invention basically meets the requirements of quantitative detection.

[0040] In summary, this invention significantly improves detection sensitivity and accuracy by optimizing chromatographic separation and mass spectrometry detection parameters. The precision and accuracy of the standard curve prepared by this invention meet the requirements of the General Rules of Pharmacopoeia 9012, with a limit of quantification of 250 pg / mL. The detection method has high sensitivity and can meet the new needs for quantitative detection of acetylcholine hydrochloride in preclinical and clinical trials.

[0041] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An analytical method for acetic acid ketidine hydrochloride in a biological matrix, characterized in that, The process includes the following steps: pretreating the biological matrix using a protein precipitation method to obtain the sample to be tested; performing liquid chromatography-tandem mass spectrometry analysis on the sample to be tested, and obtaining the content of acetylcholine hydrochloride in the biological matrix according to the standard curve of acetylcholine hydrochloride.

2. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The pretreatment includes the following steps: adding the biological matrix to a precipitant containing an internal standard solution, vortexing and mixing thoroughly, centrifuging at 3800~4500 r / min for 8~12 minutes at 4℃, taking the supernatant and adding it to deionized water, mixing thoroughly, and obtaining the sample to be tested.

3. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 2, characterized in that, The volume ratio of the biological matrix to the precipitant is 1:9, and the volume ratio of the supernatant to the deionized water is 1:

1.

4. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 2, characterized in that, The precipitant is acetonitrile, and the internal standard solution is tolbutamide solution.

5. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The biological matrix is ​​any one of plasma, serum, whole blood, excrement, or tissue homogenate.

6. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry using an AB Sciex 7500 Triple Quad instrument and an ACQUITY UPLC® BEH C18 1.7 μm 2.1 mm × 50 mm column.

7. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry. The first mobile phase was an ultrapure aqueous solution containing 0.1% formic acid, and the second mobile phase was an acetonitrile solution containing 0.1% formic acid.

8. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry. The chromatographic conditions used included: column temperature of 40℃; injection plate temperature of 4℃; injection volume of 5 µL; run time of 5 min; and gradient elution at a flow rate of 0.4 mL / min.

9. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 8, characterized in that, The gradient elution procedure includes: from 0.0 min to 0.5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%; from 0.5 min to 3.5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%; from 3.5 min to 4.2 min, the volume percentage of the first mobile phase is 5% and the volume percentage of the first mobile phase is 95%; from 4.2 min to 4.3 min, the volume percentage of the first mobile phase is 5% and the volume percentage of the first mobile phase is 95%; and from 4.3 min to 5 min, the volume percentage of the first mobile phase is 95% and the volume percentage of the first mobile phase is 5%.

10. The analytical method for acetylchloride hydrochloride in a biological matrix according to claim 1, characterized in that, The sample to be tested was analyzed by liquid chromatography-tandem mass spectrometry. The mass spectrometry conditions used included: electrospray ionization source; positive ion multiple reaction monitoring mode; positive ion mode; curtain gas: 40 psi; collision gas: 10 psi; ion voltage: 5500 V; ion source temperature: 550℃; nebulizer gas: 35 psi; auxiliary heating gas: 70 psi; inlet voltage: 10 V; collision cell outlet voltage: 13 V.

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