A method for quantitatively determining the concentration of dapsone in human plasma by high performance liquid chromatography tandem mass spectrometry

Abstract

The application provides a method for quantitatively detecting the concentration of dapsone in human plasma by using liquid chromatography-tandem mass spectrometry (LC-MS / MS). By optimizing the chromatography and mass spectrometry conditions, the detection specificity and sensitivity are improved. The protein precipitation pretreatment and rapid chromatography separation technology are adopted, the analysis time is shortened, the sampling amount is reduced, and the automation adaptability is good. The method can be used for efficient and accurate quantification, and is suitable for clinical pharmacokinetic research and treatment monitoring.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of daprestat concentration in human plasma. Background Technology

[0002] Daprodustat is a novel oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) that promotes erythropoiesis by stabilizing hypoxia-inducible factor (HIF) and is used to treat anemia associated with chronic kidney disease (CKD). Its structural formula is as follows:

[0003] .

[0004] Currently, there are no publicly reported detection methods suitable for determining the concentration of dapoxetine in human plasma, either domestically or internationally. Existing literature primarily addresses dapoxetine detection techniques in areas such as raw material quality control and residual solvent detection. For example, the method described in the literature "UPLC–MS-Based Procedures to Detect Prolyl-hydroxylase Inhibitors of HIF in Urine" is mainly applicable to the screening and confirmation of nine hypoxia-inducible factor prolyl hydroxylase inhibitors in urinary matrix. This method employs liquid-liquid extraction and solid-phase extraction, resulting in lengthy analysis times, high detection limits, and significant baseline fluctuations at the peak position, making it unsuitable for the quantitative analysis of dapoxetine in human plasma.

[0005] With the widespread clinical application of dapoxetine, there is an urgent market need for a sensitive, accurate, efficient, and robust method for the quantitative detection of dapoxetine, suitable for large-scale bioequivalence trial sample analysis, clinical therapeutic drug monitoring, and personalized dosing regimen development. This invention provides a method for the quantitative detection of dapoxetine concentration in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method offers advantages such as small sample size, simple pretreatment, high sensitivity, and high specificity, providing crucial technical support for generic drug consistency evaluation, pharmacokinetic studies of original drugs, drug development, and clinical applications both domestically and internationally. Summary of the Invention

[0006] This invention provides a method for the quantitative detection of dapoxetine concentration in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS). By optimizing chromatographic and mass spectrometric conditions, the method improves detection specificity and sensitivity. Employing protein precipitation pretreatment and rapid chromatographic separation techniques shortens analysis time, reduces sample volume, and offers good automation adaptability. This method enables efficient and accurate quantification and is suitable for clinical pharmacokinetic studies and treatment monitoring.

[0007] This invention provides a method for quantitatively determining the concentration of dapoxetine in human plasma, comprising the following steps:

[0008] (1) Sample pretreatment;

[0009] (2) Detection was performed using liquid chromatography-mass spectrometry;

[0010] (3) Preparation of standard curve;

[0011] (4) Determination of dapoxetine concentration in plasma;

[0012] In step (1), the sample pretreatment step includes: taking an appropriate amount of the sample to be tested, adding an appropriate amount of internal standard working solution, after protein precipitation, mixing and centrifuging, taking the supernatant, adding diluent and shaking well, and using it as the sample to be injected; all the above processes are carried out at room temperature.

[0013] The protein precipitant is selected from methanol, acetonitrile, organic solvents containing formic acid or acetic acid, preferably acetonitrile, and the amount of precipitant added is at least twice the volume of the sample to be tested.

[0014] The diluent is 0.1% (v / v) formic acid. By adjusting the pH to less than 3, the target analyte is kept in a molecular state, making the sample analysis more stable.

[0015] The preparation process of the internal standard working solution is as follows:

[0016] a. Preparation of internal standard stock solution: Accurately weigh an appropriate amount of internal standard reference, add a certain volume of solvent 1, and dissolve by ultrasonic shaking to obtain an internal standard stock solution with a concentration of 1.00 mg / ml; all stock solutions and working solutions are aliquoted and stored at -20℃.

[0017] b. Preparation of internal standard working solution: Dilute the internal standard stock solution with solvent 2 to prepare an internal standard working solution of 15.0 ng / ml; do not add internal standard working solution to blank samples, use solvent 2 instead.

[0018] The internal standard reference is selected from tolbutamide, ennasostat, and dapoxetine-d8, preferably dapoxetine-d8, and its structure is as follows:

[0019]

[0020] Solvent 1 is dimethyl sulfoxide and methanol in a ratio of 30~70:70~30 (v / v), preferably 50:50 (v / v); solvent 2 is methanol and water in a ratio of 50~90:10~50 (v / v), preferably 80:20 (v / v).

