CN115541778B - Detection method for measuring apremilast concentration in human plasma - Google Patents
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Abstract
The invention relates to the technical field of medicine analysis, in particular to a detection method for determining apremilast concentration in human blood plasma. The method for detecting the concentration of apremilast in the blood plasma comprises the following steps: mixing plasma containing apremilast and acetonitrile solution containing apremilast-d 5 for precipitation reaction, respectively injecting the obtained supernatant into a liquid chromatograph-mass spectrometer for multi-reaction monitoring reaction, and calculating the apremilast content in the plasma by an internal standard method according to a standard curve, wherein the standard curve takes the concentration of apremilast as an abscissa and the peak area ratio of apremilast and apremilast-d 5 as an ordinate. The invention carries out the detection of apremilast in blood plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, can be used for detecting the concentration of apremilast in organisms, and provides a rapid, accurate and stable detection method.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for detecting apremilast concentration in blood plasma.
Background
Apremilast is an oral cyclic adenosine monophosphate (cAMP) specific phosphodiesterase-4 (PDE 4) small molecule inhibitor. Inhibition of PDE4 can raise intracellular cAMP levels, and PDE4 degradation of cAMP can lead to immune cell activation and proinflammatory cytokine release. Inhibition of PDE4 may therefore be able to reduce PDE 4-mediated inflammatory responses. Apriste tablet, marketed in U.S. at 21, 2014, with approved indications being active psoriatic arthritis, moderate to severe plaque psoriasis and canker sores associated with Behcet's disease for phototherapy or systemic treatment. In month 9 2014, the FDA approved apremilast for the treatment of moderate to severe plaque psoriasis. Because of its promising therapeutic results and safety, the drug molecule is becoming very popular in dermatology. According to research reports, at present, the domestic method for measuring the blood concentration of the apremilast in the human body has not been studied, but the measurement of the blood plasma drug concentration in the living body has important effects on the aspects of evaluating the drug curative effect, analyzing the pharmacological effect and the like, so a detection method for measuring the apremilast concentration in the blood plasma is developed, and the research on the pharmacokinetics and the human bioequivalence of the apremilast in the hollow and postprandial states of a healthy subject in China is carried out, so that the method not only can provide basis for the consistency evaluation of the apremilast related preparation, but also has wide economic and social benefits.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a method for detecting apremilast concentration in blood plasma. The invention carries out the detection of apremilast in blood plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, can be used for detecting the concentration of apremilast 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:
the invention provides a method for detecting apremilast concentration in blood plasma, which comprises the following steps:
mixing plasma containing apremilast and acetonitrile solution containing apremilast-d 5 for precipitation reaction, carrying out vortex mixing for 5 min, placing in a 4 ℃ desk type high-speed refrigerated centrifuge (96-well plate), centrifuging for 10min at 4750 r/min, taking 100 [ mu ] L of supernatant from each well, transferring to another 96-well plate, diluting with 100 [ mu ] L of 10% acetonitrile aqueous solution in each well, carrying out vortex mixing for 5 min, carrying out liquid chromatography detection, and calculating the apremilast content in the plasma by using an internal standard method according to a standard curve, wherein the standard curve takes the concentration of apremilast as an abscissa and the peak area ratio of apremilast to apremilast-d 5 as an ordinate.
