Method for quantitatively determining concentration of dotenorad in blood plasma

Abstract

The invention provides a method for quantitatively determining the concentration of dotenorad in blood plasma, protein precipitation pretreatment is adopted, the detection time of dotenorad is within 3 minutes under the chromatographic condition adopted by the test, dotenorad and an internal standard peak thereof are good in shape, no impurity peak interferes in determination, and a baseline is stable. The linear range of a plasma standard curve of the method is 2.00-300ng / ml, and the selectivity, specificity, system residue, lower limit of quantification, the standard curve, precision, accuracy, matrix effect, extraction recovery rate, stability and the like of the method are investigated. Data of each inspection item meet relevant standards, the stability of a biological sample and a standard solution can be ensured under controllable conditions, and the method is suitable for measuring the concentration of the dotenorad in the plasma in batches.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for quantitatively determining the concentration of polytinoride in plasma. Background Art

[0002] Dotinarud is the first highly selective urate transporter 1 (URAT1) inhibitor approved in China. It can effectively lower blood uric acid levels by inhibiting URAT1 and is suitable for the treatment of gout and hyperuricemia. Its structural formula is as follows:

[0003]

[0004] Currently, there are no published literature or patent reports on methods for determining the concentration of polytinorabine in biological samples. The present invention provides a detection method for quantitatively determining the concentration of polytinorabine in plasma using LC-MS / MS, which can provide data support for pharmacokinetic studies of polytinorabine, evaluate the efficacy and safety of polytinorabine, and optimize drug treatment plans. Summary of the Invention

[0005] The present invention provides a method for quantitatively detecting the concentration of polytinoride in plasma based on liquid chromatography-mass spectrometry (LC-MS / MS). By optimizing chromatographic and mass spectrometric conditions, highly specific and sensitive detection is achieved. Combined with protein precipitation pretreatment and rapid chromatographic separation technology, the single sample analysis time is short, the sampling volume is small, and the automation compatibility is strong. It can be quantified efficiently and accurately, and is suitable for clinical pharmacokinetic research and treatment monitoring.

[0006] The present invention provides a method for quantitatively determining the concentration of polytinoride in plasma, which is characterized by comprising the following steps:

[0007] (1) Sample pretreatment;

[0008] (2) Liquid chromatography-mass spectrometry detection;

[0009] (3) Preparation of standard curve;

[0010] (4) determination of plasma polytinoride concentration;

[0011] In the 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, mixing and centrifuging after protein precipitation, taking the supernatant, adding the diluent and shaking it, and using it as the sample to be injected; the above process is carried out at room temperature;

[0012] The protein precipitation is performed by adding acetonitrile to precipitate the protein, and the amount of acetonitrile added is at least 1.5 times the volume of the sample to be tested; the diluent is methanol, water, and formic acid, and the ratio thereof is 40-60:40-60:0.1 (v / v), preferably 50:50:0.1 (v / v).

[0013] The preparation process of the internal standard working solution is:

[0014] a. Preparation of internal standard stock solution: Accurately weigh an appropriate amount of internal standard reference substance, add a certain volume of solvent 1 and shake to dissolve to obtain an internal standard stock solution with a concentration of 1.00 mg / ml;

[0015] b. Preparation of internal standard working solution: Dilute the internal standard stock solution with solvent 2 to a 20.0 ng / ml internal standard working solution; do not add the internal standard working solution to the blank sample, but use solvent 2 instead.

[0016] The internal standard reference substance is polytinorel-d4 or polytinorel impurity I-1, preferably polytinorel-d4; the structures of polytinorel-d4 and polytinorel impurity I-1 are as follows:

[0017]

[0018] The solvent 1 is dimethyl sulfoxide and methanol, and the ratio thereof is 40-60:40-60 (v / v), preferably 50:50 (v / v); the solvent 2 is methanol and water, and the ratio thereof is 70-90:10-30 (v / v), preferably 80:20 (v / v).