[0021] Furthermore, the preprocessing step specifically includes:

[0022] (1) Add 50.0 μl of the sample to be tested to a 96-well plate or centrifuge tube, and then add 50.0 μl of internal standard working solution; (2) Add 300 μl of acetonitrile to precipitate the protein; (3) Vortex at 2000 rpm for 10 minutes, and then centrifuge for 20 minutes (8℃, 3100g); (4) Add 150 μl of supernatant to a 96-well plate and dilute with 150 μl of 0.1% (v / v) formic acid solution. After dilution, shake the sample at 1000 rpm for 30 minutes. For biological matrix samples, centrifuge for 10 minutes (8℃, 3100g) before LC-MS / MS determination. Pure solution samples can be directly injected for analysis after shaking.

[0023] In step (2), the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase is: mobile phase A: organic solvent or organic reagent containing acid, mobile phase B: water or aqueous solution containing acid, isocratic elution or gradient elution.

[0024] The C18 column of the chromatographic column is specifically selected from octadecylsilane-bonded silica gel, ethylene-bridged hybrid particles, solid-core silica gel particles, etc., preferably a chromatographic column with a diameter of less than 100 mm, more preferably an octadecylsilane-bonded silica gel column, and most preferably an Agilent ZORBAX Eclipse XDB-C18 (50 mm × 2.1 mm, 3.5 μm) or a chromatographic column with equivalent performance.

[0025] The mobile phase A is selected from methanol, acetonitrile, methanol containing formic acid or acetic acid, acetonitrile containing formic acid or acetic acid, preferably acetonitrile containing formic acid or acetic acid, more preferably acetonitrile containing acetic acid, wherein the concentration of acetic acid is 0.02~0.1% (v / v), preferably 0.05% (v / v).

[0026] The mobile phase B is selected from formic acid, acetic acid, ammonium formate, and ammonium acetate, preferably an acetic acid solution, wherein the concentration of acetic acid is 0.02~0.1% (v / v), preferably 0.05% (v / v).

[0027] The preferred elution method is gradient elution, and the gradient elution table is as follows:

[0028]

[0029] Furthermore, the liquid chromatography conditions also include:

[0030] Flow rate: 0.500 ml / min;

[0031] Injection volume: 6.00 μl;

[0032] Column temperature: 45℃.

[0033] The mass spectrometry conditions include: the parent ion (Q1) in the mass spectrometry parameters is 392.3, the daughter ion (Q3) is selected from 290.9, 100.0, 348.3, preferably 290.9; the declustering voltage is selected from -80 to -130V, preferably -120V.

[0034] Specifically, the mass spectrometry conditions are as follows:

[0035] Mass spectrometry was performed using an AB Sciex Triple Quad 4500 mass spectrometer, a tandem quadrupole mass spectrometer, an electrospray negative ion source, and multiple reaction monitoring (MRM) mode. The parameters were: collision activation dissociation: 9 psi; curtain gas: 35 psi; spray gas: 55 psi; auxiliary heating gas: 55 psi; ionization voltage: -4500 V; temperature: 450 °C; inlet voltage: -10 V; collision outlet voltage: -15 V. The MRM monitoring method parameters were as follows: dapoxetine. m / z 392.3→290.9, declustering voltage: -120V, collision energy: -22V; residence time: 110ms; internal standard: m / z 400.4→299.0, declustering voltage: -140V, collision energy: -24V; residence time: 110ms; each mass spectrometry parameter can be optimized according to the instrument used.

[0036] In step (3), the standard curve preparation steps include: preparing the standard curve sample solution, preparing the sample to be injected according to the method in step (1), detecting according to the method in step (2), and recording the peak area corresponding to each concentration of dapoxetine; using the peak area ratio of dapoxetine to the internal standard as the vertical axis, the concentration of dapoxetine as the horizontal axis, and 1 / X as the horizontal axis. 2 Using the weighting coefficients, a linear weighted least squares regression equation for dapoxetine was prepared;

[0037] The preparation of the standard curve sample solution includes the following steps:

[0038] Accurately weigh an appropriate amount of dapoxetine reference standard and place it in a weighing bottle. Add a certain volume of solvent 1 and sonicate to dissolve and mix thoroughly to obtain a dapoxetine stock solution with a concentration of 1.00 mg / ml. Dilute the dapoxetine stock solution with solvent 2 to prepare standard curve working solutions of 0.200, 0.400, 1.00, 2.00, 6.00, 20.0, 50.0, 120, and 150 ng / ml. Add 10.0 μl of each concentration of standard curve working solution to 190 μl of blank plasma and vortex to mix thoroughly to obtain standard curve sample solutions of different concentrations.

