CN115541778B - A kind of detection method for measuring the concentration of apremilast in human plasma - Google Patents

Abstract

The invention relates to the technical field of drug analysis, in particular to a detection method for measuring the concentration of apremilast in human blood plasma. The detection method of apremilil concentration in the plasma comprises the following steps: mixing the acetonitrile solution containing the plasma of apremilast and apremilast-d5 to carry out the precipitation reaction, and injecting the supernatant of gained into liquid chromatography respectively ‑Mass spectrometer, carry out multiple reaction monitoring reaction, use internal standard method to calculate the content of Apremilast in plasma with standard curve, described standard curve takes the concentration of Apremilast as abscissa, takes Apremilast and Apremilast The ratio of the peak areas of ster-d5 is the ordinate. The invention detects the apremilast in the blood plasma based on the precipitation method and liquid mass spectrometry technology, can be used to detect the concentration of the apremilast in the living body, and provides a fast, accurate and stable detection method.

Description

A kind of detection method for measuring the concentration of apremilast in human plasma

technical field

The invention relates to the technical field of drug analysis, in particular to a method for detecting the concentration of apremilast in plasma.

Background technique

Apremilast is an oral, small-molecule inhibitor of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase-4 (PDE4). Inhibition of PDE4 increases intracellular cAMP levels, and degradation of cAMP by PDE4 leads to immune cell activation and release of pro-inflammatory cytokines. Therefore, inhibition of PDE4 may be able to reduce PDE4-mediated inflammatory response. Apremilast tablets were approved for marketing in the United States on March 21, 2014. The approved indications are active psoriatic arthritis, moderate to severe plaque psoriasis suitable for phototherapy or systemic treatment and related Oral ulcers associated with Behcet's disease. In September 2014, the FDA approved apremilast for the treatment of moderate to severe plaque psoriasis. This drug molecule has become very popular in dermatology due to its promising therapeutic results and safety profile. According to research reports, at present, domestic methods for determining the blood concentration of apremilast in the human body have not yet been studied, and the determination of its plasma drug concentration in the living body plays a very important role in evaluating the efficacy of the drug and analyzing the pharmacological effects. Therefore, the development of A detection method for determining the concentration of apremilast in plasma, the pharmacokinetics and human bioequivalence studies of apremilast in Chinese healthy subjects under fasting and postprandial conditions, can not only provide It provides a basis for the consistency evaluation of special related preparations, and has extensive economic and social benefits.

Contents of the invention

In order to solve the technical problems in the prior art, the object of the present invention is to provide a method for detecting the concentration of apremilast in plasma. The invention detects the apremilast in the blood plasma based on the precipitation method and liquid mass spectrometry technology, can be used to detect the concentration of the apremilast in the living body, and provides a fast, accurate and stable detection method.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for detecting the concentration of apremilast in plasma, comprising the following steps:

The plasma containing apremilast and the acetonitrile solution of apremilast-d5 were mixed for precipitation reaction, vortexed for 5 min, placed in a desktop high-speed refrigerated centrifuge (96-well plate) at 4 °C, and centrifuged at 4750 r/min for 10 min , take 100 µL of the supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, perform liquid chromatography-mass spectrometry detection, and use the internal standard method to calculate the standard curve The content of apremilil in plasma, the standard curve takes the concentration of apremilast as the abscissa, and the peak area ratio of apremilil and apremilil-d5 as the ordinate.

Further, the high-performance liquid chromatography conditions detected by liquid chromatography-mass spectrometry include: the chromatographic column is a Phenomenex Kinetex C18 (2.1×50 mm, 2.6 µm) chromatographic column, gradient elution is used, and 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 program is 0~0.5 min, the volume percentage of mobile phase A is kept at 63%, and the volume content of mobile phase B is kept at 37%; 0.5~1.3 min, mobile phase A The volume percentage of mobile phase A decreased from 63% to 0.0%, and the volume percentage of mobile phase B increased from 37% to 100%; 1.3~2.4 min, the volume percentage of mobile phase A remained at 0.0%, and the volume percentage of mobile phase B The volume content remained at 100%; 2.4~2.41 min, the volume percentage of mobile phase A increased from 0.0% to 63%, and the volume percentage of mobile phase B decreased from 100% to 37%; 2.41~3 min, the mobile phase The volume percentage of A was kept at 63%, the volume content of mobile phase B was kept at 37%, the flow rate was 0.45 mL/min, the column temperature was 40°C, and the injection volume was 5 µL;