[0019] Furthermore, the pre-processing step specifically includes:

[0020] (1) Add 40.0 μl of the sample to be tested to a 96-well plate or centrifuge tube, and then add 50.0 μl of the internal standard working solution; (2) Add 300 μl of acetonitrile to precipitate the protein; (3) Vortex at 2000 rpm for 10 minutes, and centrifuge for 20 minutes (8°C, 3100g); (4) Add 100 μl of the supernatant to a 96-well plate and dilute with 200 μl of methanol: water: formic acid (50:50:0.1, v / v). After dilution, shake the sample at 1000 rpm for 30 minutes. Biological matrix samples need to be centrifuged for 10 minutes (8°C, 3100g) before LC-MS / MS analysis. Pure solution samples can be directly injected after shaking.

[0021] In the step (2), the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; the mobile phase is: mobile phase A: an organic solvent or an acid-containing organic reagent, mobile phase B: an acidic reagent, and gradient elution;

[0022] The chromatographic column is preferably Agilent ZORBAX Eclipse XDB-C18 (50 mm × 2.1 mm, 3.5 μm), Agilent ZORBAX Polaris 5C18 (50 mm × 2.0 mm, 5.0 μm), or a chromatographic column with equivalent performance; the mobile phase A is selected from one or more of methanol, acetonitrile, methanol containing formic acid or acetic acid, and acetonitrile containing formic acid or acetic acid, preferably acetonitrile; the mobile phase B is selected from one or two of formic acid, acetic acid, ammonium formate, and ammonium acetate, preferably an acetic acid-ammonium acetate mixed solution, wherein the acetic acid concentration is 0.1% (v / v) and the ammonium acetate concentration is 10 mmol / L; the gradient elution table is as follows:

[0023] Time (min) Phase A (%) Phase B (%) 0.00 30-40 60-70 0.80 90-98 2-10 1.80 90-98 2-10 2.00 30-40 60-70 3.00 30-40 60-70

[0024] Preferably, the gradient elution table is:

[0025] Time (min) Phase A (%) Phase B (%) 0.00 35 65 0.80 95 5 1.80 95 5 2.00 35 65 3.00 35 65 ;

[0026] Furthermore, the liquid chromatography conditions also include:

[0027] Flow rate: 0.500ml / min;

[0028] Injection volume: 2.00 μl;

[0029] Column temperature: 45℃.

[0030] The mass spectrometry conditions include: the parent ion (Q1) in the mass spectrometry parameters is 356, and the daughter ion (Q3) is selected from 159.9, 203.8, and 168.0, preferably 159.9.

[0031] Specifically, the mass spectrometry conditions are:

[0032] The mass spectrometer was equipped with a Sciex Triple Quad 5500 mass spectrometer, a tandem quadrupole mass spectrometer, an electrospray negative ion source, and a multiple reaction monitoring (MRM) analysis mode; collision activated dissociation: 7 psi; curtain gas: 30 psi; spray gas: 55 psi; auxiliary heating gas: 55 psi, ionization voltage: -4500 V, temperature: 450°C; inlet voltage: -10 V; collision outlet voltage: -13 V; MRM monitoring method parameters are as follows: dotinorel m / z 356.0→159.9, declustering voltage: -120 V, collision energy: -33 V; internal standard: m / z 360.1→160.0, declustering voltage: -120 V, collision energy: -33 V; each mass spectrometer parameter can be optimized according to the instrument used.

[0033] In the step (3), the step of preparing the standard curve comprises: preparing a standard curve sample solution, preparing a sample to be injected according to the method of step (1), detecting according to the method of step (2), and recording the peak area corresponding to each concentration of polytinol; taking the peak area ratio of polytinol to the internal standard as the vertical axis, taking the concentration of polytinol as the horizontal axis, and taking 1 / X 2 is the weight coefficient, and a linear weighted least squares regression equation for dotinorel is prepared;

[0034] Wherein, the preparation of the standard curve sample solution comprises the following steps:

[0035] Accurately weigh an appropriate amount of polytinorel reference substance and place it in a weighing bottle. Add a certain volume of solvent 1 to dissolve and mix to obtain a polytinorel stock solution with a concentration of 1.00 mg / ml; dilute the polytinorel stock solution with solvent 2 to 40, 80, 200, 400, 1200, 2400, 4800 and 6000 ng / ml standard curve working solutions; add 10 μl of the standard curve working solution of each concentration to 190 μl of blank plasma, and vortex mix to obtain standard curve sample solutions of different concentrations.