[0039] Solvent 1 is selected as dimethyl sulfoxide and methanol in a ratio of 30~70:70~30 (v / v), preferably 50:50 (v / v); solvent 2 is methanol and water in a ratio of 50~90:10~50 (v / v), preferably 80:20 (v / v).

[0040] In step (4), the steps for determining the concentration of dapoxetine in plasma include: preparing the sample to be injected according to step (1), detecting it according to the method in step (2), recording the peak area corresponding to dapoxetine, substituting the peak area ratio of dapoxetine and internal standard into the established standard curve, and calculating the concentration of dapoxetine in the plasma to be tested.

[0041] This invention provides a method for quantitatively analyzing the concentration of daprestat in human plasma, which can be used for clinical pharmacokinetic sample detection.

[0042] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0043] (1) Sample processing optimization: The protein precipitation pretreatment method is simpler and faster than liquid-liquid extraction, solid-phase extraction and other pretreatment methods. The extraction recovery rate is about 100%, which can achieve high-throughput analysis while greatly reducing reagents and consumables.

[0044] (2) High sensitivity: The lower limit of quantification of daprestat is 0.200 ng / ml, which can meet the needs of clinical trials;

[0045] (3) High specificity: Dapoxetine-d8 is used as an isotope internal standard, which is completely consistent with the chromatographic retention and mass spectrometry detection behavior of dapoxetine, and the analyte and the internal standard do not interfere with each other;

[0046] (4) High selectivity: The blank matrix does not interfere with the internal standard and analyte. Optimized mass spectrometry parameters make the baseline at the peak position stable and do not affect the accurate quantification at all.

[0047] (5) High analytical efficiency: Optimized chromatographic separation conditions, with an analysis time of only 3.00 minutes;

[0048] (6) Wide linear range: 0.200-150 ng / ml, good residual behavior, and can be adapted to a wide range of biological sample concentrations in clinical studies. Attached Figure Description

[0049] Figure 1 This is the ion scanning diagram of dapoxetine in Example 3.

[0050] Figure 2 This is a scan of the daughter ion of the internal standard in Example 3.

[0051] Figure 3The interference spectrum of the internal standard for the analyte in Example 4.

[0052] Figure 4 This is the blank matrix spectrum from the selectivity test in Example 5.

[0053] Figure 5 The sample chromatogram is shown in the lower limit of quantitation test of Example 5.

[0054] Figure 6 This is the interference spectrum of the internal standard on the analyte in the specificity test of Example 5.

[0055] Figure 7 This is the interference spectrum of the analyte to the internal standard in the specificity test of Example 5.

[0056] Figure 8 This is the linear graph in Example 8. Detailed Implementation

[0057] The specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0058] The equipment and instruments used in the following embodiments are shown in the table below.

[0059]

[0060] The sources of raw materials and auxiliary materials used in the following examples are shown in the table below.

[0061]

[0062] The solutions used in the following examples were prepared as follows:

[0063] 1. Preparation of stock solution

[0064] Preparation of dapoxetine stock solution: Accurately weigh an appropriate amount of dapoxetine reference standard and place it in a weighing bottle. After conversion, add a certain volume of dimethyl sulfoxide: methanol (50:50, v / v) and sonicate to dissolve and mix well to obtain a dapoxetine stock solution with a concentration of 1.00 mg / ml.

[0065] Preparation of internal standard stock solution: Accurately weigh an appropriate amount of dapoxetine-d8 internal standard reference, and after conversion, add a certain volume of dimethyl sulfoxide:methanol (50:50, v / v) and sonicate to dissolve and mix well to obtain dapoxetine-d8 internal standard stock solution with a concentration of 1.00 mg / ml.

[0066] 2. Preparation of working solution

[0067] Preparation of standard curve working solutions: Dilute dapoxetine stock solution to 4.00, 8.00, 20.0, 40.0, 120, 400, 1000, 2400 and 3000 ng / ml using methanol:water (80:20, v / v) as diluent.

[0068] Preparation of quality control working solutions: Dilute dapoxetine stock solution to 4.00, 12.0, 100, 720 and 2250 ng / ml using methanol:water (80:20, v / v) as diluent.

[0069] Preparation of the lower limit of quantitation working solution: Dilute the dapoxetine stock solution to a 0.200 ng / ml working solution using methanol:water (80:20, v / v) as the diluent.