Further, the mass spectrometry conditions detected by liquid chromatography-mass spectrometry include: using a triple quadrupole mass spectrometer, the ion source is an electrospray ion source, using negative ion mode, using multiple reaction monitoring scanning mode, ionization voltage: 5500 V; inlet Voltage (EP): 10 V; Collision Cell Exit Voltage (CXP): 13 V; Spray Gas (Gas1): 50; Auxiliary Heating Gas (Gas2): 55; is 461.2/257.1 (quantitative ion pair), collision energy (CE): 14 V, declustering voltage (DP): 190 V; the ion pair of Apremilast-d5 is 466.1/262.3, CE: 14 V, DP: 190V.

Further, the detection method of plasma samples containing apremilast is as follows: take a 96-well plate, add 100 µL of plasma samples containing apremilast to each well, and then add an internal standard working solution with a mass concentration of 20.0 ng/mL (internal standard The working solution diluent is acetonitrile solution)) 300 µL, vortexed for 5 min, placed in a desktop high-speed refrigerated centrifuge (96-well plate) at 4 °C, centrifuged at 4750 r/min for 10 min, and 100 µL of supernatant from each well was transferred to In another 96-well plate, add 100 μL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, and carry out liquid chromatography-mass spectrometry detection, and use the internal standard method to calculate the content of apremilast in the plasma with the standard curve, the standard curve Take the concentration of apremilast as the abscissa, and the ratio of the peak areas of apremilil and apremilast-d5 as the ordinate.

Further, the preparation method of the internal standard working solution is: weigh 1 mg of Apremilast-d5 standard substance, dissolve it with N,N-dimethylformamide, and prepare a 1 mg/mL internal standard mother solution , using acetonitrile solution to further dilute the internal standard mother solution to acetonitrile solution with a concentration of 20.0 ng/mL Apremilast-d5, which is the internal standard working solution.

Further, the standard curve is prepared by a method comprising the following steps: Weigh 1 mg of Apremilast, dissolve it with N,N-dimethylformamide to obtain a 1 mg/mL mother solution of Apremilast, and precisely pipette An appropriate amount of Apremilast mother solution was diluted with 50% acetonitrile aqueous solution to a mass concentration of 100 ng/mL Apremilast working solution, and the working solution was diluted to 40, 80, 200, 600, 2000 with 50% acetonitrile aqueous solution, For standard curve working solution of 4000, 9600 and 12000 ng/mL, take 285 µL of blank plasma and add 15 µL of standard curve working solution of different concentrations to obtain standards of 2.00, 4.00, 10.0, 30.0, 100, 200, 480 and 600 ng/mL For the curve sample, take 100 µL of the standard curve sample, add 400 µL of Apremilast-d5 acetonitrile solution (internal standard working solution) for precipitation reaction, vortex and mix for 5 min, and place in a desktop high-speed refrigerated centrifuge at 4°C (96 orifice plate), centrifuge at 4750r/min for 10 min, take 100 µL of the supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well, vortex and mix for 5 min, and perform liquid chromatography-mass spectrometry detection , using the internal standard method to process the obtained data, with the concentration of Apremilast as the abscissa, the peak area ratio of Apremilast and Apremilast-d5 as the ordinate, and 1/x ² as the weight coefficient, Linear regression was carried out by weighted least square method to obtain the standard curve.

The present invention establishes a method for detecting apremilast in plasma based on precipitation method and liquid chromatography-mass spectrometry technology, which can be used to detect the concentration of apremilast in living organisms and provide reliable analysis means for clinical and basic research. The present invention Use the protein precipitation method to process the plasma samples containing apremilast, and remove impurities and endogenous interference substances through a one-step precipitation reaction. During the separation process of high performance liquid chromatography, formic acid is added to the mobile phase to make apremilast After ionization, more hydrogenated quasi-molecular ion peaks are formed, and the abundance of corresponding characteristic fragment ions increases accordingly, which improves the detection sensitivity; in the quantitative process, Apremilast-d5 is used as the internal The quantitative detection of apremilast is realized through the specific and high-sensitivity monitoring of quasi-molecular ion peak→product ion.