[0036] The solvent 1 is dimethyl sulfoxide and methanol, and the ratio thereof is 40-60:40-60 (v / v), preferably 50:50 (v / v); the solvent 2 is methanol and water, and the ratio thereof is 70-90:10-30 (v / v), preferably 80:20 (v / v).

[0037] In the step (4), the step of determining the concentration of polytinoride in plasma comprises: preparing the sample to be injected according to the step (1), detecting according to the method of step (2), recording the peak area corresponding to polytinoride, substituting the peak area ratio of polytinoride and the internal standard into the constructed standard curve, and calculating the concentration of polytinoride in the plasma to be tested.

[0038] The present invention provides a detection method for quantitatively determining the concentration of polytinoride in plasma, which can be used for clinical pharmacokinetic sample detection.

[0039] The beneficial technical effects of the present invention are:

[0040] (1) The present invention provides a method for quantitatively analyzing the concentration of polytinol in plasma. By determining the concentration of trace polytinol in complex matrix plasma, it can provide data reference for the pharmacokinetic study of polytinol-related preparations.

[0041] (2) The plasma samples of the present invention are processed by protein precipitation method, which is simple, rapid, convenient, low-cost, and suitable for high-throughput sample analysis; the detection method of the present invention requires a small sample volume, and only tens of microliters are needed to complete the detection; the analysis time is short, and the target analyte can be detected in 3 minutes.

[0042] (3) The detection method of the present invention has high specificity, a stable baseline, and the blank matrix does not interfere with the detection; the method has high sensitivity, and the lower limit of quantification is 2.00 ng / ml, which can be optimized and reduced as needed.

[0043] (4) The detection method of the present invention uses an isotope internal standard (polytinorel-d4) to greatly improve the accuracy, precision and reliability of the analysis results; this method has been successfully applied to batch detection of polytinorel concentrations in clinical plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the daughter ion scan of polytinoride in Example 3.

[0045] Figure 2 This is the daughter ion scan of the internal standard in Example 3.

[0046] Figure 3 The internal standard interference spectrum of the analyte in Example 4

[0047] Figure 4 This is the blank matrix spectrum in the selectivity test of Example 5.

[0048] Figure 5 This is the quantitative lower limit sample spectrum in the selectivity test of Example 5.

[0049] Figure 6 This is the interference spectrum of the internal standard on the analyte in the specificity test of Example 5.

[0050] Figure 7 This is the interference spectrum of the analyte on the internal standard in the specificity test of Example 5.

[0051] Figure 8 This is the linear graph in Example 8. DETAILED DESCRIPTION

[0052] 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 understood as limiting the present invention.

[0053] Main instruments and reagents:

[0054] A Triple Quad 5500 liquid chromatograph-mass spectrometer (SCIEX); an XSE 105DU electronic balance (Mettler Toledo); a DW-HL860 cryogenic storage chamber (Zhongke Meiling Cryogenic Technology Co., Ltd.); a DW-86L828J cryogenic storage chamber (Qingdao Haier Special Electric Co., Ltd.); and a 5427R high-speed centrifuge (Eppendorf) were used. Data processing software included Analyst 1.6.3 (SCIEX); and Watson LIMS 7.5SP1 (Thermo Fisher Scientific).

[0055] Dotinorel reference substance (99.0% content, Guangzhou Jiatu Technology Co., Ltd.); Dotinorel-d4 reference substance (99.1% purity, TLC Pharmaceutical Standards Ltd.); methanol (chromatographic grade, Merck KGaA); acetonitrile (chromatographic grade, Merck KGaA); formic acid (chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.); acetic acid (chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.); ammonium acetate (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0056] Solution preparation:

[0057] 1. Preparation of stock solution

[0058] Preparation of polytinorel stock solution: Accurately weigh an appropriate amount of polytinorel reference substance and place it in a weighing bottle. After conversion, add a certain volume of dimethyl sulfoxide: methanol (50:50, v / v) to dissolve and mix to obtain a polytinorel stock solution with a concentration of 1.00 mg / ml.