[0070] Preparation of internal standard working solution: Dilute the internal standard stock solution to a working solution of 15.0 ng / ml using methanol:water (80:20, v / v) as the diluent.

[0071] 3. Sample preparation

[0072] Preparation of standard curve samples: 10.0 μl of standard curve working solution of different concentrations was added to 190 μl of blank plasma and vortexed to obtain standard curve samples of 0.200, 0.400, 1.00, 2.00, 6.00, 20.0, 50.0, 120 and 150 ng / ml.

[0073] Preparation of quality control samples: Add 10.0 μl of quality control working solution of different concentrations to 190 μl of blank plasma, vortex to mix, and obtain quality control samples of 0.600, 5.00, 36.0 and 112.5 ng / ml.

[0074] Preparation of the lower limit of quantitation sample: Add 10.0 μl of the lower limit of quantitation working solution to 190 μl of blank plasma, vortex to mix, and obtain a sample with a lower limit of quantitation of 0.200 ng / ml.

[0075] Example 1 Sample Pretreatment

[0076] Before sample injection and analysis, pretreatment is required, and protein precipitation is used for pretreatment.

[0077] ① Selection of precipitant:

[0078] The precipitant is selected from methanol, acetonitrile, and organic solvents containing formic acid or acetic acid. Methanol precipitate is not compact enough and the precipitate layer is easily absorbed, which can contaminate the mass spectrometer. Formic acid or acetic acid is no different from pure organic solvents. Acetonitrile is preferred. Adding at least twice the sample volume of acetonitrile removes most of the protein in the sample. The precipitate is compact, which facilitates the removal of the supernatant and results in good recovery.

[0079] ② pH adjustment

[0080] Add 0.1% (v / v) formic acid to the supernatant diluent to adjust the pH to less than 3 so that the target analyte is in a molecular state, making the sample analysis more stable.

[0081] Specifically, the following steps are included:

[0082] (1) Take 50.0 μl of the prepared biological sample (blank sample, standard curve and quality control sample, etc.) or the sample to be tested and add it to a 96-well plate or centrifuge tube, and then add 50.0 μl of internal standard working solution (dapoxetine-d8 internal standard working solution, concentration of 15.0 ng / ml; for blank samples, do not add internal standard working solution, use methanol:water (80:20, v / v) instead);

[0083] (2) Add 300 μl of acetonitrile to precipitate the protein;

[0084] (3) Vortex at 2000 rpm for 10 minutes, then centrifuge for 20 minutes (8℃, 3100g).

[0085] (4) Add 150 μl of supernatant to a 96-well plate and dilute with 150 μl of 0.1% (v / v) formic acid solution. After dilution, shake the sample at 1000 rpm for 30 minutes. For biological matrix samples, centrifuge for 10 minutes (8℃, 3100g) before LC-MS / MS determination. Pure solution samples can be directly injected for analysis after shaking.

[0086] Example 2 Selection of Liquid Chromatography Conditions

[0087] Inject the pretreated sample into the liquid chromatograph and record the resulting chromatogram.

[0088] ① Selection of chromatographic column

[0089] A variety of C18 columns (octadecylsilane-bonded silica gel, ethylene-bridged hybrid particles, solid-core silica gel particles, etc.) are selected. Daprusat has a large LogP value. When the column length is greater than or equal to 100 mm, it is retained too strongly on the column, making it difficult to elute. The residue is difficult to control and the matrix effect is obvious. Therefore, in order to improve the analytical efficiency, C18 columns with a length of less than 100 mm are preferred.

[0090] Furthermore, the peak shapes of ethylene-bridged hybrid particle columns (preferably Waters XBridge® C18 or equivalent columns) and solid-core silica particle columns (preferably Agilent InfinityLab Poroshell 120 EC-C18 or equivalent columns) were poor, while the octadecylsilane-bonded silica columns (preferably Agilent ZORBAXEclipse XDB - C18 or equivalent columns) showed stable baselines, good response, and good peak shapes at the retention time.

[0091] Therefore, the preferred chromatographic column is an octadecylsilane-bonded silica column, specifically, the Agilent ZORBAX Eclipse XDB-C18 or a column with equivalent performance.

[0092] ② Selection of mobile phase

[0093] Mobile phase A was selected from methanol, acetonitrile, methanol containing formic acid or acetic acid, and acetonitrile containing formic acid or acetic acid. During the optimization process, it was found that the methanol system caused dapoxetine to be retained too strongly on the chromatographic column, making it difficult to elute completely and resulting in significant residue. Therefore, methanol was excluded and the acetonitrile system was used instead. The acidic system made the response and retention time of the analyte more stable and could improve the mass spectrometry ionization efficiency. Therefore, acetonitrile containing formic acid or acetic acid was selected as mobile phase A.