Further, the high performance liquid chromatography conditions of the liquid chromatography-mass spectrometry detection include: the chromatographic column is Phenomenex Kinetex C (2.1X50 mm, 2.6mu.m), gradient elution is adopted, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile solution, the elution process is 0-0.5 min, the volume percentage content of the mobile phase A is kept 63%, and the volume content of the mobile phase B is kept 37%; 0.5-1.3 min, the volume percentage of the mobile phase A is reduced from 63% to 0.0%, and the volume percentage of the mobile phase B is increased from 37% to 100%; 1.3-2.4 min, wherein the volume percentage of the mobile phase A is kept at 0.0%, and the volume percentage of the mobile phase B is kept at 100%; 2.4-2.41 min, the volume percentage of the mobile phase A is increased from 0.0% to 63%, and the volume percentage of the mobile phase B is decreased from 100% to 37%; 2.41-3 min, wherein the volume percentage content of the mobile phase A is kept 63%, the volume content of the mobile phase B is kept 37%, the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 5 mu L;
further, the mass spectrometry conditions of the liquid chromatography-mass spectrometry detection include: using a triple quadrupole mass spectrometer, wherein an ion source is an electrospray ion source, a negative ion mode is adopted, a multi-reaction monitoring scanning mode is adopted, and ionization voltage is calculated: 5500 V, V; inlet voltage (EP): 10 V, V; collision cell exit voltage (CXP): 13 V, V; spray Gas (Gas 1): 50; auxiliary heating Gas (Gas 2): 55; air curtain gas: 35, the ion pair of apremilast is 461.2/257.1 (quantitative ion pair), collision Energy (CE): 14 V, declustering voltage (DP): 190 V, V; the ion pair of the apremilast-d 5 is 466.1/262.3, CE:14 V, DP:190 V is provided.
Further, the detection method of the plasma sample containing apremilast comprises the following steps: taking a 96-well plate, adding 100 [ mu ] L of a plasma sample containing apremilast into each well, adding 300 [ mu ] L of an internal standard working solution (an internal standard working solution diluent is acetonitrile solution) with the mass concentration of 20.0ng/mL, carrying out vortex mixing for 5 min, placing into a 4 ℃ desk-top high-speed refrigerated centrifuge (96-well plate), centrifuging for 10min at 4750 r/min, taking 100 [ mu ] L of a supernatant from each well, transferring to another 96-well plate, adding 100 [ mu ] L of a 10% acetonitrile aqueous solution into each well for dilution, carrying out vortex mixing for 5 min, carrying out liquid-solid joint detection, and calculating the content of apremilast in the plasma by using an internal standard method according to a standard curve, wherein the standard curve adopts the concentration of apremilast as an abscissa and the peak area ratio of apremilast to d5 as an ordinate.
Further, the manufacturing method of the internal standard working solution comprises the following steps: weighing 1mg apremilast-d 5 standard substance, dissolving with N, N-dimethylformamide to prepare an internal standard mother solution of 1mg/mL, and further diluting the internal standard mother solution with acetonitrile solution to obtain an acetonitrile solution with concentration of 20.0ng/mL apremilast-d 5, namely an internal standard working solution.
Further, the standard curve is prepared by a method comprising the steps of: weighing 1mg apremilast, dissolving with N, N-dimethylformamide to obtain 1mg/mL apremilast mother liquor, precisely transferring a proper amount of apremilast mother liquor, diluting with 50% acetonitrile aqueous solution to obtain 100 ng/mL apremilast working solution, diluting the working solution with 50% acetonitrile aqueous solution to obtain 40, 80, 200, 600, 2000, 4000, 9600 and 12000 ng/mL standard curve working solution, taking 285 [ mu ] L of blank plasma, adding 15 [ mu ] L of standard curve working solution with different concentrations to obtain 2.00,4.00, 10.0, 30.0, 100, 200, 480 and 600 ng/mL standard curve samples, taking 100 [ mu ] L of standard curve sample, adding 400 [ mu ] L of apremilast-d 5 acetonitrile solution (internal standard working solution), performing precipitation reaction, vortex mixing for 5 min, placing in a 4 ℃ desk-top high-speed freezing centrifuge (96 pore plate), centrifuging for 10min at 47 4750 r/min, transferring 100 [ mu ] L of supernatant to another 96 [ mu ] L of standard curve working solution, detecting the obtained standard curve working solution with the other 96 [ mu ] L of standard curve working solution, and performing a parallel detection on the obtained standard curve working solution with the different concentrations of apremilast 1, performing a parallel detection on the standard curve sample with the standard curve sample of 100 [ mu ] L, and performing a 1-D1, performing a high-time chromatography 2 As the weight coefficient, a weighted least square method is utilizedAnd (5) performing linear regression to obtain the standard curve.
