Quantitative detection method for citicoline concentration in blood

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a quantitative detection method for citicoline concentration in blood. According to the method, a blood sample is pretreated by adopting a protein precipitation method, and the concentration of citicoline in the sample is analyzed and measured by taking an electrospray ionization source as an ionization technology and adopting a high performance liquid chromatography-tandem mass spectrometry method in a multi-reaction monitoring mode. According to the quantitative detection method for the citicoline concentration in the blood, the citicoline concentration in the blood can be effectively quantified, and the method is easy to operate, stable, reliable and high in detection sensitivity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, and particularly relates to a quantitative detection method for the concentration of choline alfoscerate in blood. BACKGROUND

[0002] Choline alfoscerate sodium is a brain metabolism activator, which can enhance the function of the ascending reticular activating system, reduce cerebral vascular resistance, increase cerebral blood flow, improve cerebral blood circulation, promote cerebral material metabolism, and restore function. It is mainly used in clinic for acute craniocerebral trauma and postoperative disturbance of consciousness, and for treating Parkinson's syndrome and nerve deafness, and can also be used for functional and conscious disorders caused by other acute injuries of central nervous system, ischemic cerebral vascular disease and vascular dementia, and has certain curative effects on senile dementia, depression, delaying aging and improving learning effect and memory.

[0003] Choline alfoscerate currently has tablets, capsules and injection solutions. In order to meet the clinical needs, many manufacturers have developed new dosage forms such as choline alfoscerate granules, enteric-coated capsules, enteric-coated tablets, chewable tablets and dispersible tablets. However, there is no recognized analytical method for evaluating the concentration change of choline alfoscerate in the human body.

[0004] In order to reflect the concentration change of choline alfoscerate in the body, so as to better provide the clinical pharmacokinetic data of the product for the approval of new dosage forms, the present inventors have developed a blood drug concentration detection method based on liquid chromatography tandem mass spectrometry (LC-MS / MS), which has the advantages of simple operation, stability and reliability, and high detection sensitivity.

[0005] Currently, there is no report on detecting choline alfoscerate in blood by using liquid chromatography tandem mass spectrometry (LC-MS / MS). SUMMARY

[0006] In order to overcome the defects and deficiencies in the prior art, the present application provides a quantitative detection method for the concentration of choline alfoscerate in blood.

[0007] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0008] The present application provides a quantitative detection method for the concentration of choline alfoscerate in blood, which comprises the following steps:

[0009] (1) pretreating the blood sample to be detected to obtain a target sample solution;

[0010] (2) detecting the target sample solution by using high performance liquid chromatography-tandem mass spectrometry to obtain the content of choline alfoscerate in the blood sample,

[0011] The high performance liquid chromatography conditions are:

[0012] Column: Phenomenex, Comixsil ACRP column (3.0 x 50.0 mm, 3 μm); Column temperature: 38-42 °C; Autosampler temperature: 2-5 °C; Isocratic elution with mobile phase A and mobile phase B, mobile phase A is 0.05-1.5% formic acid in water with 50-150 mM ammonium formate, mobile phase B is 85-95% acetonitrile in water; Injection volume is 2-5 μL.

[0013] As an optional way, in the above quantitative detection method, in step (1),

[0014] The high performance liquid chromatography conditions are as follows:

[0015] Column: Phenomenex, Comixsil ACRP column; Column temperature: 40 °C; Autosampler temperature: 4 °C; Isocratic elution with mobile phase A and mobile phase B, mobile phase A is 0.1% formic acid in water with 100 mM ammonium formate, mobile phase B is 90% acetonitrile in water; Injection volume is 5 μL.

[0016] As an optional way, in the above quantitative detection method, in step (2), the ionization mode of the tandem mass spectrometry is electrospray ion source, negative ion mode, multiple reaction monitoring.

[0017] As an optional way, in the above quantitative detection method, the ion source spray voltage is 3500 V, curtain gas 30 psi, ion source temperature 500 °C, collision energy 37 V.