[0094] Experiments showed that the response of the formic acid system was lower than that of the acetic acid system, and the acetic acid system was preferred. Furthermore, in the negative ion monitoring mode, the higher the concentration of acetic acid, the lower the response of dapoxetine. Experiments determined that the preferred mobile phase A was acetonitrile containing 0.02~0.1% (v / v) acetic acid, and more preferably an acetonitrile solution containing 0.05% acetic acid.

[0095] Mobile phase B is selected from formic acid, acetic acid, ammonium formate, and ammonium acetate. Comparison shows that ammonium formate or ammonium acetate both inhibit the analyte response, while the formic acid system causes interference from impurity peaks in the internal standard channel. Therefore, acetic acid solution is preferred, with an acetic acid concentration of 0.02~0.1% (v / v), preferably 0.05% (v / v). Under these conditions, the peak shape is good, and the response and retention time are appropriate.

[0096] The elution method is selected from isocratic elution and gradient elution. During isocratic elution, due to the complex matrix, endogenous substances co-elute with the analyte, resulting in a matrix effect greater than 30%. To ensure more complete chromatographic separation, gradient elution is used. The optimized gradient elution ratio is as follows:

[0097]

[0098] Furthermore, the liquid chromatography conditions also include:

[0099] Flow rate: 0.500 ml / min;

[0100] Injection volume: 6.00 μl;

[0101] Column temperature: 45℃.

[0102] Example 3 Selection of Mass Spectrometry Conditions

[0103] Based on the above screening, we continued to screen the mass spectrometry conditions.

[0104] Precursor and daughter ion scans were performed on dapoxetine and the internal standard. Appropriate precursor and daughter ions were selected. The daughter ion scan chromatogram is shown below. Figures 1-2 Of the three quantifiable daughter ions of dapoxetine (290.9, 100.0, and 348.3), the 290.9 daughter ion showed the highest response and was suitable for baseline levels; therefore, 290.9 was selected as the optimal parameter.

[0105] The declustering voltage is selected from -80 to -130V. Unlike conventional operation, the optimal declustering voltage for scanning is -82V. However, at this time, the baseline of the blank matrix fluctuates significantly and is unstable at the main peak position, causing significant interference and affecting the integration accuracy of low concentration samples. Under the premise of ensuring the stability of the blank baseline and the response of the analyte, -120V is preferred.

[0106] The preferred mass spectrometry conditions are as follows:

[0107] The AB Sciex Triple Quad 4500 tandem quadrupole mass spectrometer, equipped with an ESI source, was used in negative ion MRM scanning mode.

[0108] Source parameters:

[0109]

[0110] Compound parameters:

[0111]

[0112] Example 4 Selection of Internal Standard

[0113] Based on the above screening, the internal standards were further screened.

[0114] Isotope internal standards can better track analytes during sample preparation, chromatographic separation, and sample detection. However, if isotope internal standards are not readily available, structural analogs can be used. This experiment selected two compounds commonly found in bioanalytical laboratories (tolbutamide and ennastidine), whose structures differ somewhat from dapoxetine but are still suitable for the quantitative analysis of dapoxetine, demonstrating the robustness of this analytical method.

[0115] Add 50.0 μl of blank plasma to a 96-well plate, add 50.0 μl of internal standard working solution (tolbutamide concentration of 200 ng / ml; enanastat concentration of 10.0 ng / ml), add 300 μl of acetonitrile to precipitate proteins, vortex at 2000 rpm for 10 minutes, and centrifuge for 20 minutes (8℃, 3100g); add 150 μl of supernatant to a 96-well plate, dilute with 150 μl of 0.1% (v / v) formic acid solution, shake well at 1000 rpm for 30 minutes, and centrifuge for 10 minutes (8℃, 3100g) as internal standard for analysis of analyte interference samples.

[0116] The sample exhibits an interfering peak at the retention time of the analyte. The area of ​​this interfering peak is no greater than 20% of the peak area of ​​the analyte in the sample at the lower limit of quantitation of the standard curve, and therefore does not affect quantitative analysis. See the example chromatogram below. Figure 3 Therefore, dapoxetine-d8 is still the preferred internal standard.

[0117] Example 5 Interference Test

[0118] The above-mentioned preferred scheme was used to conduct a validation test of the quantitative detection method of plasma daprestine.

[0119] 5.1 Selectivity

[0120] Selective samples were prepared using six batches of blank plasma from different donors, one batch of hemolyzed plasma from one donor, and one batch of high-lipid plasma from one donor.