The invention establishes a method for detecting apremilast in plasma based on a precipitation method and a liquid chromatography-mass spectrometry technology, can be used for detecting the concentration of apremilast in organisms, and provides a reliable analysis means for clinical and basic research; in the quantitative process, apremilast-d 5 is used as an internal standard, and the quantitative detection of apremilast is realized through the specific and high-sensitivity monitoring of the excimer ion peak-the daughter ion.
The beneficial effects of the invention are that
The method for measuring the apremilast concentration in the human plasma has the advantages of high sensitivity, accuracy, reliability, good selectivity, small matrix effect, rapidness, simplicity, convenience and the like. According to the method, acetonitrile is used for carrying out protein precipitation on a plasma sample, and an internal standard working solution is directly prepared into a precipitator, so that the operation steps are reduced, the operation is simplified, the consumption of organic reagents is low, and the clinical applicability and the working efficiency are greatly improved; 0.1% formic acid water and 0.1% acetonitrile formate 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 addition, to reduce the matrix effect of the assay, the supernatant after centrifugation of the protein pellet was subjected to a 1:1 dilution (dilution: 10% acetonitrile water). 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. The test provides data reference for the bioequivalence study of the apremilast related preparation, is suitable for the blood concentration detection of apremilast and the pharmacokinetics study, and provides basis for the consistency evaluation of the apremilast preparation.
Drawings
FIG. 1 is a chromatogram of apremilast in 600 ng/mL (limit of quantitation) apremilast-containing plasma after addition of an internal standard;
FIG. 2 is a chromatogram of an internal standard substance in 600 ng/mL (limit of quantitation) of apremilast-containing plasma after addition of an internal standard;
FIG. 3 is a chromatogram of apremilast in blank plasma;
FIG. 4 is a chromatogram of an internal standard substance in blank plasma.
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.
Example 1
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 A10 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)
Apremilast control (purity 99.7%, beijing Mannheim Ha Ge Biotechnology Co., ltd.); apremilast-d 5 control (chemical purity 98.7%, isotopic internal standard purity 99.5%, beijing Mannheim Ha Ge Biotechnology Co., ltd.); 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: apremilast tablet (specification: 30 mg, lot: 02007001); reference formulation: apremilast tablet (specification: 30 mg, lot number: H02366A, manufactured by Cegene InternationaSar).
2. Experimental methods and results
1) Solution preparation
Preparing a standard substance solution: accurately weighing 1mg apremilast standard, and dissolving with N, N-Dimethylformamide (DMF) to obtain 1mg/mL apremilast mother liquor. Accurately measuring 1 mL apremilast mother liquor, placing the proper amount in a10 mL volumetric flask, and adding 50% acetonitrile water solution to the scale, so that the mass concentration is 100 mu g/mL apremilast working solution. The apremilast working solution was diluted with 50% acetonitrile in water to 40, 80, 200, 600, 2000, 4000, 9600, and 12000 ng/mL standard curve working solution.
Preparing an internal standard working solution: about 1mg apremilast-d 5 standard is precisely weighed, and dissolved by a certain amount of N, N-Dimethylformamide (DMF) to prepare an internal standard mother liquor with the concentration of 1 mg/mL. The internal standard mother liquor was further diluted to 20.0. 20.0ng/mL of internal standard working solution with acetonitrile solution.
2) Pretreatment of plasma samples containing apremilast
Taking 2 mL deep-hole 96-well plates, adding 100 mu L of plasma sample containing apremilast into each well, adding 300 mu L of internal standard working solution with the mass concentration of 20.0ng/mL, mixing for 5 min by vortex, centrifuging for 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 10% acetonitrile aqueous solution into a new 96-well plate for dilution, mixing for 5 min by vortex, adding the mixed solution into a sample bottle inserted with an internal cannula after vortex mixing, and starting a liquid mass spectrometer detection program to detect apremilast.
