CN118032984A - LC-MS/MS method for simultaneously measuring concentration of memantine and donepezil in human plasma - Google Patents

Abstract

The invention relates to the technical field of medicine analysis, in particular to an LC-MS/MS method for simultaneously measuring the concentration of memantine and donepezil in human blood plasma; the method comprises the following steps: adding 0.2-0.25 mol/L sodium carbonate into a plasma sample containing memantine and donepezil for treatment, adding an internal standard working solution II for precipitation reaction, respectively injecting the supernatant into a liquid chromatograph-mass spectrometer for multi-reaction monitoring reaction, and calculating the content of memantine and donepezil in the plasma according to a standard curve; the standard curve of the memantine content takes the concentration of memantine as an abscissa and the peak area ratio of memantine to memantine-d 6 as an ordinate; the standard curve of the donepezil content takes the concentration of donepezil as an abscissa and takes the peak area ratio of donepezil and donepezil-d 7 as an ordinate; the invention detects memantine and donepezil in blood plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, and has the advantages of rapidness, accuracy and stability.

Description

LC-MS/MS method for simultaneously measuring concentration of memantine and donepezil in human plasma

Technical Field

The invention relates to the technical field of medicine analysis, in particular to an LC-MS/MS method for simultaneously measuring the concentration of memantine and donepezil in human blood plasma.

Background

Alzheimer's Disease (AD) is hidden, most families in China lack knowledge about the disease, and patients have the conditions of untimely treatment or no hospital treatment at all. The data show that the treatment rate of Chinese mild AD patients is 14%, the treatment rate of moderate AD patients is 25%, and the treatment rate of severe AD patients is less than 34%. The U.S. Food and Drug Administration (FDA) currently approves only 5 drugs for the treatment of AD, namely tacrine, donepezil, rivastigmine, galantamine and memantine, the first 4 being acetylcholinesterase inhibitors. The European neurological Association (EFNS) and the American society of psychiatry (APA) guidelines uniformly recommend that FDA approved acetylcholinesterase inhibitors (AChEI, such as donepezil, rivastigmine and galantamine) be first line therapeutic agents for AD with glutamate N-methyl-aspartate (NMDA) receptor antagonists (memantine). AChEIs is effective in treating cognitive and non-cognitive symptoms in mild, moderate AD patients, and there is also study support AChEls for treatment of severe AD patients. Memantine treatment is effective in treating cognitive and non-cognitive symptoms in patients with severe AD, and the treatment effect of non-cognitive symptoms (agitation, delusions) is superior to other symptoms, and guidelines indicate that memantine can also be used in the treatment of patients with mild AD. EFNS and the APA guidelines indicate that combined AChEI and memantine treatment is more effective than AChEI alone, and that the combination of both has a synergistic effect, memantine (memantine) and donepezil (donepezil) in combination, is an effective treatment regimen for moderate to severe alzheimer's disease patients.

On 12 months and 23 days 2014, forest Laboratories (purchased from avis) and a compound new drug Namzaric (memantine Ji Huanshi capsule hydrochloride) of Adamas pharmaceutical company are marketed in the united states, and are suitable for moderate to severe alzheimer's patients who are receiving treatment with memantine hydrochloride and donepezil hydrochloride and have stable conditions, and the specifications are 7 mg/10 mg, 14 mg/10 mg, 21 mg/10 mg, 28 mg/10 mg. Namzaric is a once daily oral capsule consisting of a fixed dose of memantine and donepezil. The capsule may be opened and the contents sprinkled on the food to facilitate patients who may have dysphagia. Namzaric help to ease the burden of daily medication on the patient and improve patient compliance and compliance (i.e., compliance).

At present, the method for simultaneously measuring the blood concentration of memantine and donepezil in human bodies in China has not been studied, and the conventional organic solvent plasma protein precipitation method has low precipitation efficiency, so that the extraction recovery rate is low, and the detection sensitivity of the method cannot meet the requirement of the lower limit of quantification; the determination of the plasma drug concentration in the living body plays an important role in evaluating the drug curative effect, analyzing the pharmacological action and the like, so that a method for simultaneously determining the concentration of memantine and donepezil in human plasma is developed, and the study of the pharmacokinetics and human bioequivalence of memantine and donepezil in the body of a Chinese healthy subject in a fasting and postprandial state is carried out, so that the method not only can provide basis for evaluating the consistency of related preparations of memantine and donepezil, but also has wide economic benefit and social benefit.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide an LC-MS/MS method for simultaneously measuring the concentration of memantine and donepezil in human blood plasma. The invention carries out the detection of memantine and donepezil in blood plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, can be used for detecting the concentration of memantine and donepezil in organisms, and provides a rapid, accurate and stable detection method.