[0121] Eight different batches of blank plasma (50.0 μl each) were added to a 96-well plate. 50.0 μl of methanol:water (80:20, v / v) was added, followed by 300 μl of acetonitrile to precipitate the protein. The plate was vortexed at 2000 rpm for 10 minutes, then centrifuged for 20 minutes (8℃, 3100 g). 150 μl of the supernatant was added to the 96-well plate and diluted with 150 μl of 0.1% (v / v) formic acid solution. The plate was shaken at 1000 rpm for 30 minutes and then centrifuged for 10 minutes (8℃, 3100 g). This was used as the selective blank matrix sample for analysis.

[0122] Eight different batches of blank plasma were used to prepare 50.0 μl of the limit of quantitation (LOQ) sample, which was then added to a 96-well plate. 50.0 μl of internal standard working solution was added, and 300 μl of acetonitrile was added to precipitate the protein. The plate was vortexed at 2000 rpm for 10 minutes and then centrifuged for 20 minutes (8℃, 3100g). 150 μl of the supernatant was added to the 96-well plate, diluted with 150 μl of 0.1% (v / v) formic acid solution, shaken at 1000 rpm for 30 minutes, and then centrifuged for 10 minutes (8℃, 3100g). This sample was used as the LQ sample for selective analysis.

[0123] Six different sources of blank normal matrix, one source of blank high-lipid matrix, and one source of blank hemolyzed matrix showed no significant interference with the analyte and internal standard. Example chromatograms are shown below. Figures 4-5 .

[0124] 5.2 Specificity

[0125] Add 50.0 μl of the 150 ng / ml standard curve sample to a 96-well plate, add 50.0 μl of methanol:water (80:20, v / v), add 300 μl of acetonitrile to precipitate the protein, vortex at 2000 rpm for 10 minutes, and centrifuge for 20 minutes (8℃, 3100g); add 150 μl of the supernatant to the 96-well plate, dilute with 150 μl of 0.1% (v / v) formic acid solution, shake well at 1000 rpm for 30 minutes, and centrifuge for 10 minutes (8℃, 3100g). This supernatant is used as the analyte for analysis of the interfering samples.

[0126] Add 50.0 μl of blank plasma to a 96-well plate, add 50.0 μl of internal standard working solution, add 300 μl of acetonitrile to precipitate proteins, vortex at 2000 rpm for 10 minutes, and centrifuge for 20 minutes (8℃, 3100g); add 150 μl of supernatant to a 96-well plate, dilute with 150 μl of 0.1% (v / v) formic acid solution, shake well at 1000 rpm for 30 minutes, and centrifuge for 10 minutes (8℃, 3100g) as internal standard for analysis of analyte interference samples.

[0127] The results showed no interference between the analyte and the internal standard, and did not affect the quantitative analysis. An example chromatogram can be found below. Figures 6-7 .

[0128] Example 6: Matrix Effect Test

[0129] 10.0 μl of 12.0 ng / ml and 2250 ng / ml quality control working solutions were added to six batches of blank plasma from different donors, one batch of hemolyzed plasma from one donor, and one batch of high-lipid plasma from one donor, and diluted with 190 μl to prepare matrix effect samples of 0.600 ng / ml and 112.5 ng / ml. 50.0 μl of matrix effect sample was added to a 96-well plate, along with 50.0 μl of internal standard working solution and 300 μl of acetonitrile to precipitate proteins. The plate was vortexed at 2000 rpm for 10 minutes, centrifuged for 20 minutes (8℃, 3100 g), and 150 μl of the supernatant was added to the 96-well plate. The plate was diluted with 150 μl of 0.1% (v / v) formic acid solution, shaken at 1000 rpm for 30 minutes, and centrifuged for 10 minutes (8℃, 3100 g). This was used as the matrix effect sample for analysis (three samples were prepared for each concentration).

[0130] Six blank normal matrix samples, one blank high-lipid matrix sample, and one blank hemolyzed matrix sample from different sources showed good matrix effects. The results are shown in Table 1.

[0131] Table 1 Matrix effect test

[0132]

[0133] Example 7 Recovery Test

[0134] Quality control working solutions of 12.0 ng / ml, 720 ng / ml, and 2250 ng / ml were diluted with methanol:water (80:20, v / v) to 0.600, 36.0, and 112.5 ng / ml, respectively, to obtain extraction recovery solutions. 120 μl of blank plasma was placed in a centrifuge tube, 900 μl of acetonitrile was added, and the mixture was vortexed at 2000 rpm for 10 minutes, followed by centrifugation for 20 minutes (8℃, 3100 g). Take 50.0 μl of the extraction recovery solution sample, add 50.0 μl of the internal standard working solution, then add 300 μl of the supernatant prepared in the above steps, vortex to mix, take 150 μl and add it to a 96-well plate, dilute with 150 μl of 0.1% (v / v) formic acid solution, shake at 1000 rpm for 30 minutes, centrifuge for 10 minutes (8℃, 3100g), and use it as the unextracted sample, labeled as the extraction recovery A sample (prepare six samples in parallel for each concentration).