[0018] Preferably, as an optional way, in the above quantitative detection method, the ion source parameters are as follows: ion source spray voltage (IS): 3500 V;

[0019] curtain gas (CUR): 30 psi;

[0020] ion source gas 1 (GS1): 50 psi;

[0021] ion source gas 2 (GS2): 55 psi;

[0022] ion source temperature (TEM): 500 °C;

[0023] resolution Q1 (Resolution Q1): Unit;

[0024] resolution Q3 (Resolution Q3): Unit;

[0025] collision gas (CAD): Medium;

[0026] de-clustering voltage (DP): 70 V;

[0027] Inlet voltage (EP): 10 V;

[0028] Outlet voltage (CXP): 10 V;

[0029] Collision energy (CE): 37 V;

[0030] Pause time (MR Pause): 10.0000 msec.

[0031] As an optional mode, in the quantitative detection method, the blood sample is a human plasma sample.

[0032] Preferably, the blood sample is an EDTA-K2 anticoagulated human plasma sample.

[0033] As an optional mode, in the quantitative detection method, the isocratic elution condition is time 0-3 min; flow rate 0.3-0.5 mL / min; the ratio of mobile phase A to mobile phase B is (20-50) %:(50-80) % in terms of volume percentage.

[0034] As an optional mode, in the quantitative detection method, the isocratic elution condition is time 0-3 min; flow rate 0.4 mL / min; the ratio of mobile phase A to mobile phase B is 30 %:70 % in terms of volume percentage.

[0035] As an optional mode, in the quantitative detection method, the method for pretreating the blood sample to be tested comprises the following steps: collecting the blood sample to be tested, adding the internal standard substance, choline d9, vortexing, adding the precipitant, methanol, vortexing, centrifuging, taking the supernatant, adding purified water, and mixing to obtain the target sample solution.

[0036] As an optional mode, in the quantitative detection method, the concentration of the internal standard substance, choline d9, is 5000 ng / mL, and the volume ratio of the blood sample to the internal standard solution is 1:1.

[0037] As an optional mode, in the quantitative detection method, the amount of methanol added is 3-5 times the total volume of the sample, and the amount of purified water added is 1-3 times the total volume of the supernatant.

[0038] Preferably, the amount of methanol added is 4 times the total volume of the sample, and the amount of purified water added is 2 times the total volume of the supernatant.

[0039] More preferably, in the quantitative detection method, the method for pretreating the blood sample to be detected comprises the following steps: collecting the blood sample to be detected, adding an internal standard choline d9 with a concentration of 5000 ng / mL, the volume ratio of the blood sample to the internal standard solution being 1:1, vortexing for at least 3 min, adding a precipitant methanol, the amount of methanol added being 4 times the total volume of the sample, vortexing for at least 10 min, centrifuging (at a temperature of 4 DEG C, 4000 rpm for 10 min), taking the supernatant, adding purified water, the amount of purified water added being 2 times the total volume of the supernatant, and vortexing for at least 3 min to obtain the target sample solution.

[0040] As an optional mode, in the quantitative detection method, the detection concentration range of the quantitative detection method is 50.0 to 50000 ng / mL.

[0041] As an optional mode, in the quantitative detection method, the retention time of choline is about 1.35 min, and the retention time of the internal standard choline d9 is about 1.32 min.

[0042] As an optional mode, in the quantitative detection method, the mass-to-charge ratio (m / z) of choline is 489.3 / 264.1, and the mass-to-charge ratio (m / z) of the internal standard choline d9 is 498.3 / 273.1.

[0043] Compared with the prior art, the present application has the following advantages and positive effects:

[0044] In the present application, the blood sample is pretreated by using a protein precipitation method, in a multiple reaction monitoring mode, an electrospray ion source is used as an ionization technology, and a high performance liquid chromatography-tandem mass spectrometry method is used to analyze and determine the concentration of choline in the sample. The quantitative detection method for the concentration of choline in blood plasma established in the present application can effectively quantitatively detect the concentration of choline in blood plasma, and the method is simple, stable and reliable, and has high detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0045] Fig. 1 The ion scan diagram of the measured substance choline in the quantitative detection method for the concentration of choline in blood plasma of the present application.

[0046] Fig. 2 The blank matrix spectrum of the quantitative detection method for the concentration of choline in blood plasma of the present application.