[0059] Preparation of internal standard stock solution: Accurately weigh an appropriate amount of polytinol-d4 internal standard reference substance, convert it, add a certain volume of dimethyl sulfoxide: methanol (50:50, v / v) and shake to dissolve it to obtain a polytinol-d4 stock solution with a concentration of 1.00 mg / ml.

[0060] 2. Preparation of working solution

[0061] Preparation of standard curve working solution: Dilute the polytinol stock solution with methanol: water (80:20, v / v) to prepare standard curve working solutions of 40.0, 80.0, 200, 400, 1200, 2400, 4800 and 6000 ng / ml.

[0062] Preparation of quality control working solution: Dilute the dotinorel stock solution to 120, 600, 2000 and 4500 ng / ml quality control working solutions using methanol: water (80:20, v / v) as diluent.

[0063] Preparation of the lower limit of quantitation working solution: Dilute the dotinorel stock solution to a 40.0 ng / ml lower limit of quantitation working solution using methanol: water (80:20, v / v) as the diluent.

[0064] Preparation of internal standard working solution: Dilute the internal standard stock solution to 20.0 ng / ml internal standard working solution using methanol: water (80:20, v / v) as diluent.

[0065] 3. Sample Preparation

[0066] 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 the mixture was vortexed to obtain standard curve samples of 2.00, 4.00, 10.0, 20.0, 60.0, 120, 240, and 300 ng / ml.

[0067] Preparation of quality control samples: 10.0 μl of quality control working solution of different concentrations was added to 190 μl of blank plasma, and vortexed to mix, to obtain quality control samples of 6.00, 30.0, 100, and 225 ng / ml.

[0068] Preparation of the lower limit of quantification sample: add 10.0 μl of the lower limit of quantification working solution to 190 μl of blank plasma and vortex to mix to obtain a lower limit of quantification sample of 2.00 ng / ml.

[0069] Example 1 Sample pretreatment

[0070] Before sample injection and analysis, pretreatment is required. This involves protein precipitation, using acetonitrile as the precipitant. Adding at least 1.5 times the sample volume of acetonitrile removes most of the protein in the sample, resulting in a compact precipitate that facilitates transfer of the supernatant and a good recovery rate. Methanol, an organic reagent, is added to the supernatant diluent to increase the elution strength of the injection solution slightly above that of the initial mobile phase, reducing carryover. This includes the following steps:

[0071] (1) Take 40.0 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 (dotinorex-d4 internal standard working solution, concentration is 20.0 ng / ml. The blank sample does not add the internal standard working solution, but uses methanol: water (80:20, v / v) instead);

[0072] (2) adding acetonitrile to precipitate the protein;

[0073] (3) Vortex at 2000 rpm for 10 min and centrifuge for 20 min (8°C, 3100 g);

[0074] (4) 100 μl of supernatant was added to a 96-well plate and diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v). The diluted sample was shaken at 1000 rpm for 30 minutes. Biological matrix samples were centrifuged for 10 minutes (8°C, 3100 g) before LC-MS / MS analysis. Pure solution samples were shaken and directly injected for analysis.

[0075] Example 2 Selection of liquid chromatography conditions

[0076] The sample pretreated as above was injected into the liquid chromatograph, and the obtained chromatogram was recorded.

[0077] In terms of chromatographic column selection, Agilent ZORBAX Eclipse XDB-C18 and Agilent ZORBAX Polaris5 C18 performed well in peak retention time, response, and peak shape under the same conditions.

[0078] In terms of mobile phase selection, mobile phase A compared methanol and acetonitrile, and acetonitrile had stronger elution ability, appropriate retention time of the analyte and internal standard, and good peak shape; mobile phase B compared 0.1% (v / v) acetic acid solution, 0.1% (v / v) formic acid solution, and 0.1% (v / v) acetic acid-10mmol / L ammonium acetate, among which 0.1% (v / v) acetic acid-10mmol / L ammonium acetate had good peak shape, appropriate response and retention time.