In order to achieve the above object, the present invention provides the following technical solutions:

An LC-MS/MS method for simultaneously determining the concentration of memantine and donepezil in human plasma, said method comprising the steps of:

Adding sodium carbonate into a plasma sample containing memantine and donepezil for treatment, vortex mixing for 1min, adding an internal standard working solution II into the mixture for precipitation reaction, vortex mixing for 5min, placing the mixture into a 4 ℃ table-type high-speed refrigerated centrifuge (96-well plate), centrifuging for 10 min per minute by 4750 r/min, taking 100 mu L of supernatant from each well, transferring the supernatant to another 96-well plate, adding 100 mu L of 0.1% formic acid aqueous solution into each well for dilution, vortex mixing for 5min, and performing liquid-solid combination detection to calculate the content of memantine and donepezil in the plasma by using an internal standard method according to a standard curve; the standard curve of the memantine content takes the concentration of memantine as an abscissa and the peak area ratio of memantine to memantine-d 6 as an ordinate; the standard curve of donepezil content is on the abscissa of donepezil concentration and on the ordinate of the peak area ratio of donepezil to donepezil-d 7.

Further, the internal standard working solution II is a methanol solution containing memantine-d 6 and donepezil-d 7.

Further, the high performance liquid chromatography conditions of the liquid chromatography-mass spectrometry detection include: the column was an ACQUITY UPLC-BEH C18 (2.1X150 mm,1.7 μm) column, gradient elution was performed, mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was acetonitrile, and the elution procedure was: 0-1.70 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; 1.70-1.75 min, the volume percentage of the mobile phase A is reduced from 80% to 65%, and the volume percentage of the mobile phase B is increased from 20% to 35%; 1.75-2.20 min, the volume percentage of the mobile phase A is kept 65%, and the volume percentage of the mobile phase B is kept 35%; 2.20-2.90 min, the volume percentage of the mobile phase A is reduced from 65% to 20%, and the volume percentage of the mobile phase B is increased from 35% to 80%; 2.90-3.50 min, wherein the volume percentage of the mobile phase A is kept 20%, and the volume percentage of the mobile phase B is kept 80%; 3.50-3.70 min, the volume percentage of the mobile phase A is reduced from 20% to 5%, and the volume percentage of the mobile phase B is increased from 80% to 95%; 3.70-4.00 min, wherein the volume percentage of the mobile phase A is kept 5%, and the volume percentage of the mobile phase B is kept 95%; 4.00-4.20 min, the volume percentage of the mobile phase A is increased from 5% to 80%, and the volume percentage of the mobile phase B is decreased from 95% to 20%; 4.20-4.50 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 15 mu L.

Further, the mass spectrometry conditions of the liquid chromatography-mass spectrometry detection include: using a triple quadrupole mass spectrometer, wherein the ion source is an electrospray ion source, a positive ion mode is adopted, a multi-reaction monitoring scanning mode is adopted, and ionization voltage is calculated: 5500 V, V; inlet voltage: 10 V, V; collision cell exit voltage: 13 V, V; spraying gas: 50; auxiliary heating gas: 55; air curtain gas: 35; collision energy of the memantine: 18V; collision energy of memantine-d 6: 20V; collision energy of the donepezil: 41V; collision energy of donepezil-d 7: 60V; the declustering voltage of memantine: 68V; declustering voltage of memantine-d 6: 95V; the declustering voltage of donepezil: 70V; the declustering voltage of donepezil-d 7: 150V.

Further, the quantitative ion pairs of memantine are 180.2- > 163.2, respectively, the quantitative ion pair of donepezil is 380.3- > 91.1, the ion pair of memantine-d 6 is 186.2- > 169.2, and the ion pair of donepezil-d 7 is 387.2- > 98.1.

Further, the manufacturing method of the internal standard working solution II comprises the following steps: respectively precisely weighing memantine-d 6 and donepezil-d 7 standard substances, dissolving the standard substances with N, N-dimethylformamide to prepare a memantine-d 6 internal standard mother solution of 1 mg/mL and a donepezil-d 7 internal standard mother solution of 1 mg/mL, and mixing the memantine-d 6 internal standard mother solution and the donepezil-d 7 internal standard mother solution according to a volume ratio of 1:1 to obtain a mixed internal standard working solution I, and diluting the mass concentration of the mixed internal standard working solution I to 1 ng/mL by using a methanol solution to obtain an internal standard working solution II, namely the methanol solution containing memantine-d 6 and donepezil-d 7.

Further, the method for pretreating plasma containing memantine and donepezil employs the following steps: 100 μl of plasma samples containing memantine and donepezil were removed and placed in a 2mL 96-well plate, to which 0.2-0.25 mol/liter sodium carbonate solution 50 μl was added, and vortexed to mix 1 min.