[0047] Fig. 3 The lowest quantitative limit spectrum of the quantitative detection method for the concentration of choline in blood plasma of the present application.

[0048] Fig. 4 The standard curve diagram of the quantitative detection method for the concentration of choline in blood plasma of the present application. DETAILED DESCRIPTION

[0049] The application will be further described in connection with specific examples. It should be understood that the specific examples described herein are intended to be illustrative only and are not intended to limit the scope of the application.

[0050] Unless otherwise indicated, technical or conditions in the examples were carried out according to the techniques or conditions described in the literature or according to the manufacturer's instructions. Unless otherwise indicated, reagents or instruments were commercially available and were used according to the manufacturer's instructions.

[0051] Unless otherwise indicated, experimental methods in the following examples were carried out according to the conventional methods. Unless otherwise indicated, experimental materials in the following examples were commercially available.

[0052] Example 1: Establishment of a quantitative detection method for the concentration of choline alfoscerate in plasma

[0053] 1. Experimental methods and materials

[0054] 1.1 Test substance

[0055] Choline alfoscerate (from China Institute for Food and Drug Control, batch number 140675-202107).

[0056] 1.2 Reference substance

[0057] Internal standard: choline alfoscerate-d9 (from Toronto Research Chemicals, batch number 24-MJC-81-1).

[0058] 1.3 Blank matrix

[0059] Blank matrix, blank human plasma with anticoagulant EDTA-K2 and whole blood (to investigate the stability of whole blood) from Chinese healthy volunteers, 0.1% formic acid aqueous solution of 5% human serum albumin.

[0060] 1.4 Reagents

[0061] All solvents and reagents were chromatographically pure (HPLC), mass spectrometrically pure (LC-MS grade), and were used without further purification. Water was deionized and purified using an ELGA water purification system.

[0062] 1.5 Main instruments

[0063]

[0064] 1.6 Analysis conditions 1.6.1 Liquid chromatography conditions

[0065]

[0066] 1.6.2 Mass spectrometry conditions

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[0068]

[0069] 1.7 Preparation of standard curve, quality control sample, internal standard stock solution and working solution 1.7.1 Preparation procedure of mobile phase:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] 1.8 Data processing

[0076] 1.8.1 Chromatogram acquisition and concentration calculation method

[0077] Data acquisition, integration, regression (weight factor w = 1 / x 2 ) and concentration calculation were performed using software Analyst Version 1.6.3 (Applied Biosystems / Sciex), all by automatic integration method.

[0078] 1.8.2 Concentration unit and decimal point retention method

[0079] The peak area was retained as an integer, the retention time was retained as three decimal places, the ratio of the peak area of the analyte to the internal standard was retained as six decimal places, the concentration was expressed in ng / mL, the result was retained as three significant figures, and the deviation and precision, etc. (all values expressed in percentage) were reported as one decimal place.

[0080] 1.8.3 Data acquisition and processing software

[0081]

[0082] 1.8.4 Calculation method of data in the table

[0083] The interference of endogenous substances in the blank matrix on the analyte and internal standard (%) = peak area value of the analyte (internal standard) in the blank matrix sample / average peak area of the analyte (internal standard) in the valid LLOQ standard curve sample x 100;

[0084] Interference of internal standard to analyte (%) = (average peak area of internal standard in the upper limit of quantification sample without adding internal standard) / (average peak area of internal standard in the lower limit of quantification sample of the effective LLOQ calibration curve) x 100;

[0085] Interference of internal standard to analyte (%) = (average peak area of internal standard in the upper limit of quantification sample without adding internal standard) / (average peak area of internal standard in the lower limit of quantification sample of the effective LLOQ calibration curve) x 100;

[0086] Matrix effect factor = (peak area of analyte in the extracted blank matrix sample, which needs to be deducted from the average peak area of endogenous substances in the corresponding blank matrix) / (average peak area of analyte in the sample of the same concentration solution);

[0087] Stability (bias) of stock solution: average stability (%) = (average peak area ratio of stability investigation sample - average peak area ratio of control sample) / average peak area ratio of control sample x 100;

[0088] Stability (bias) of working solution: average stability (%) = (average peak area ratio of stability investigation sample - average peak area ratio of control sample) / average peak area ratio of control sample x 100;

[0089] Stability of matrix sample: accuracy % = (average measured concentration - background concentration) / theoretical concentration x 100;

[0090] Some representative graphs of the quantitative detection method of the concentration of choline alfoscerate in plasma of the present application are shown in Figs. 1 to 4 .