[0079] To ensure a more complete chromatographic separation effect, a gradient elution method was used. The initial ratio of mobile phase B during gradient elution has an impact on the residue. After testing, the residues at initial ratios of 85%, 70%, and 65% of mobile phase B were 12%, 3.2%, and 1.5%, respectively. That is, the residue was lower when the initial ratio was 65%, so this ratio was selected. The optimized specific gradient elution ratio is:

[0080] Time (min) Phase A (%) Phase B (%) 0.00 35 65 0.80 95 5 1.80 95 5 2.00 35 65 3.00 35 65

[0081] Furthermore, the liquid chromatography conditions also include:

[0082] Flow rate: 0.500ml / min;

[0083] Injection volume: 2.00 μl;

[0084] Column temperature: 45℃.

[0085] Example 3 Selection of mass spectrometry conditions

[0086] Based on the above screening, the mass spectrometry conditions were further screened.

[0087] Scan the parent ion and daughter ion of Dotinol and internal standard, select the appropriate parent ion and daughter ion, and the daughter ion scan is shown in Figure 1-Figure 2 Among the three daughter ions (159.9, 203.8, and 168.0) of dotinorel that can be used for quantification, the daughter ion 159.9 has the highest response. Therefore, 159.9 is selected as the most preferred parameter. The preferred mass spectrometry conditions are as follows:

[0088] A Sciex Triple Quad 5500 tandem quadrupole mass spectrometer was used with an ESI source and negative ion MRM scanning mode.

[0089] Source parameters:

[0090]

[0091] Compound parameters:

[0092]

[0093] Example 4 Selection of internal standard

[0094] On the basis of the above screening, continue to screen the internal standard.

[0095] Isotope internal standards can better track the analyte during sample preparation, chromatographic separation and sample detection. However, if isotope internal standards are not easy to obtain, structural analogues can be used. The structural analogue selected in this experiment (Dotinorel Impurity I-1) has the same key chemical structure and functional groups as the analyte, with only one oxygen difference. It is not the main metabolite of Dotinorel and is also suitable for the quantitative analysis of Dotinorel.

[0096] 50.0 μl of blank plasma was added to a 96-well plate, 50.0 μl of internal standard working solution (the concentration of polytinorel impurity I-1 was 5.00 ng / ml) was added, 300 μl of acetonitrile was added to precipitate the protein, vortexed at 2000 rpm for 10 minutes, and centrifuged for 20 minutes (8°C, 3100 g); 150 μl of supernatant was added to a 96-well plate, diluted with 150 μl of 0.2% (v / v) formic acid solution, shaken at 1000 rpm for 30 minutes, and centrifuged for 10 minutes (8°C, 3100 g) to be used as an internal standard for the analysis of the interference sample of the analyte.

[0097] The sample has an interference peak at the retention time of the analyte. The interference peak area is no more than 20% of the analyte peak area of the standard curve quantitative lower limit, which does not affect the quantitative analysis. See the example spectrum for details. Figure 3 Therefore, polytinorex-d4 is still preferred as the internal standard.

[0098] Example 5 Interference Test

[0099] The above preferred scheme was used to conduct a validation test of the quantitative detection method of plasma polytinoride.

[0100] 5.1 Selectivity

[0101] 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 hyperlipidemia plasma from one donor.

[0102] 40.0 μl of blank plasma from 8 different batches was added to a 96-well plate, 50.0 μl of methanol:water (80:20, v / v) was added, and then 300 μl of acetonitrile was added to precipitate the protein. The plate was vortexed at 2000 rpm for 10 minutes and centrifuged for 20 minutes (8°C, 3100 g). 100 μl of the supernatant was added to a 96-well plate and diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v). The plate was shaken at 1000 rpm for 30 minutes and centrifuged for 10 minutes (8°C, 3100 g) for injection as a selective blank matrix sample for analysis.