Further, the precipitation reaction includes the steps of: mixing the pretreated plasma sample containing memantine and donepezil with 450 mu L of internal standard working solution II, vortex mixing 5min, placing in a4 ℃ desk-top high-speed refrigerated centrifuge, centrifuging 10min at 4750 r/min, taking 100 mu L of supernatant from each hole, transferring to another 96-well plate, diluting 100 mu L of 0.1% formic acid aqueous solution in each hole, vortex mixing 5min, and injecting into a liquid chromatograph-mass spectrometer for liquid chromatography-mass spectrometry detection.

Further, the standard curve is prepared by a method comprising the steps of:

(1) And weighing memantine and donepezil standard substances, respectively dissolving the memantine standard substances with N, N-dimethylformamide to obtain a memantine mother solution with the concentration of 1 mg/mL and donepezil Ji Muye with the concentration of 1 mg/mL, and mixing the memantine mother solution with the donepezil Ji Muye according to the volume ratio of 1:1, mixing to obtain a mixed solution, diluting the mass concentration of the mixed solution to 20 mug/mL by using a 50% methanol aqueous solution to obtain a mixed working solution, and diluting the mixed working solution to 4.00,8.00, 20.0, 100.00, 200.00, 600.00, 900.00 and 1000.00 and ng/mL standard curve mixed working solution by using the 50% methanol aqueous solution;

(2) Taking 285 mu L of blank plasma, and respectively adding 15 mu L of standard curve mixed working solutions with different concentrations obtained in the step (1) to obtain 0.20,0.40,1.00,5.00, 10.00, 30.00, 45.00 and 50.00 ng/mL standard curve samples;

(3) Respectively carrying out precipitation reaction on the standard curve sample obtained in the step (2) and 400 mu L of methanol solution containing memantine-d 6 and donepezil-d 7, carrying out vortex mixing for 5min, placing the mixture in a 4 ℃ table type high-speed refrigerated centrifuge, centrifuging for 10min at 4750 r/min, taking 100 mu L of supernatant from each hole, transferring the supernatant to another 96-well plate, diluting each hole by adding 100 mu L of 0.1% formic acid aqueous solution, and carrying out liquid chromatography-mass spectrometry detection by vortex mixing for 5 min;

(4) Calculating the contents of memantine and donepezil in the blood plasma according to a standard curve by an internal standard method respectively; the standard curve of the memantine content takes the concentration of memantine as an abscissa and the peak area ratio of memantine to memantine-d 6 as an ordinate; the standard curve of the donepezil content takes the concentration of donepezil as an abscissa and takes the peak area ratio of donepezil and donepezil-d 7 as an ordinate; and (3) taking 1/x ² as a weight coefficient, and carrying out linear regression by using a weighted least square method to obtain the standard curve.

The invention establishes a method for detecting the concentration of memantine and donepezil in blood plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, can be used for simultaneously detecting the concentration of memantine and donepezil in a living body, provides a reliable analysis means for clinical and basic research, carries out pretreatment on the blood plasma, can separate part of memantine and donepezil combined with protein, can remove endogenous interferents through one-step precipitation reaction, adds formic acid into a mobile phase in the separation process of high performance liquid chromatography, leads the concentration of memantine and donepezil to form more hydrogenated excimer ion peaks after ionization, and increases the abundance of corresponding characteristic fragment ions, thereby improving the detection sensitivity; in the quantitative process, memantine-d 6 and donepezil-d 7 are used as internal standard substances, and the quantitative detection of memantine and donepezil is realized through the specific and high-sensitivity monitoring of excimer ion peak-daughter ion.

The beneficial effects are that: 1. the invention provides a method for simultaneously measuring the concentration of memantine and donepezil in human blood plasma; according to the method, sodium carbonate is used for pretreatment of plasma, then methanol is used for protein precipitation of a plasma sample, an internal standard working solution II is directly prepared into a precipitator, organic reagent consumption is low, and detection accuracy, clinical applicability and working efficiency are greatly improved; 0.1% formic acid aqueous solution and acetonitrile are selected as mobile phases, so that the sensitivity is improved, the peak position is stable, and the single-needle detection time is shortened; the method uses an isotope labeled internal standard, so that the durability of the method is greatly improved; in order to reduce the matrix effect of this assay, the supernatant after centrifugation of the protein pellet was subjected to 1:1 dilution (dilution: 0.1% aqueous formic acid). The method is verified by methodology to meet the analysis requirement of biological samples, the passing rate of the reanalysis of the actual samples is 100 percent, and the method is successfully applied to the clinical blood concentration detection and has reliable results.

