Method for detecting concentration of lumepirone tosylate in plasma

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the concentration of lumepirone tosylate in plasma. The detection method comprises the following steps: (1) preparing an experimental solution; (2) liquid chromatography-tandem mass spectrometry determination and analysis: chromatographic conditions are as follows: a gradient elution mode is adopted, the temperature of an automatic sampler is 0-10 DEG C, the column temperature is 35-45 DEG C, a mobile phase A is a mixed aqueous solution of 0.05-0.15% formic acid and 5-20mM ammonium formate, a mobile phase B is acetonitrile, and the flow velocity is 0.3-0.6 mL / min; mass spectrometry conditions are as follows: an electrospray positive ion mode is adopted. The accuracy, the precision, the matrix effect, the stability and the like of the method all meet related requirements of methodology verification, and the system adaptability, the specificity and the reproducibility of the method are good.

Description

Technical Field

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

[0002] Lumiperone tosylate is a new drug for the treatment of schizophrenia in adults approved by the FDA on December 20, 2019. Its mechanism of action in the treatment of schizophrenia is still unclear, but it may be achieved by jointly antagonizing the activity of central 5-HT2A receptors and postsynaptic D2 receptors.

[0003] The marketed dosage form of lumepirone tosylate is capsules. The original research company is Intra-Cellular Therapies Inc. CDE has published the reference preparation. When conducting BE studies on generic drugs and other new drug development, it is necessary to test the concentration of lumepirone tosylate in the blood. However, there is no literature reporting the method for determining the concentration of lumepirone tosylate in the blood.

[0004] For example, Chinese patent CN118566395B discloses a method, application and system for determining the impurity content in lumepirone tosylate orodisintegrating tablets, which belongs to the technical field of drug impurity analysis. The method for determining the impurity content in lumepirone tosylate orodisintegrating tablets provided by the invention can distinguish the contents of 6 impurities in lumepirone tosylate orodisintegrating tablets, and the minimum separation degree between each impurity can be up to 1.5, and the quantitative limit concentration of each impurity can be as low as 0.84 μg / mL. This method can simply, accurately and efficiently evaluate the impurity content in lumepirone tosylate orodisintegrating tablets, which is of great significance for the quality control of lumepirone tosylate orodisintegrating tablets. Although the invention discloses a method for detecting the impurity content in lumepirone tosylate orodisintegrating tablets by high performance liquid chromatography, its research is limited to in vitro, and the impurity content is detected, and the concentration of lumepirone tosylate in plasma is not detected.

[0005] For example, US20240033262A1 discloses a drug capsule comprising lumatipirone in free form or in a pharmaceutically acceptable salt form, optionally in combination with one or more additional therapeutic agents, and also relates to a method for its preparation and a method for treating or preventing a disease. The method uses lumatipirone, which provides dopamine D1 receptor-dependent enhancement of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) currents, and is associated with the activation of mammalian rapamycin target (mTOR) (eg, mTORC1) signaling pathways. This batch of capsule preparations was used for a Phase I human clinical trial to determine the plasma pharmacokinetic parameters of a single 60 mg oral dose of lumatipirone tosylate in volunteers with schizophrenia. However, the invention does not disclose a method for detecting the concentration of lumatipirone tosylate in plasma.

[0006] Therefore, it is urgent to develop a method for detecting the concentration of lumepirone tosylate in plasma so as to better apply lumepirone tosylate in clinical practice. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention provides a method for detecting the concentration of lumepirone tosylate in plasma.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In one aspect, the present invention provides a method for detecting the concentration of lumepirone tosylate in plasma, comprising the following steps:

[0010] (1) Prepare experimental solutions;

[0011] (2) Liquid chromatography-tandem mass spectrometry analysis:

[0012] Liquid chromatography-tandem mass spectrometry was used to inject the test sample for detection, where:

[0013] Chromatographic conditions: Mobile phase A is a mixed aqueous solution of 0.05-0.15% formic acid and 5-20 mM ammonium formate, mobile phase B is acetonitrile, flow rate is 0.3-0.6 mL / min, gradient elution is adopted, and the gradient elution program is as follows:

[0014] Time / min A％ B％ 0.01 55 45 050 55 45 200 25 75 201 10 90 2.50 10 90 3.00 55 45 5.50 55 45

[0015] Mass spectrometry conditions: electrospray positive ionization mode was used.