[0103] 40.0 μl of the lower limit of quantification sample prepared from 8 different batches of blank plasma was added to a 96-well plate, 50.0 μl of the internal standard working solution was added, 300 μl of acetonitrile was added to precipitate the protein, and the sample was vortexed at 2000 rpm for 10 minutes and centrifuged for 20 minutes (8°C, 3100 g). 100 μl of the supernatant was added to a 96-well plate, diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v), shaken at 1000 rpm for 30 minutes, and centrifuged for 10 minutes (8°C, 3100 g) for injection and analysis as the selective lower limit of quantification sample.

[0104] Six blank normal matrices from different sources, one blank high-fat matrix, and one blank hemolytic matrix from different sources showed no significant interference with the analytes and internal standards. See the example spectra for details. Figure 4-Figure 5 .

[0105] 5.2 Specificity

[0106] 40.0 μl of 300 ng / ml standard curve sample was added to a 96-well plate, 50.0 μl of methanol: water (80:20, v / v) was added, 300 μl of acetonitrile was added to precipitate the protein, vortexed at 2000 rpm for 10 minutes, and centrifuged for 20 minutes (8°C, 3100 g); 100 μl of supernatant was added to a 96-well plate, diluted with 200 μl of methanol: water: formic acid (50:50:0.1, v / v), shaken at 1000 rpm for 30 minutes, and centrifuged for 10 minutes (8°C, 3100 g). This was used as the internal standard interference sample for analysis.

[0107] 40.0 μl of blank plasma was added to a 96-well plate, 50.0 μl of internal standard working solution was added, 300 μl of acetonitrile was added to precipitate the protein, vortexed at 2000 rpm for 10 min, and centrifuged for 20 min (8°C, 3100 g); 100 μl of supernatant was added to a 96-well plate, diluted with 200 μl of methanol: water: formic acid (50:50:0.1, v / v), shaken at 1000 rpm for 30 min, and centrifuged for 10 min (8°C, 3100 g). This was used as an internal standard to analyze the interfering samples of the analyte.

[0108] Results: There was no interference between the analyte and the internal standard, which did not affect the quantitative analysis. See the example spectrum for details. Figure 6-Figure 7 .

[0109] Example 6 Matrix effect test

[0110] Matrix effect samples at concentrations of 6.00 ng / ml and 225 ng / ml were prepared by diluting 10.0 μl of the 120 ng / ml and 4.50 μg / ml quality control working solutions with 190 μl of blank plasma from six different donors, hemolyzed plasma from one donor, and hyperlipidemia plasma from one donor, respectively. 40.0 μl of the matrix effect sample was added to a 96-well plate, and 50.0 μl of the internal standard working solution was added. Protein was precipitated with 300 μl of acetonitrile, vortexed at 2000 rpm for 10 minutes, and centrifuged for 20 minutes (8°C, 3100 g). 100 μl of the supernatant was added to a 96-well plate and diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v). The sample was shaken at 1000 rpm for 30 minutes and centrifuged for 10 minutes (8°C, 3100 g). This was then injected as a matrix effect sample (triplicate samples were prepared for each concentration).

[0111] The matrix effects of six blank normal matrices from different sources, one blank high-fat matrix, and one blank hemolytic matrix were good. The results are shown in Table 1.

[0112] Table 1 Matrix effect test

[0113]

[0114]

[0115]

[0116] Example 7 Recovery Test

[0117] Quality control solutions of 4.50 μg / ml, 2.00 μg / ml, and 120 ng / ml were diluted with methanol:water (80:20, v / v) to 6.00, 100, and 225 ng / ml, respectively, to serve as extraction recovery solution samples. 120 μl of blank plasma was placed in a centrifuge tube, 900 μl of acetonitrile was added, and the tube was vortexed at 2000 rpm for 10 minutes. The tube was then centrifuged for 20 minutes (8°C, 3100 g) and set aside. Take 40.0 μl of the extraction recovery solution sample, add 50.0 μl of the internal standard working solution, then add 300 μl of the supernatant to be used in the above step, vortex mix, take 100 μl and add it to a 96-well plate, dilute with 200 μl of methanol: water: formic acid (50:50:0.1, v / v), shake at 1000 rpm for 30 minutes, centrifuge for 10 minutes (8°C, 3100g), as the unextracted sample, marked as the extraction recovery A sample (six samples were prepared in parallel for each concentration).