2. The method for measuring the contents of memantine and donepezil in human plasma has the advantages of high sensitivity, accuracy, reliability, good selectivity, small matrix effect, rapidness, simplicity, convenience and the like; the invention provides data reference for bioequivalence study of the related preparations of the memantine and the donepezil, is suitable for blood concentration detection of the memantine and the donepezil and pharmacokinetics study thereof, and provides basis for consistency evaluation of the preparations of the memantine and the donepezil.

Drawings

FIG. 1 is a chromatogram of memantine and donepezil in plasma containing quantitative upper limit memantine and donepezil after addition of an internal standard; FIG. 1a is a chromatogram of memantine; FIG. 1b is a chromatogram of donepezil;

FIG. 2 is a chromatogram of memantine-d 6 and donepezil-d 7 in plasma containing upper limit amounts of memantine-d 6 and donepezil-d 7 after addition of an internal standard; FIG. 2a is a chromatogram of memantine-d 6; FIG. 2b is a chromatogram of donepezil-d 7;

FIG. 3 is a chromatogram of memantine and donepezil in blank plasma; FIG. 3a is a chromatogram of memantine; FIG. 3b is a chromatogram of donepezil;

FIG. 4 is a chromatogram of memantine-d 6 and donepezil-d 7 in blank plasma; FIG. 4a is a chromatogram of memantine-d 6; FIG. 4b is a chromatogram of donepezil-d 7.

Detailed Description

In the following description, specific details of the invention are set forth in order to provide a thorough understanding of the invention. The terminology used in the description of the invention herein is for the purpose of describing the advantages and features of the invention only and is not intended to be limiting of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The medicines or reagents used in the present invention are used according to the product instructions or by the conventional methods of use in the art unless specifically stated. The technical scheme of the invention is further described according to the attached drawings and the specific embodiments.

Examples

1. Instruments and reagents

1) Analytical instrument

AB SCIEX Tripe Quad 5500 liquid chromatography-mass spectrometry (AB SCIEX company, usa); 3K15 bench high-speed refrigerated centrifuge (Sigma, germany); allegra X-15R bench top high-speed cryocentrifuge (96-well plate, beckmann Coulter, U.S.A.); MDF-U5412N medical incubator (Japanese pine Co.); milli-Q ADVANTAGE A ultra-pure water unit (Millipore Co., U.S.A.); DG-2500R Multitube vortex mixer (Shanghai Bajia Co., ltd.). Data processing software: analyst 1.6.3 (AB SCIEX Co., USA); watson LIMS 7.5 SPS1 (Sieimer, inc. of United states).

2) Reagent(s)

Memantine hydrochloride control (purity 99.4%, bePure); donepezil hydrochloride control (purity 99.9%, shanghai' an spectrum bright world); memantine-d 6 hydrochloride control (chemical purity 99.9%, isotopic internal standard purity 97.8%, ALTA SCIENTIFIC); donepezil hydrochloride-d 7 control (chemical purity 99.9%, isotopic internal standard purity 98.0%, ALTA SCIENTIFIC); methanol, acetonitrile (chromatographic purity, merck, germany); formic acid (chromatographic purity, ACS encyclopedia chemical in the united states); n, N-dimethylformamide (chromatographic purity, sameifeishi technologies (china) technologies). Test formulation: memantine hydrochloride Ji Huanshi capsules (specification: each granule contains memantine hydrochloride 28 mg and donepezil hydrochloride 10 mg); reference formulation: memantine hydrochloride Ji Huanshi capsules (trade name: namzaric) (specification: each containing Memantine hydrochloride 28 mg and donepezil hydrochloride 10 mg,Allergan Sales LLC are approved by XX Co., ltd.).

2. Experimental methods and results

1) Solution preparation

Preparing a standard substance solution: memantine and donepezil standards were precisely weighed and dissolved in N, N-Dimethylformamide (DMF) to give 1mg/mL memantine mother liquor and 1mg/mL donepezil Ji Muye, respectively. Respectively precisely measuring 200 mu L of memantine mother solution and 200 mu L of donepezil mother solution, placing into a 10 mL volumetric flask, and adding 50% methanol aqueous solution to the scale to enable the mass concentration to be 20 mu g/mL of the memantine and donepezil mixed working solution. The memantine and donepezil mixed working solution was diluted with 50% aqueous methanol to 4.00,8.00, 20.00, 100.00, 200.00, 600.00, 900.00 and 1000.00 ng/mL standard curve mixed working solution.

Preparing an internal standard working solution: about 1 mg memantine-d 6 and donepezil-d 7 standard substances are precisely weighed, a certain amount of N, N-Dimethylformamide (DMF) is used for dissolution, 1 mg/mL of memantine-d 6 internal standard mother solution and 1 mg/mL of donepezil-d 7 internal standard mother solution are prepared respectively, 10 mu L of memantine mother solution and 10 mu L of donepezil mother solution are respectively taken and placed in a 10 mL volumetric flask, 1000 ng/mL of mixed internal standard working solution I is obtained, and the mixed internal standard working solution I is further diluted into 1 ng/mL of internal standard working solution II by methanol solution, namely methanol solution containing memantine-d 6 and donepezil-d 7.