[0091] Example 2: Methodological verification of the quantitative detection method of the concentration of choline alfoscerate in plasma

[0092] The methodological verification results of the method of the present application meet the acceptance indicators of linearity, accuracy, precision, selectivity (interference), recovery rate, matrix effect, dilution reliability, residual in the process of batch sample detection, stability and the like specified in the method of the present application. The method of the present application is suitable for LC-MS / MS determination of the concentration of choline alfoscerate in human plasma (EDTA-K2) sample, and the sample usage is 50.0 μL, and the concentration range is 50.0 to 50000 ng / mL. The specific method verification results are shown in Tables 1-10.

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Table 10: Long term stability test conditions of the test substances in the biological matrix at -80°C: stability samples were tested after 65 days of storage at -80°C.

[0103]

[0104] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A method for quantitative determination of the concentration of cytidine diphosphate choline in blood, characterized in that: The detection method comprises the following steps: (1) pretreating the blood sample to be detected to obtain a target sample solution; (2) detecting the target sample solution by using high performance liquid chromatography-tandem mass spectrometry to obtain the content of choline in the blood sample, The high performance liquid chromatography conditions are as follows: The chromatographic column is a Phenomenex Comixsil ACRP column, the column temperature is 38-42 DEG C, the sample injector temperature is 2-5 DEG C, isocratic elution is performed by using mobile phase A and mobile phase B, the mobile phase A is 0.05-1.5% formic acid aqueous solution containing 50-150 mM ammonium formate, the mobile phase B is 85-95% acetonitrile aqueous solution, and the sample injection volume is 2-5 μL.

2. The quantitative detection method according to claim 1, characterized in that: In step (1), The high performance liquid chromatography conditions are as follows: The chromatographic column is a Phenomenex Comixsil ACRP column, the column temperature is 40 DEG C, the sample injector temperature is 4 DEG C, isocratic elution is performed by using mobile phase A and mobile phase B, the mobile phase A is 0.1% formic acid aqueous solution containing 100 mM ammonium formate, the mobile phase B is 90% acetonitrile aqueous solution, and the sample injection volume is 5 μL.

3. The method of claim 1, wherein: In step (2), the ionization mode of the tandem mass spectrometry is electrospray ion source, negative ion mode and multiple reaction monitoring.

4. The quantitative detection method according to claim 3, characterized in that: The ion source spray voltage is 3500 V, the curtain gas is 30 psi, the ion source temperature is 500 DEG C, and the collision energy is 37 V.

5. The method of claim 1, wherein: The blood sample is a human plasma sample.

6. The method of claim 1, wherein: The isocratic elution conditions are as follows: time 0-3 min, flow rate 0.3-0.5 mL / min, and the ratio of mobile phase A to mobile phase B is (20-50)%:(50-80)% in terms of volume percentage.

7. The quantitative detection method according to claim 6, characterized in that: The isocratic elution conditions are as follows: time 0-3 min, flow rate 0.4 mL / min, and the ratio of mobile phase A to mobile phase B is 30%:70% in terms of volume percentage.

8. The method of claim 1, wherein: The method for pretreating the blood sample to be detected comprises the following steps: collecting the blood sample to be detected, adding internal standard choline d9, vortexing, adding precipitant methanol, vortexing, centrifuging, taking the supernatant, adding purified water, and mixing to obtain the target sample solution.

9. The quantitative detection method according to claim 8, characterized in that: The concentration of the internal standard choline d9 is 5000 ng / mL, and the volume ratio of the blood sample to the internal standard solution is 1:

1.

10. The method of claim 1, wherein: The detection concentration range of the quantitative detection method is 50.0-50000 ng / mL.

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