3) High performance liquid chromatography conditions
The conditions of the high performance liquid chromatography for the liquid chromatography-mass spectrometry detection comprise: the chromatographic column is Phenomenex Kinetex C (2.1X50 mm, 2.6mu.m), gradient elution is adopted, the mobile phase A is an aqueous solution containing 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile solution, the elution process is 0-0.5 min, the volume percentage content of the mobile phase A is kept 63%, and the volume content of the mobile phase B is kept 37%; 0.5-1.3 min, the volume percentage of the mobile phase A is reduced from 63% to 0.0%, and the volume percentage of the mobile phase B is increased from 37% to 100%; 1.3-2.4 min, wherein the volume percentage of the mobile phase A is kept at 0.0%, and the volume percentage of the mobile phase B is kept at 100%; 2.4-2.41 min, the volume percentage of the mobile phase A is increased from 0.0% to 63%, and the volume percentage of the mobile phase B is decreased from 100% to 37%; 2.41-3.0 min, the volume percentage content of the mobile phase A is kept 63%, the volume content of the mobile phase B is kept 37%, the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 5 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 an ion source is an electrospray ion source, a negative ion mode is adopted, a multi-reaction monitoring scanning mode is adopted, and ionization voltage is calculated: 5500 V, V; inlet voltage (EP): 10 V, V; collision cell exit voltage (CXP): 13 V, V; spray Gas (Gas 1): 50; auxiliary heating Gas (Gas 2): 55; air curtain gas: 35, the ion pair of apremilast is 461.2→ 257.1 (quantitative ion pair), collision Energy (CE): 14 V, declustering voltage (DP): 190 V, V; ion pairs of the apremilast-d 5 are 466.1-262.3, CE:14 V, DP:190 V is provided.
5) Establishment of a Standard Curve
Taking 285 [ mu ] L of blank plasma, adding 15 [ mu ] L of standard curve working solutions with different concentrations to obtain standard curve samples of 2.0,4.0, 10.0, 30.0, 100.0, 200.0, 480.0 and 600.0 ng/mL, taking 100 [ mu ] L of the standard curve samples, and adding 400. Carrying out precipitation reaction on an acetonitrile solution (internal standard working solution) of [ mu ] L apremilast-d 5, carrying out vortex mixing for 5 min, placing in a 4 ℃ desk type high-speed refrigerated centrifuge (96-well plate), centrifuging for 10min at 4750 r/min, taking 100 [ mu ] L of supernatant from each well, transferring to another 96-well plate, diluting with a 10% acetonitrile aqueous solution of 100 [ mu ] L of each well, carrying out liquid chromatography detection after vortex mixing for 5 min, processing obtained data by using an internal standard method, taking the concentration of apremilast as an abscissa, and the peak area ratio of apremilast and apremilast-d 5 as an ordinate, and taking the ratio of peak areas of apremilast and apremilast-d 5 as 1/x 2 For the weight coefficient, linear regression is performed by using a weighted least square method to obtain a standard curve asy=0.0147982 x+ -0.00436850 (n=9), linear range 2.00-60000 ng/mL, correlation coefficient r 2 0.9977, the quantitative cut-off line was 2.00 ng/mL.
6) Residue of
Residual was assessed by injecting a blank matrix sample (blank plasma) after injection of the high concentration sample, fig. 1 is a chromatogram of apremilast in 600 ng/mL (limit of quantitation) apremilast-containing plasma after addition of an internal standard, with a retention time of 1.17 min for the analyte (apremilast); FIG. 2 shows a chromatogram of an internal standard substance in 600 ng/mL (limit of quantitation) apremilast-containing plasma after addition of an internal standard, with a retention time of 1.15 min for the internal standard (apremilast-d 5), which gave good response values and peak shapes.
FIG. 3 is a chromatogram of apremilast in blank plasma; FIG. 4 is a chromatogram of an internal standard substance in blank plasma. Comparing fig. 1 and 2 with fig. 3 and 4, it was found that the chromatograms of the blank plasma samples showed no interference peaks in the vicinity of the chromatographic peaks of apremilast and apremilast-d 5, indicating that the method has little residue and no effect on the quantification of both analyte (apremilast) and internal standard (apremilast-d 5).