2) Pretreatment of plasma samples containing memantine and donepezil

Taking 2 mL deep-hole 96-well plates, adding 100 mu L of plasma samples containing memantine and donepezil into each well, adding 50 mu L of sodium carbonate solution of 0.2 mol/L into each well, and vortex mixing for 1 min; adding 450 mu L of internal standard working solution II into the solution, vortex mixing 5min, centrifuging 10min (4750 r/min) in a 4 ℃ desk-top high-speed refrigerated centrifuge (96-well plate), taking 100 mu L of supernatant from each well, transferring to another 2 mL deep-hole 96-well plate, adding 100 mu L of 0.1% formic acid aqueous solution into the new 96-well plate for dilution, vortex mixing 5min, and starting a liquid chromatography-mass spectrometer detection program to detect memantine and donepezil after vortex mixing.

3) High performance liquid chromatography conditions

The conditions of the high performance liquid chromatography for the liquid chromatography-mass spectrometry detection comprise: the column was an ACQUITY UPLC-BEH C18 (2.1X150 mm,2.6 μm) column, gradient elution was performed, mobile phase A was an aqueous solution containing 0.1% formic acid, mobile phase B was an acetonitrile solution, and the elution procedure was: 0-1.70 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; 1.70-1.75 min, the volume percentage of the mobile phase A is reduced from 80% to 65%, and the volume percentage of the mobile phase B is increased from 20% to 35%; 1.75-2.20 min, the volume percentage of the mobile phase A is kept 65%, and the volume percentage of the mobile phase B is kept 35%; 2.20-2.90 min, the volume percentage of the mobile phase A is reduced from 65% to 20%, and the volume percentage of the mobile phase B is increased from 35% to 80%; 2.90-3.50 min, wherein the volume percentage of the mobile phase A is kept 20%, and the volume percentage of the mobile phase B is kept 80%; 3.50-3.70 min, the volume percentage of the mobile phase A is reduced from 20% to 5%, and the volume percentage of the mobile phase B is increased from 80% to 95%; 3.70-4.00 min, wherein the volume percentage of the mobile phase A is kept 5%, and the volume percentage of the mobile phase B is kept 95%; 4.00-4.20 min, the volume percentage of the mobile phase A is increased from 5% to 80%, and the volume percentage of the mobile phase B is decreased from 95% to 20%; 4.20-4.50 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 15 mu L.

TABLE 1 liquid phase gradient elution procedure

4) Triple quadrupole mass spectrometry detection conditions

Mass spectrometry conditions for the liquid chromatography-mass spectrometry detection include: using a triple quadrupole mass spectrometer, wherein the ion source is an electrospray ion source, a positive ion mode is adopted, a multi-reaction monitoring scanning mode is adopted, and ionization voltage is calculated: 5500 V, V; inlet voltage: 10 V, V; collision cell exit voltage: 13 V, V; spraying gas: 50; auxiliary heating gas: 55; air curtain gas: 35, the ion pairs of memantine and donepezil are 180.2→163.2, 380.3 →91.1 (quantitative ion pair), respectively, collision energy: (memantine: 18V; donepezil: 41V), declustering voltage: (Memantine: 68V; donepezil: 70V); the ion pairs of memantine-d 6 and donepezil-d 7 are 186.2-169.2 and 387.2-98.1 respectively, and the collision energy is as follows: (memantine-d 6:20V; donepezil-d 7: 60V), declustering voltage: (Memantine-d 6:95V; donepezil-d 7: 150V).

5) Establishment of a Standard Curve

Taking 285 mu L of blank plasma, adding 15 mu L of standard curve mixed working solution with different concentrations to obtain 0.20,0.40,1.00,5.00, 10.00, 30.00, 45.00 and 50.00 ng/mL standard curve samples, carrying out precipitation reaction on the standard curve samples, memantine-d 6 with the concentration of 1 ng/mL and a methanol solution (internal standard working solution II) of donepezil-d 7, vortex mixing 5 min, placing in a4 ℃ desk type high-speed refrigerated centrifuge (96 pore plate), centrifuging 4750 r/min for 10 min, taking 100 mu L of supernatant from each pore, transferring to another 96 pore plate, adding 100 mu L of 0.1% formic acid aqueous solution into each pore for dilution, the obtained data are processed by an internal standard method after vortex mixing of 5 min, the peak area ratio of memantine and donepezil to memantine-d 6 and donepezil-d 7 respectively is taken as an ordinate, 1/x ² is taken as a weight coefficient, linear regression is carried out by a weighted least square method, a standard curve is obtained, wherein y=0.269218 x+ -0.00559551 (n=10), the linear range is 0.20-50.00 ng/mL, the correlation coefficient r ² is 0.9991, and the quantitative reduction line is 0.20 ng/mL.