Beneficial effects of the present invention

The method for determining the concentration of apremilast in human blood plasma established by the invention has the advantages of high sensitivity, accuracy and reliability, good selectivity, small matrix effect, quickness and convenience, and the like. This method uses acetonitrile to carry out protein precipitation on plasma samples, and the internal standard working solution is directly prepared into the precipitant, which reduces the operation steps, makes the operation more simplified, and consumes less organic reagents, which greatly improves the clinical applicability and work efficiency; the selection of 0.1 % formic acid water and 0.1% formic acid acetonitrile as the mobile phase, which improves the sensitivity, stabilizes the peak position, and shortens the detection time of a single needle; the method uses an isotope-labeled internal standard, and the durability of the method is greatly improved; in addition, in order to reduce the analysis The matrix effect of the method, the supernatant after centrifugation of the protein pellet was diluted 1:1 (diluent: 10% acetonitrile water). The method was verified by methodology to meet the requirements of biological sample analysis, and the pass rate of actual sample reanalysis was 100%. This method was successfully applied to clinical blood drug concentration detection, and the results were reliable. This test provides a data reference for the bioequivalence study of related preparations of Apremilast, and is suitable for the blood concentration detection and pharmacokinetic research of Apremilast, and provides a basis for the consistency evaluation of Apremilast preparations .

Description of drawings

Fig. 1 is the chromatogram of apremilast in plasma containing 600 ng/mL (limit of quantification) apremilast after adding internal standard;

Fig. 2 is the chromatogram of internal standard substance in the plasma containing 600 ng/mL (limit of quantification) apremilast after adding internal standard;

Fig. 3 is the chromatogram of apremilast in blank plasma;

Figure 4 is a chromatogram of the internal standard substance in blank plasma.

Detailed ways

Specific details of the invention are further set forth in the ensuing description for a full understanding of the invention. The terms used in the description of the present invention are only used to describe the advantages and features of the present invention, and are not intended to limit the present invention.

Unless otherwise defined, all professional and scientific terms used in the present invention have the same meaning as understood by those skilled in the technical field of the present invention. Unless otherwise specified, the medicines or reagents used in the present invention are used in accordance with product instructions or conventional methods in the field. The technical solution of the present invention will now be further described according to the accompanying drawings and specific embodiments of the specification.

Example 1

1. Instruments and reagents

1) Analytical instrument

AB SCIEX Tripe Quad 5500 liquid chromatography-mass spectrometry system (AB SCIEX, USA); 3K15 desktop high-speed refrigerated centrifuge (Germany Sigma); Allegra X-15R desktop high-speed refrigerated centrifuge (96-well plate, Beckman Library, USA) Walter Company); MDF-U5412N medical low temperature box (Panasonic Company of Japan); Milli-Q Advantage A10 ultrapure water device (Millipore Company of the United States); DG-2500R multi-tube vortex mixer (Shanghai Bajiu Industrial Co., Ltd.). Data processing software: Analyst 1.6.3 (AB SCIEX, USA); Watson LIMS 7.5 SPS1 (Thermo Fisher, USA).

2) Reagent

Apremilast reference substance (purity 99.7%, Beijing Manhag Biotechnology Co., Ltd.); Apremilast-d5 reference substance (chemical purity 98.7%, isotope internal standard purity 99.5%, Beijing Manhage Biotechnology Co., Ltd. ); methanol, acetonitrile (chromatographically pure, Merck, Germany); formic acid (chromatographically pure, American ACS Enke Chemicals); N, N-dimethylformamide (chromatographically pure, Thermo Fisher Scientific (China) Technology Co., Ltd. Ltd). Test preparation: Apremilast tablet (specification: 30 mg, batch number: 02007001); reference preparation: apremilast tablet (specification: 30 mg, batch number: H02366A, produced by Cegene International Sar).

2. Experimental methods and results

1) Solution preparation

Preparation of standard solution: Accurately weigh 1 mg of apremilast standard substance and dissolve it in N,N-dimethylformamide (DMF) to obtain 1 mg/mL apremilast mother solution. Precisely measure 1 mL of Apremilast mother liquor and place it in a 10 mL volumetric flask, add 50% acetonitrile aqueous solution to the mark, so that the mass concentration is 100 μg/mL Apremilast working solution. Dilute the apremilast working solution with 50% acetonitrile aqueous solution to 40, 80, 200, 600, 2000, 4000, 9600, and 12000 ng/mL standard curve working solution.

Preparation of internal standard working solution: Accurately weigh about 1 mg of apremilast-d5 standard substance, dissolve it in a certain amount of N,N-dimethylformamide (DMF), and prepare a 1 mg/mL internal standard mother solution. The internal standard mother solution was further diluted to 20.0 ng/mL internal standard working solution with acetonitrile solution.