[0016] Preferably, the specific operation steps of preparing the experimental solution in step (1) are as follows:

[0017] First, prepare the required lumepirone tosylate internal standard working solution, standard curve working solution and quality control working solution respectively;

[0018] Then add the internal standard working solution to the plasma sample, then continue to add acetonitrile to the plasma sample for vortexing, and take the supernatant after centrifugation to obtain the test solution;

[0019] Further preferably, the preparation of the internal standard working solution is:

[0020] Take an appropriate amount of lumepirone ditosylate-d4 reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone ditosylate-d4 internal standard stock solution; then dilute it to obtain a lumepirone tosylate internal standard working solution of corresponding concentration;

[0021] Preparation of Lumiperone Tosylate Standard Solution:

[0022] Take an appropriate amount of lumepirone tosylate reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone tosylate standard stock solution, which is then diluted to obtain a lumepirone tosylate standard working solution of the corresponding concentration;

[0023] Preparation of quality control working solution:

[0024] Take an appropriate amount of lumepirone tosylate reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone tosylate quality control stock solution; then dilute it to obtain a lumepirone tosylate quality control working solution of corresponding concentration;

[0025] Among them, the needle washing solution of the liquid phase system: methanol: water = 40-50:60-50 (v / v).

[0026] The vortexing is carried out at 1200 rpm for 5 minutes, and the centrifugation is carried out at 4000 g centrifugal force for 5 minutes.

[0027] Preferably, the dilution in step (1) is performed using a methanol aqueous solution with a volume fraction of 45%-55%.

[0028] Preferably, the concentration of lumepirone ditosylate-d4 in the preparation of the internal standard working solution described in step (1) is 50-100 ng / mL.

[0029] Preferably, the concentration of lumepirone tosylate in the preparation of the lumepirone tosylate standard curve working solution described in step (1) is 20-8000 ng / mL.

[0030] Preferably, the chromatographic column packing is C18, with specifications of 3.5 μm, 2.1×100 mm, the automatic sampler temperature is 0-10° C., and the column temperature is 35-45° C.

[0031] Preferably, in the electrospray positive ion mode in step (2), the collision voltages of lumepirone tosylate and its internal standard are 30-40 V, respectively, and lumepirone tosylate and lumepirone ditosylate-d4 are detected simultaneously.

[0032] Further preferably, the collision voltages of lumepirone tosylate and its internal standard in the electrospray positive ion mode in step (2) are 36 V and 35 V, respectively.

[0033] Preferably, in the electrospray positive ion mode described in step (2), the quantitative parent ion of lumepirone tosylate is 394.4 m / z, and the quantitative daughter ion is 165.0; the quantitative parent ion of deuterated lumepirone tosylate is 398.4 m / z, and the quantitative daughter ion is 168.9.

[0034] Preferably, the mass spectrometry conditions also include: electrospray voltage of 5000-6000 V, curtain gas pressure of 35-45 psi, nebulization pressure of 35-45 psi, and ion source temperature of 500-600°C.

[0035] Further preferably, the mass spectrometry conditions also include: an electrospray voltage of 5500 V, a curtain gas pressure of 40 psi, and a nebulization pressure of 40 psi.

[0036] On the other hand, the present invention also provides the use of the above detection method in detecting the concentration of lumepirone tosylate in blood.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The detection method provided by the present invention has high accuracy and sensitivity, strong specificity, and good system adaptability, specificity and reproducibility;

[0039] (2) The detection method provided by the present invention lays a foundation for the detection of the concentration of lumepirone tosylate in vivo and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The chromatogram obtained for Comparative Example 1;

[0041] Figure 2 The chromatogram obtained for Comparative Example 2;

[0042] Figure 3 The chromatogram obtained for Comparative Example 3;

[0043] Figure 4 The product ion mass spectra of lumepirone tosylate and its internal standard (A represents the chromatogram of lumepirone tosylate, and B represents the chromatogram of lumepirone ditosylate-d4);

[0044] Figure 5 is the chromatogram of lumepirone tosylate and its internal standard at LLQC concentration (I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4);