6) Residue of

Evaluating the residue by injecting a blank matrix sample after injection of an upper limit sample of the dose, fig. 1 is a chromatogram of memantine and donepezil in plasma containing upper limit memantine and donepezil after addition of an internal standard, fig. 1a is a chromatogram of memantine with a retention time of 1.15 min; FIG. 1b is a chromatogram of donepezil with a retention time of 1.42 min and FIG. 2 is a chromatogram of memantine-d 6 and donepezil-d 7 in plasma containing the upper limit of quantitation of memantine-d 6 and donepezil-d 7 after addition of an internal standard. FIG. 2a is a chromatogram of memantine-d 6 with a retention time of 1.13 min for memantine-d 6; FIG. 2b is a chromatogram of donepezil-d 7 with a retention time of 1.41 min for donepezil-d 7.

FIG. 3 is a chromatogram of memantine and donepezil in blank plasma; FIG. 3a is a chromatogram of memantine; FIG. 3b is a chromatogram of donepezil; FIG. 4 is a chromatogram of memantine-d 6 and donepezil-d 7 in blank plasma; FIG. 4a is a chromatogram of memantine-d 6; FIG. 4b is a chromatogram of donepezil-d 7.

The results showed that the chromatograms of the blank plasma samples showed no interference peaks in the vicinity of the chromatographic peaks of memantine, donepezil, memantine-d 6 and donepezil-d 7, indicating that the method had little residue and had no effect on the quantification of both the analytes (memantine and donepezil) and the internal standard (memantine-d 6 and donepezil-d 7).

7) Precision and accuracy investigation

Quality control samples containing 0.20, 0.60, 3.00, 25.00, 40.00 ng/mL memantine and donepezil were prepared according to the standard curve sample preparation method, 6 samples were prepared independently for each concentration, and detection was continued for 3 days. The concentration of the quality control sample was calculated according to the standard curve prepared on the same day, and further the precision and accuracy of the batch and the batch were calculated, and the results are shown in table 2. As can be seen from the data in Table 2, the accuracy and precision of the method disclosed by the invention are within + -15% in batches and between batches, which shows that the method is accurate and repeatable and is suitable for popularization.

TABLE 2 accuracy and precision test results

8) Selectivity of

Blank substrate samples and quantitative lower limit samples were prepared from blank plasma of 6 subjects, and the blank substrate samples were treated according to the item "pretreatment of plasma samples containing memantine and donepezil" except that 400 μl of methanol solution was used instead of the internal standard working solution II, and the quantitative lower limit samples were treated according to the item "pretreatment of plasma samples containing memantine and donepezil". The results show that the response of the blank matrix interfering components of 6 subjects in the study at the retention times of memantine and donepezil and memantine-d 6 and donepezil-d 7 are all 0, indicating that the method has good selectivity and can distinguish the endogenous components in the matrix from the endogenous components in the matrix of memantine and donepezil and memantine-d 6 and donepezil-d 7.

9) Investigation of extraction recovery and matrix Effect

Recovery rate: the quality control samples containing 0.6 ng/mL,25.0 ng/mL and 40.0 ng/mL of memantine and donepezil were treated as "pretreatment of plasma samples containing memantine and donepezil", respectively, the peak areas A are shown in Table 3, and the peak areas A of the internal standards are also shown in Table 3; taking 100 mu L of blank plasma, adding 400 mu L of methanol precipitant, uniformly mixing and centrifuging, taking 100 mu L of supernatant to a new 2mL deep hole 96-well plate, adding 100 mu L of mixed reference substances into the new 96-well plate, wherein the mixed reference substances are as follows: 0.12 ng/mL,5.00 ng/mL,8.00 ng/mL memantine and donepezil, 0.800 ng/mL memantine-d 6 and donepezil-d 7 internal standards; vortex mixing 5min, mixing evenly, and then sampling and detecting to obtain peak area B. The ratio of peak areas A to B was the recovery rate, and the results are shown in Table 3, in which LQC in Table 3 is the recovery rate detection result of the quality control sample with the concentration of 0.6 ng/mL after extraction and not extraction, MQC in Table 3 is the recovery rate detection result of the quality control sample with the concentration of 25.0 ng/mL after extraction and not extraction, and HQC in Table 3 is the recovery rate detection result of the quality control sample with the concentration of 25.0 ng/mL after extraction and not extraction.