[0135] Quality control working solutions of 12.0 ng / ml, 720 ng / ml, and 2250 ng / ml were diluted with blank plasma to 0.600, 36.0, and 112.5 ng / ml, respectively, as extraction recovery matrix samples. 50.0 μl of the extraction recovery matrix sample was added to a 96-well plate, along with 50.0 μl of internal standard working solution and 300 μl of acetonitrile to precipitate the protein. The plate was vortexed at 2000 rpm for 10 minutes, then centrifuged for 20 minutes (8℃, 3100 g). 150 μl of the supernatant was added to the 96-well plate, diluted with 150 μl of 0.1% (v / v) formic acid solution, mixed at 1000 rpm for 30 minutes, and centrifuged for 10 minutes (8℃, 3100 g). This was used as the extracted sample and labeled as recovery sample B (six samples were prepared in parallel for each concentration).

[0136] The recovery rate was good, and the results are shown in Tables 2 and 3.

[0137] Table 2. Analyte Extraction and Recovery Test

[0138]

[0139] Table 3 Internal standard extraction recovery test

[0140]

[0141] Example 8 Linearity Test

[0142] Add 50.0 μl of the prepared standard curve sample to a 96-well plate or centrifuge tube, then add 50.0 μl of internal standard working solution, add 300 μl of acetonitrile to precipitate proteins, vortex at 2000 rpm for 10 minutes, centrifuge for 20 minutes (8℃, 3100g), and add 150 μl of the supernatant to a 96-well plate. Dilute with 150 μl of 0.1% (v / v) formic acid solution. After dilution, shake well at 1000 rpm for 30 minutes, centrifuge for 10 minutes (8℃, 3100g), and then inject for analysis. Plot the concentration of dapoxetine on the x-axis and the peak area ratio of dapoxetine to the internal standard on the y-axis, and use the weighted least squares method (weight 1 / X). 2 The standard curve is obtained by performing regression calculations on the straight line.

[0143] Dapoxetine showed good linearity in the range of 0.200–150 ng / ml; see example chromatogram. Figure 8 .

[0144] Example 9 Precision and Accuracy Test

[0145] Add 50.0 μl of the prepared limit of quantitation (LOQ) and quality control samples to a 96-well plate or centrifuge tube, then add 50.0 μl of internal standard working solution, and add 300 μl of acetonitrile to precipitate the protein. Vortex at 2000 rpm for 10 minutes, then centrifuge for 20 minutes (8℃, 3100g). Add 150 μl of the supernatant to a 96-well plate and dilute with 150 μl of 0.1% (v / v) formic acid solution. After dilution, shake well at 1000 rpm for 30 minutes, centrifuge for 10 minutes (8℃, 3100g), and then inject for analysis. Calculate the concentration of dapoxetine using the standard curve, and calculate its intra-batch accuracy and precision. Process four analytical batches, and calculate the inter-batch precision and accuracy of dapoxetine.

[0146] Dapoxetine showed good intra- and inter-batch precision and accuracy, as shown in Table 4.

[0147] Table 4. Intra-batch and inter-batch precision and accuracy

[0148]

[0149] Note: The measured concentration is "average ± SD".

[0150] Example 10 Stability Test

[0151] The stability of plasma matrix samples was investigated using low-concentration control samples (0.600 ng / ml) and high-concentration control samples (112.5 ng / ml) after 92 days of freezing at -20℃, 92 days of freezing at -80℃, four freeze-thaw cycles, 23 hours and 50 minutes of pretreatment at room temperature, 22 hours and 28 minutes of posttreatment at room temperature, and 72 hours and 20 minutes of posttreatment in an autosampler (set temperature: 8℃). Six replicates were used for each concentration, and all results were stable. The results are shown in Table 5.

[0152] Table 5 Stability of matrix samples

[0153]

[0154] Note: The measured concentration is "average ± SD".

[0155] The stability deviations of whole blood matrix samples were investigated using low-concentration quality control samples (0.600 ng / ml) and high-concentration quality control samples (112.5 ng / ml) after 2 hours of room temperature storage. The deviations were 2.0% and 2.4%, respectively. After 2 hours of ice-water bath storage, the deviations were 2.9% and 0.8%, respectively. All results were stable, as shown in Table 6.