2) Pretreatment of plasma samples containing apremilast

Take a 2 mL deep-well 96-well plate, add 100 µL of plasma samples containing apremilast to each well, and add 300 µL of internal standard working solution with a mass concentration of 20.0 ng/mL, vortex and mix for 5 min, and place at 4 °C on a bench-top high-speed Centrifuge in a refrigerated centrifuge (96-well plate) for 10min (4750 r/min), take 100 µL supernatant from each well, transfer to another 2 mL deep-well 96-well plate, add 10% acetonitrile to the new 96-well plate Dilute with 100 µL of aqueous solution, vortex mix for 5 min, add the mixed solution into the injection bottle with the inner insert tube after vortex mixing, and start the liquid chromatography-mass spectrometer detection program to detect Apremilast.

3) High performance liquid chromatography conditions

The HPLC conditions for LC-MS detection include: the chromatographic column is Phenomenex Kinetex C18 (2.1×50 mm, 2.6 μm) chromatographic column, gradient elution is used, mobile phase A is aqueous solution containing 0.1% formic acid, mobile phase B It is 0.1% formic acid acetonitrile solution, the elution program is 0~0.5 min, the volume percentage content of mobile phase A is kept at 63%, and the volume content of mobile phase B is kept at 37%; 0.5~1.3 min, the volume percentage content of mobile phase A is content decreased from 63% to 0.0%, and the volume percentage of mobile phase B increased from 37% to 100%; 1.3~2.4 min, the volume percentage of mobile phase A remained at 0.0%, and the volume content of mobile phase B remained at 100%. %; 2.4~2.41 min, the volume percentage of mobile phase A increased from 0.0% to 63%, and the volume percentage of mobile phase B decreased from 100% to 37%; 2.41~3.0 min, the volume percentage of mobile phase A The component content was kept at 63%, the volume content of mobile phase B was kept at 37%, the flow rate was 0.45 mL/min, the column temperature was 40°C, and the injection volume was 5 µL;

Table 1 Liquid phase gradient elution program

4) Triple quadrupole mass spectrometry detection conditions

The mass spectrometry conditions for liquid chromatography-mass spectrometry detection include: using a triple quadrupole mass spectrometer, the ion source is an electrospray ion source, using negative ion mode, using multiple reaction monitoring scanning mode, ionization voltage: 5500 V; entrance voltage (EP): 10 V; Collision cell outlet voltage (CXP): 13 V; Spray gas (Gas1): 50; Auxiliary heating gas (Gas2): 55; Curtain gas: 35. Quantitative ion pair), collision energy (CE): 14 V, declustering potential (DP): 190 V; the ion pair of Apremilast-d5 is 466.1→262.3, CE: 14 V, DP: 190 V.

5) Establishment of standard curve

Take 285 µL of blank plasma, add 15 µL of standard curve working solution of 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, take 100 µL of the standard curve sample, Add 400 µL of acetonitrile solution of Apremilast-d5 (internal standard working solution) to carry out precipitation reaction, vortex and mix for 5 min, put in a desktop high-speed refrigerated centrifuge (96-well plate) at 4°C, and centrifuge at 4750 r/min for 10 min , take 100 µL of supernatant from each well, transfer to another 96-well plate, add 100 µL of 10% acetonitrile aqueous solution to each well to dilute, vortex and mix for 5 min, then perform liquid chromatography-mass detection, and use the internal standard method to process the obtained data , with the concentration of Apremilast as the abscissa, the ratio of the peak areas of Apremilast and Apremilast-d5 as the ordinate, and 1/x ² as the weight coefficient, use the weighted least squares method to carry out linear regression, find The obtained standard curve was y =0.0147982*x+-0.00436850 (n=9), the linear range was 2.00-60000 ng/mL, the correlation coefficient ^r2 was 0.9977, and the lower limit of quantification was 2.00 ng/mL.

6) residual

The residue was evaluated by injecting a blank matrix sample (blank plasma) after injecting a high-concentration sample. Figure 1 is the chromatogram of apremilast in plasma containing 600 ng/mL (quantitative limit) apremilast after adding an internal standard , the retention time of the analyte (Apremilast) was 1.17 min; Figure 2 is the chromatogram of the internal standard substance in plasma containing 600 ng/mL (limit of quantification) Apremilast after adding the internal standard, the internal standard ( Apremilast-d5) had a retention time of 1.15 min, and both obtained good response values and peak shapes.