TABLE 3 extraction recovery Experimental results

Matrix effect: taking blank matrixes (blank matrix 1, blank matrix 2, blank matrix 3, blank matrix 4, blank matrix 5 and blank matrix 6) of 6 subjects, respectively 100 mu L of 1 hyperlipidemia blank matrix and 1 hemolysis blank matrix, adding 400 mu L of methanol precipitant, uniformly mixing and centrifuging, taking 100 mu L of supernatant to a new 2 mL deep hole 96-well plate, adding 100 mu L of mixed reference substance into the new 96-well plate, vortex mixing 5min, mixing, carrying out sample injection detection, analyzing object peak area C and internal standard peak area D. Taking 100 mu L of pure water, adding 400 mu L of methanol precipitant, uniformly mixing and centrifuging, taking 100 mu L of supernatant to a new 2 mL deep hole 96 hole plate, adding 100 mu L of mixed reference substance to the new 96 hole plate, vortex mixing 5min, uniformly mixing, detecting sample injection, and analyzing the peak area E of an object and the peak area F of an internal standard. The matrix efficacy was evaluated using the Coefficient of Variation (CV) of the internal standard normalized matrix factor. The matrix factor is the ratio of the peak area in the presence of matrix to the corresponding peak area without matrix, i.e. the matrix factors for memantine and donepezil are C/E, and the matrix factors for memantine-D6 and donepezil-D7 are D/F. The internal standard normalized matrix factor was further calculated by dividing the matrix factors of memantine and donepezil by the matrix factors of memantine-d 6 and donepezil-d 7, and the coefficient of variation of the internal standard normalized matrix factor was calculated, and the results are shown in table 4.

Table 4 6 matrix effects of lots of plasma blank matrices of different origins, hyperlipidemia blank matrices and hemolysis blank matrices

From the above results, it can be seen that the quantitative determination method of memantine and donepezil in plasma based on precipitation method and liquid chromatography-mass spectrometry technology disclosed by the invention can rapidly and stably determine the concentration of memantine and donepezil in biological samples.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be comprehended within the scope of the present invention.

Claims (8)

1. An LC-MS/MS method for simultaneous determination of memantine and donepezil concentrations in human plasma, said method comprising the steps of:

Adding sodium carbonate into a plasma sample containing memantine and donepezil for pretreatment, adding an internal standard working solution II into the plasma sample for precipitation reaction, respectively injecting the obtained supernatant into a liquid chromatograph-mass spectrometer for multi-reaction monitoring reaction, and respectively calculating the content of memantine and donepezil in the plasma by using an internal standard method according to a standard curve; the standard curve of the memantine content takes the concentration of memantine as an abscissa and the peak area ratio of memantine to memantine-d 6 as an ordinate; the standard curve of the donepezil content takes the concentration of donepezil as an abscissa and takes the peak area ratio of donepezil and donepezil-d 7 as an ordinate;

The internal standard working solution II is a methanol solution containing memantine-d 6 and donepezil-d 7.

2. The method according to claim 1, wherein in the multi-reaction monitoring reaction, the quantitative ion pair of memantine is 180.2 to 163.2, the quantitative ion pair of donepezil is 380.3 to 91.1, the ion pair of memantine-d 6 is 186.2 to 169.2, and the ion pair of donepezil-d 7 is 387.2 to 98.1, respectively.

3. The method of claim 1, wherein the liquid phase conditions in the liquid chromatograph-mass spectrometer are: the chromatographic column is an ACQUITY UPLC-BEH C18 (2.1X150 mm,1.7 mu m) chromatographic column, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution, gradient elution is adopted, and the elution procedure is as follows: 0-1.7 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; 1.70-1.75 min, the volume percentage of the mobile phase A is reduced from 80% to 65%, and the volume percentage of the mobile phase B is increased from 20% to 35%; 1.75-2.20 min, the volume percentage of the mobile phase A is kept 65%, and the volume percentage of the mobile phase B is kept 35%; 2.20-2.90 min, the volume percentage of the mobile phase A is reduced from 65% to 20%, and the volume percentage of the mobile phase B is increased from 35% to 80%; 2.90-3.50 min, wherein the volume percentage of the mobile phase A is kept 20%, and the volume percentage of the mobile phase B is kept 80%; 3.50-3.70 min, the volume percentage of the mobile phase A is reduced from 20% to 5%, and the volume percentage of the mobile phase B is increased from 80% to 95%; 3.70-4.00 min, wherein the volume percentage of the mobile phase A is kept 5%, and the volume percentage of the mobile phase B is kept 95%; 4.00-4.20 min, the volume percentage of the mobile phase A is increased from 5% to 80%, and the volume percentage of the mobile phase B is decreased from 95% to 20%; 4.20-4.50 min, wherein the volume percentage of the mobile phase A is kept 80%, and the volume percentage of the mobile phase B is kept 20%; the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 15 mu L.