[0156] The stability deviation of 1.00 mg / ml daprostat stock solution after 31 days at -20°C was 2.1%, and the stability deviation after 23 hours and 54 minutes at room temperature was 0.6%. The stability deviations of 4.00 ng / ml and 11.25 μg / ml daprostat working solutions after 19 days at -20°C were -1.8% and -0.3%, respectively. The stability deviations of daprostat plasma samples after 23 hours and 15 minutes at room temperature were -4.8% and 0.8%, respectively. All results showed that the solutions were stable.

Claims

1. A method for quantitatively determining the concentration of daprestat in human plasma, characterized in that, Includes the following steps: (1) Sample pretreatment; (2) Detection was performed using liquid chromatography-mass spectrometry; (3) Preparation of standard curve; (4) Determination of daprestat concentration in plasma; In step (1), the sample pretreatment step includes: taking an appropriate amount of the sample to be tested, adding an appropriate amount of internal standard working solution, after protein precipitation, mixing and centrifuging, taking the supernatant, adding diluent and shaking well, as the sample to be injected. In step (2), the chromatographic conditions for liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase is: mobile phase A: organic solvent or organic reagent containing acid, mobile phase B: water or aqueous solution containing acid, gradient elution; The mass spectrometry conditions include: the parent ion (Q1) is 392.3, the daughter ion (Q3) is selected from 290.9, 100.0, and 348.3, and the declustering voltage is selected from -80 to -130V.

2. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 1, characterized in that, The chromatographic column is octadecylsilane-bonded silica gel, the mobile phase A is selected from methanol, acetonitrile, methanol containing formic acid or acetic acid, acetonitrile containing formic acid or acetic acid, and the mobile phase B is selected from formic acid, acetic acid, ammonium formate, and ammonium acetate.

3. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 2, characterized in that, The chromatographic column is an Agilent ZORBAX Eclipse XDB-C18 (50 mm × 2.1 mm, 3.5 μm) or a column with equivalent performance; the mobile phase A is an acetonitrile solution containing 0.02~0.1% (v / v) acetic acid, and the mobile phase B is a 0.02~0.1% (v / v) acetic acid solution.

4. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 1, characterized in that, The gradient elution table is as follows: 。 5. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 1, characterized in that, The mass spectrometry conditions were as follows: an AB Sciex Triple Quad 4500 mass spectrometer, tandem quadrupole mass spectrometer, electrospray negative ion source, and multiple reaction monitoring (MRM) mode were used; collision activation dissociation: 9 psi; curtain gas: 35 psi. Spray gas: 55 psi; auxiliary heating gas: 55 psi; ionization voltage: -4500V; temperature: 450℃; inlet voltage: -10V; collision outlet voltage: -15V; MRM monitoring method parameters are as follows: dapoxetine m / z 392.3→290.9, declustering voltage: -120V, collision energy: -22V; residence time: 110ms; internal standard: m / z 400.4→299.0, declustering voltage: -140V, collision energy: -24V; residence time: 110ms.

6. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 1, characterized in that, In step (1), the protein precipitant is selected from methanol, acetonitrile, organic solvents containing formic acid or acetic acid, and the amount of protein precipitant added is at least twice the volume of the sample to be tested; the diluent is water or formic acid.

7. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 1, characterized in that, The preparation process of the internal standard working solution is as follows: a. Preparation of internal standard stock solution: Accurately weigh an appropriate amount of internal standard reference, add a certain volume of solvent 1, and dissolve by ultrasonic shaking to obtain an internal standard stock solution with a concentration of 1.00 mg / ml; all stock solutions and working solutions are aliquoted and stored at -20℃. b. Preparation of internal standard working solution: Dilute the internal standard stock solution with solvent 2 to prepare an internal standard working solution of 15.0 ng / ml; do not add internal standard working solution to blank samples, use solvent 2 instead.

8. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 7, characterized in that, The internal standard reference is selected from tolbutamide, ennastamine, and dapoxetine-d8; solvent 1 is dimethyl sulfoxide and methanol; solvent 2 is methanol and water.

9. The method for quantitatively determining the concentration of daprestat in human plasma according to claim 8, characterized in that, The internal standard reference is dapoxetine-d8; solvent 1 is dimethyl sulfoxide and methanol in a ratio of 30~70:70~30 (v / v); solvent 2 is methanol and water in a ratio of 50~90:10~50 (v / v).

10. The method for quantitative determination of dapoxetine concentration in human plasma according to claim 1 is used for clinical pharmacokinetic sample detection.

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