[0118] Quality control solutions at 120 ng / ml, 2.00 μg / ml, and 4.50 μg / ml were diluted with blank plasma to 6.00, 100, and 225 ng / ml, respectively, to serve as extraction recovery matrix samples. 40.0 μl of the extraction recovery matrix sample was placed in a 96-well plate, 50.0 μl of the internal standard working solution was added, and 300 μl of acetonitrile was added to precipitate the protein. The sample was vortexed at 2000 rpm for 10 minutes and centrifuged for 20 minutes (8°C, 3100 g). 100 μl of the supernatant was added to a 96-well plate and diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v). The sample was shaken at 1000 rpm for 30 minutes and centrifuged for 10 minutes (8°C, 3100 g). This was used as the post-extraction sample, labeled Recovery B. (Six samples were prepared in parallel for each concentration.)

[0119] The recovery rate was good, and the results are shown in Tables 2 and 3.

[0120] Table 2 Extraction recovery test of analytes

[0121]

[0122] Table 3 Internal standard extraction recovery test

[0123]

[0124]

[0125] Example 8 Linearity Test

[0126] Take 40.0 μl of the prepared standard curve sample and add it to a 96-well plate or centrifuge tube, then add 50.0 μl of the internal standard working solution, add 300 μl of acetonitrile to precipitate the protein, vortex at 2000 rpm for 10 minutes, centrifuge for 20 minutes (8°C, 3100g), take 100 μl of the supernatant and add it to a 96-well plate, and dilute with 200 μl of methanol: water: formic acid (50:50:0.1, v / v). The diluted sample is shaken at 1000 rpm for 30 minutes, centrifuged for 10 minutes (8°C, 3100g), and then injected for analysis. With the concentration of polytinoride as the horizontal axis X and the peak area ratio of polytinoride to the internal standard as the vertical axis Y, the weighted least squares method (weight is 1 / X) is used. 2 ) is used for regression calculation, and the obtained straight line equation is the standard curve.

[0127] Dotinol has a good linear relationship in the range of 2.00~300ng / ml. Figure 8 .

[0128] Example 9 Precision Accuracy Test

[0129] 40.0 μl of the prepared lower limit of quantitation and quality control samples were added to a 96-well plate or centrifuge tube. 50.0 μl of the internal standard working solution was then added. Protein was precipitated with 300 μl of acetonitrile. The samples were vortexed at 2000 rpm for 10 minutes and centrifuged for 20 minutes (8°C, 3100 g). 100 μl of the supernatant was then added to a 96-well plate and diluted with 200 μl of methanol:water:formic acid (50:50:0.1, v / v). The diluted samples were shaken at 1000 rpm for 30 minutes, centrifuged for 10 minutes (8°C, 3100 g), and then injected for analysis. The concentration of dotinorel was calculated using the standard curve, and the intra-assay accuracy and precision were calculated. Three analytical batches were processed over three consecutive days to calculate the inter-assay precision and accuracy of dotinorel.

[0130] The intra- and inter-batch precision and accuracy of dotinoreplast were good, as shown in Table 4.

[0131] Table 4 Intra-batch and inter-batch precision and accuracy

[0132]

[0133] Example 10 Stability Test

[0134] Low-concentration quality control samples (6.00 ng / ml) and high-concentration quality control samples (225 ng / ml) were used to investigate the stability of plasma matrix samples after 30-day storage at -20°C, 30-day storage at -80°C, four freeze-thaw cycles, 19 hours and 33 minutes of pre-treatment at room temperature, 18 hours and 34 minutes of post-treatment at room temperature, and 69 hours and 45 minutes of post-treatment automatic sample injection (set temperature: 8°C). Six replicates were performed for each concentration, and the results were all stable. The results are shown in Table 5.

[0135] Table 5 Matrix sample stability

[0136]

[0137] Low-concentration quality control samples (6.00 ng / ml) and high-concentration quality control samples (225 ng / ml) were used to investigate the stability of whole blood matrix samples. The stability deviations of the low- and high-concentration quality control samples were -1.6% and -1.4%, respectively, after being placed at room temperature for 2 hours. The stability deviations of the low- and high-concentration quality control samples were 2.7% and -0.5%, respectively, after being placed in an ice-water bath for 2 hours. The results were all stable. The results are shown in Table 6.