4. The method of claim 1, wherein the mass spectrometry conditions in the liquid chromatograph-mass spectrometer are: using a triple quadrupole mass spectrometer, wherein the ion source is an electrospray ion source, a positive ion mode is adopted, a multi-reaction monitoring scanning mode is adopted, and ionization voltage is calculated: 5500 V, V; inlet voltage: 10 V, V; collision cell exit voltage: 13 V, V; spraying gas: 50; auxiliary heating gas: 55; air curtain gas: 35; collision energy of the memantine: 18 V, V; collision energy of memantine-d 6: 20 V, V; collision energy of the donepezil: 41 V, V; collision energy of donepezil-d 7: 60 V, V; the declustering voltage of memantine: 68 V, V; declustering voltage of memantine-d 6: 95 V, V; the declustering voltage of donepezil: 70 V, V; the declustering voltage of donepezil-d 7: 150 V is provided.

5. The method according to claim 1, wherein the pretreatment of plasma containing memantine and donepezil is performed by: 100 μl of plasma samples containing memantine and donepezil were removed and placed in a2 mL 96-well plate to which 0.2-0.25 mol/liter sodium carbonate solution 50 μl was added and vortexed to mix 1 min.

6. The method according to claim 1, wherein the precipitation reaction comprises the steps of: mixing the pretreated plasma sample containing memantine and donepezil with 450 mu L of internal standard working solution II, vortex mixing 5min, placing in a4 ℃ desk-top high-speed refrigerated centrifuge, centrifuging 10min at 4750 r/min, taking 100 mu L of supernatant from each hole, transferring to another 96-well plate, diluting 100 mu L of 0.1% formic acid aqueous solution in each hole, vortex mixing 5min, and injecting into a liquid chromatograph-mass spectrometer for liquid chromatography-mass spectrometry detection.

7. The method according to claim 1, wherein the preparation method of the internal standard working solution II is as follows: respectively weighing memantine-d 6 standard substances and donepezil-d 7 standard substances, dissolving the memantine-d 6 standard substances with N, N-dimethylformamide to prepare 1mg/mL of memantine-d 6 internal standard mother solution and 1mg/mL of donepezil-d 7 internal standard mother solution, and mixing the memantine-d 6 internal standard mother solution and the donepezil-d 7 internal standard mother solution according to a volume ratio of 1:1, mixing to obtain a mixed internal standard working solution I, and diluting the mass concentration of the mixed internal standard working solution I to 1 ng/mL by using a methanol solution to obtain an internal standard working solution II.

8. The method of claim 1, wherein the standard curve is produced by a method comprising the steps of:

(1) And weighing memantine and donepezil standard substances, respectively dissolving the memantine standard substances with N, N-dimethylformamide to prepare 1 mg/mL of memantine mother solution and 1 mg/mL of donepezil Ji Muye, and mixing the memantine mother solution and the donepezil Ji Muye according to a volume ratio of 1:1, mixing to obtain a mixed solution, diluting the mass concentration of the mixed solution to 20 mug/mL by using a 50% methanol aqueous solution to obtain a mixed working solution, and diluting the mixed working solution to 4.00,8.00, 20.00, 100.00, 200.00, 600.00, 900.00 and 1000.00 and ng/mL standard curve mixed working solution by using the 50% methanol aqueous solution;

(2) Taking 285 mu L of blank plasma, and respectively adding 15 mu L of standard curve mixed working solutions with different concentrations obtained in the step (1) to obtain 0.20,0.40,1.00,5.00, 10.00, 30.00, 45.00 and 50.00 ng/mL standard curve samples;

(3) Respectively carrying out precipitation reaction on the standard curve sample obtained in the step (2) and 400 mu L of the methanol solution containing memantine-d 6 and donepezil-d 7, carrying out vortex mixing for 5min, putting the mixture into a 4 ℃ desk type high-speed refrigerated centrifuge, centrifuging for 10 min at 4750 r/min, taking 100 mu L of supernatant from each hole, transferring the supernatant to another 96-well plate, diluting each hole by adding 100 mu L of 0.1% formic acid aqueous solution, and carrying out liquid chromatography-mass spectrometry detection by vortex mixing for 5min;

(4) Calculating the contents of memantine and donepezil in the blood plasma according to a standard curve by an internal standard method respectively; the standard curve of the memantine content takes the concentration of memantine as an abscissa and the peak area ratio of memantine to memantine-d 6 as an ordinate; the standard curve of the donepezil content takes the concentration of donepezil as an abscissa and takes the peak area ratio of donepezil and donepezil-d 7 as an ordinate; and (3) taking 1/x ² as a weight coefficient, and carrying out linear regression by using a weighted least square method to obtain the standard curve.