7) Precision and accuracy investigation
According to the preparation method of standard curve samples, quality control samples containing 2.0, 6.0, 50.0 and 450 ng/mL apremilast are respectively prepared, 6 samples are independently prepared for each concentration, and continuous detection is carried out 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 tables 2 and 3. 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
Taking blank plasma of 6 subjects to prepare a blank matrix sample and a quantitative lower limit sample, wherein the blank matrix sample is treated according to the 'pretreatment of plasma sample containing apremilast' item, and the rest steps are treated according to the 'pretreatment of plasma sample containing apremilast' item except for using 300 mu L acetonitrile solution to replace an internal standard working solution. The results show that the responses of the blank matrix interfering components of 6 subjects in the study at the retention time of apremilast and apremilast-d 5 are all 0, and the method has good selectivity and can distinguish the apremilast from the apremilast-d 5 and endogenous components in the matrix.
9) Investigation of extraction recovery and matrix Effect
Recovery rate: treating the quality control sample according to the pretreatment item of plasma sample containing apremilast to obtain a peak area A; taking 100 mu L of blank plasma, adding 300 mu L of acetonitrile precipitant, uniformly mixing and centrifuging, taking supernatant 100 to a new 2 mL deep hole 96-well plate, adding 100 mu L of mixed reference substance to the new 96-well plate, vortex mixing for 5 min, and detecting after uniform mixing to obtain a peak area B. The ratio of peak area A to B was the recovery and the results are shown in Table 3.
TABLE 3 extraction recovery Experimental results
Matrix effect: taking blank matrixes of 6 subjects, 1 hyperlipidemia blank matrix and 1 hemolysis 100 mu L respectively, adding 300 mu L acetonitrile precipitant, taking supernatant 100 mu L to a new 2 mL deep hole 96-well plate after uniform mixing and centrifugation, adding 100 mu L mixed reference substance into the new 96-well plate, vortex mixing for 5 min, sampling and detecting after uniform mixing, analyzing object peak area C and internal standard peak area D. Taking 100 mu L of pure water, adding 300 mu L of acetonitrile 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 to the new 96-well plate, vortex mixing for 5 min, detecting after uniform mixing, 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 factor of apremilast is C/E and the matrix factor of apremilast-D5 is D/F. The internal standard normalized matrix factor was further calculated by dividing the matrix factor of apremilast by the matrix factor of apremilast-d 5, and the coefficient of variation of the internal standard normalized matrix factor was calculated, the results are shown in table 4.
Table 46 matric effects of different sources of plasma, high fat and hemolysis
From the results, the specific amount of apremilast detection method in the plasma based on the precipitation method and the liquid chromatography-mass spectrometry technology disclosed by the invention can rapidly and stably detect the concentration of apremilast in a biological sample.