[0138] The stability deviation of 1.00 mg / ml stock solution after being placed at -20°C for 31 days was 0.1%, and the stability deviation was 0.3% after being placed at room temperature for 23 hours and 46 minutes. The stability deviations of 40.0 ng / ml and 22.5 μg / ml working solutions after being placed at -20°C for 31 days were -2.2% and -3.2%, respectively, and the stability deviations were 0.4% and 0.2%, respectively, after being placed at room temperature for 22 hours and 46 minutes. The results were all stable.

Claims

1. A method for quantitatively determining the concentration of polytinoride in plasma, characterized in that: The following steps are involved: (1) Sample pretreatment; (2) Liquid chromatography-mass spectrometry detection; (3) Preparation of standard curve; (4) determination of plasma polytinorel concentration; Wherein, in the 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, mixing and centrifuging after protein precipitation, taking the supernatant, adding the diluent and shaking it, and using it as the sample to be injected; In the step (2), the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler; the mobile phase is: mobile phase A: an organic solvent or an organic reagent containing an acid, mobile phase B: an acidic reagent, and gradient elution; the mass spectrometry conditions are as follows: the parent ion in the mass spectrometry parameters is 356, and the daughter ions are selected from 159.9, 203.8, and 168.

0.

2. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The chromatographic column is selected from Agilent ZORBAX Eclipse XDB-C18 and Agilent ZORBAX Polaris 5C18.

3. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The mobile phase A is selected from one or more of methanol, acetonitrile, methanol containing formic acid or acetic acid, and acetonitrile containing formic acid or acetic acid, and the mobile phase B is selected from one or two of formic acid, acetic acid, ammonium formate, and ammonium acetate.

4. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 3, wherein: The mobile phase A is acetonitrile, and the mobile phase B is a mixed solution of acetic acid and ammonium acetate, wherein the concentration of acetic acid is 0.1% (v / v) and the concentration of ammonium acetate is 10 mmol / L.

5. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The gradient elution table is: 。 6. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The mass spectrometry conditions are as follows: mass spectrometry was performed using a Sciex Triple Quad 5500 mass spectrometer, a tandem quadrupole mass spectrometer, an electrospray negative ion source, and a multiple reaction monitoring (MRM) analysis mode; collision activated dissociation: 7 psi; curtain gas: 30 psi; Spray gas: 55 psi; auxiliary heating gas: 55 psi, ionization voltage: -4500 V, temperature: 450 ° C; inlet voltage: -10 V; Collision exit voltage: -13 V; MRM monitoring method parameters are as follows: dotinorel m / z 356.0→159.9, declustering voltage: -120 V, collision energy: -33 V; Internal standard: m / z 360.1→160.0, declustering voltage: -120 V, collision energy: -33 V.

7. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The protein precipitation in step (1) is performed by adding acetonitrile to precipitate the protein, and the amount of acetonitrile added is at least 1.5 times the volume of the sample to be tested; the diluent is methanol, water, and formic acid.

8. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 1, wherein: The preparation process of the internal standard working solution is: a. Preparation of internal standard stock solution: Accurately weigh an appropriate amount of internal standard reference substance, add a certain volume of solvent 1 and shake to dissolve to obtain an internal standard stock solution with a concentration of 1.00 mg / ml; b. Preparation of internal standard working solution: Dilute the internal standard stock solution with solvent 2 to a 20.0 ng / ml internal standard working solution; do not add the internal standard working solution to the blank sample, but use solvent 2 instead.

9. The method for quantitatively determining the concentration of polytinoride in plasma according to claim 8, characterized in that: The internal standard reference substance is polytinorel-d4 or polytinorel impurity I-1; the solvent 1 is dimethyl sulfoxide and methanol, and the solvent 2 is methanol and water.

10. Use of the method for quantitatively determining the concentration of polytinoride in plasma according to claim 1 for clinical pharmacokinetic sample testing.

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