Fig. 3 is the chromatogram of apremilast in blank plasma; Fig. 4 is the chromatogram of internal standard substance in blank plasma. Comparing Fig. 1 and Fig. 2 with Fig. 3 and 4, it is found that the chromatogram of the blank plasma sample has no interference peaks near the chromatographic peaks of Apremilast and Apremilast-d5, indicating that the residue of this method is small, There was no effect on the quantification of the analyte (Apremilast) and the internal standard (Apremilast-d5).

7) Inspection of precision and accuracy

According to the standard curve sample preparation method, quality control samples containing 2.0, 6.0, 50.0, and 450 ng/mL apremilast were prepared respectively, and 6 samples were independently prepared for each concentration, and tested continuously for 3 days. According to the standard curve prepared on the same day, the concentration of the quality control sample was calculated, and then the intra-assay and inter-assay precision and accuracy were calculated. The results are shown in Table 2 and Table 3. It can be seen from the data in Table 2 that the intra-assay and inter-assay accuracy and precision of the method disclosed in the present invention are within the range of ±15%, indicating that the method is accurate and repeatable, and is suitable for promotion.

Table 2 Accuracy and precision experimental results

8) Optional

Take the blank plasma of 6 subjects to prepare the blank matrix sample and the lower limit of quantification sample. Except for the blank matrix sample, 300 μL acetonitrile solution was used to replace the internal standard working solution, and the rest of the steps were as in the "Plasma Sample Pretreatment Containing Apremilast" item. Samples were processed, samples with the lower limit of quantitation were processed according to the item "Plasma Sample Pretreatment Containing Apremilast". The results showed that the responses of the blank matrix interference components of the 6 subjects in this study were all 0 at the retention time of Apremilast and Apremilast-d5, indicating that the method had good selectivity and could distinguish Apremilast Tetra and apremilast-d5 and endogenous components in the stroma.

9) Investigation of extraction recovery and matrix effect

Recovery rate: The quality control sample was processed according to the item "Plasma Sample Pretreatment Containing Apremilast" to obtain the peak area A; take 100 µL of blank plasma, add 300 µL of acetonitrile precipitant, mix and centrifuge, and take the supernatant 100 to A new 2 mL deep-well 96-well plate was added, and 100 µL of the mixed reference substance was added to the new 96-well plate, vortexed for 5 min, mixed evenly and injected for detection, and the peak area B was obtained. The ratio of the peak area A to B is the recovery rate, and the results are shown in Table 3.

Table 3 extraction recovery experimental results

Matrix effect: Take 100 µL each of the blank matrix, 1 hyperlipidemia blank matrix, and 1 hemolysis of 6 subjects, add 300 µL of acetonitrile precipitant, mix well and centrifuge, and transfer 100 µL of the supernatant to a new 2 mL Deep-well 96-well plate, then add 100 μL mixed reference substance to a new 96-well plate, vortex mix for 5 min, mix well and inject sample for detection, get analyte peak area C and internal standard peak area D. Take 100 µL of pure water, add 300 µL of acetonitrile precipitant, mix well and centrifuge, transfer 100 µL of the supernatant to a new 2 mL deep-well 96-well plate, then add 100 µL of the mixed control substance to the new 96-well plate, vortex Rotate and mix for 5 min, inject the sample for detection after mixing, and obtain the peak area E of the analyte and the peak area F of the internal standard. The matrix effect was evaluated by the coefficient of variation (CV) of the matrix factor normalized by the internal standard. The matrix factor is the ratio of the peak area in the presence of matrix to the corresponding peak area without matrix, that is, the matrix factor of Apremilast is C/E, and the matrix factor of Apremilast-d5 is D/F. Further, by dividing the matrix factor of Apremilast by the matrix factor of Apremilast-d5, the matrix factor normalized by the internal standard was calculated, and then the coefficient of variation of the matrix factor normalized by the internal standard was calculated. The results are shown in Table 4 .

Table 4 Matrix effect of 6 batches of different sources of plasma, high fat and hemolysis

It can be seen from the above results that the quantitative detection method for apremilast in plasma based on the precipitation method and liquid chromatography-mass spectrometry technology disclosed by the present invention can quickly and stably detect the concentration of apremilast in biological samples.

The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. It should be pointed out that those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection 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.