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 (4)
1. A method for detecting apremilast concentration in blood plasma, which is characterized by comprising the following steps:
mixing a plasma sample containing apremilast and an acetonitrile solution containing apremilast-d 5 for protein precipitation reaction, diluting the obtained supernatant, injecting the diluted supernatant into a liquid chromatograph-mass spectrometer, detecting by adopting a multi-reaction monitoring scanning mode, and calculating the concentration of apremilast in the plasma by an internal standard method according to a standard curve, wherein the standard curve takes the concentration of apremilast as an abscissa and the peak area ratio of apremilast and apremilast-d 5 as an ordinate;
the specific operation of carrying out protein precipitation reaction by mixing is as follows: taking 2 mL deep-hole 96-well plates, adding 100 mu L of plasma sample containing apremilast into each well, adding 300 mu L of internal standard working solution with the mass concentration of 20.0ng/mL, mixing for 5 min by vortex, centrifuging for 10min at 4750 r/min in a 4 ℃ 96-well plate table type high-speed refrigerated centrifuge, taking 100 mu L of supernatant from each well, transferring to another new 2 mL deep-hole 96-well plate, adding 100 mu L of 10% acetonitrile aqueous solution into the mixture for dilution, mixing for 5 min by vortex, and starting a liquid chromatography-mass spectrometer detection program to detect apremilast in the plasma;
the standard curve is prepared by a method comprising the following steps: weighing 1mg apremilast, dissolving with N, N-dimethylformamide to obtain a apremilast mother solution of 1mg/mL, diluting with a 50% acetonitrile aqueous solution to obtain a apremilast working solution with the mass concentration of 100 ng/mL, diluting the working solution with a 50% acetonitrile aqueous solution to obtain standard curve working solutions of 40, 80, 200, 600, 2000, 4000, 9600 and 12000 ng/mL, taking 285 [ mu ] L of blank plasma, adding 15 [ mu ] L of standard curve working solutions with different concentrations, and obtaining standard curve samples of 2.0,4.0, 10.0, 30.0, 100.0, 200.0, 480.0 and 600.0 ng/mL; taking 100 mu L of the standard curve sample, adding 400 mu L of an apremilast-d 5 acetonitrile solution for precipitation reaction, vortex mixing for 5 min, placing in a 4 ℃ desk type high-speed refrigerated centrifuge, centrifuging for 10min at 4750/r/min, taking 100 mu L of a supernatant from each hole, transferring to another 96-well plate, adding 100 mu L of a 10% acetonitrile aqueous solution into each hole for dilution, vortex mixing for 5 min for liquid chromatography detection analysis, processing the obtained data by using an internal standard method, taking the concentration of apremilast as an abscissa, and the peak area ratio of apremilast and apremilast-d 5 as an ordinate, and taking the ratio of 1/x 2 Performing linear regression by using a weighted least square method as a weight coefficient to obtain the standard curve;
the internal standard working solution is an acetonitrile solution of apremilast-d 5, and the preparation method comprises the following steps: 1mg Apremilast-d 5 standard is weighed, dissolved by N, N-dimethylformamide to prepare an internal standard mother solution with the concentration of 1mg/mL, and the internal standard mother solution is further diluted by acetonitrile solution to obtain the acetonitrile solution with the concentration of 20.0ng/mL Apremilast-d 5.
2. The method of claim 1, wherein the quantitative ion pair of apremilast is 461.2- > 257.1 and the ion pair of apremilast-d 5 is 466.1- > 262.3 in the detection using the multi-reaction monitoring scanning mode.
3. The method according to claim 1, wherein the liquid phase conditions in the liquid chromatograph-mass spectrometer are: the chromatographic column is Phenomenex Kinetex C (2.1X50 mm, 2.6mum), the mobile phase A is aqueous solution containing 0.1% formic acid, the mobile phase B is 0.1% formic acid acetonitrile solution, gradient elution is adopted, and the elution procedure is as follows: 0-0.5 min, wherein the volume percentage of the mobile phase A is kept 63%, and the volume percentage of the mobile phase B is kept 37%; 0.5-1.3 min, the volume percentage of the mobile phase A is reduced from 63% to 0.0%, and the volume percentage of the mobile phase B is increased from 37% to 100%; 1.3-2.4 min, wherein the volume percentage of the mobile phase A is kept at 0.0%, and the volume percentage of the mobile phase B is kept at 100%; 2.4-2.41 min, the volume percentage of the mobile phase A is increased from 0.0% to 63%, and the volume percentage of the mobile phase B is decreased from 100% to 37%; 2.41-3.0 min, the volume percentage content of the mobile phase A is kept 63%, the volume content of the mobile phase B is kept 37%, the flow rate is 0.45 mL/min, the column temperature is 40 ℃, and the sample injection amount is 5 mu L.
4. The method according to claim 1, wherein the mass spectrometry conditions in the liquid chromatograph-mass spectrometer are: using a triple quadrupole mass spectrometer, wherein an ion source is an electrospray ion source, a negative ion mode, a multi-reaction monitoring scanning mode and ionization voltage: 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: 14 V, declustering voltage: 190 V is provided.
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