[0045] Figure 6 The chromatograms of lumeperone tosylate and its internal standard at STD8 concentration (I represents the chromatogram of lumeperone tosylate, and II represents the chromatogram of lumeperone ditosylate-d4);

[0046] Figure 7 The chromatograms of lumepirone tosylate and its internal standard in blood samples of SD rats after oral administration of lumepirone tosylate (I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4);

[0047] Figure 8 The chromatograms of lumepirone tosylate and its internal standard in blood samples of SD rats injected with the long-acting preparation of lumepirone tosylate (I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4);

[0048] Fig. 9 This is the standard curve for the determination of lumepirone tosylate in SD rat plasma. DETAILED DESCRIPTION

[0049] The technical scheme of the present invention is clearly and completely described below with specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The materials, reagents, etc. used, if not otherwise specified, are reagents and materials available from commercial sources.

[0050] 750 μL of SD rat whole blood sample was centrifuged at 4°C and 3500 rpm for 10 min, and the obtained supernatant was the plasma sample; in the embodiments and comparative examples of the present invention, the whole blood sample was prepared by taking venous blood from the retroorbital venous plexus of SD rats and adding it into an anticoagulant tube containing sodium heparin / EDTA.

[0051] Example 1 A method for detecting the concentration of lumepirone tosylate in plasma

[0052] The details are as follows:

[0053] (1) Preparation of experimental solution:

[0054] Take an appropriate amount of lumepirone ditosylate-d4 reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone ditosylate-d4 internal standard stock solution with a concentration of 100 μg / mL.

[0055] Take 75 μL of the internal standard stock solution and dilute it with a 50% by volume methanol aqueous solution to prepare a 750 ng / mL internal standard working solution.

[0056] Take an appropriate amount of lumepirone tosylate reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone tosylate reference solution with a concentration of 1.000 mg / mL.

[0057] The standard stock solution was prepared into a 200 μg / mL mother solution with a 50% volume fraction of methanol aqueous solution, and further diluted with a 50% volume fraction of methanol aqueous solution according to the table below to obtain a corresponding concentration of lumepirone tosylate standard working solution.

[0058] Table 1 Standard curve working solution preparation method and final concentration statistics

[0059]

[0060] Take an appropriate amount of lumepirone tosylate reference substance, accurately weigh it, and dissolve it in methanol to prepare a lumepirone tosylate quality control stock solution with a concentration of 1.000 mg / mL.

[0061] A 200 μg / mL mother solution was prepared with a 50% by volume methanol aqueous solution quality control stock solution, and further diluted with a 50% by volume methanol aqueous solution according to the table below to obtain a corresponding concentration of lumepirone tosylate quality control working solution.

[0062] Table 2 Quality control working solution preparation method and final concentration statistics

[0063]

[0064] The needle washing solution of the liquid phase system is: methanol: water = 50:50 (v / v).

[0065] Take 100 μL of SD rat plasma sample into a centrifuge tube, add 10 μL of internal standard working solution and 300 μL of acetonitrile, vortex at 1200 rpm for 5 min, centrifuge at 4000 g for 5 min, and take the supernatant to obtain the test solution;

[0066] (2) Liquid chromatography-tandem mass spectrometry analysis:

[0067] Take 2 μL of the above biological matrix sample, use liquid chromatography-tandem mass spectrometry, with the concentration of lumepirone tosylate as the horizontal axis and the peak area ratio of lumepirone tosylate to the internal standard as the vertical axis to prepare a calibration curve; take the test solution and measure it on the machine under the same conditions to calculate the concentration of lumepirone tosylate in plasma, wherein the liquid chromatography conditions and mass spectrometry conditions during the determination are as follows:

[0068] Liquid chromatography conditions:

[0069] Chromatographic column: Xselect HSS T3 (3.5 μm, 2.1 × 100 mm);

[0070] Mobile phase A: 0.1% formic acid 10 mM ammonium formate in water;

[0071] Mobile phase B: acetonitrile

[0072] Flow rate: 0.5 mL / min;

[0073] Running time: 5.5min;

[0074] Column temperature: 40°C;

[0075] Injection volume: 2 μL;

[0076] Injection chamber temperature: 4°C;

[0077] Gradient elution program:

[0078] Table 3 Gradient elution program

[0079] Time / min A％ B％ 0.01 55 45 0.50 55 45 200 25 75 201 10 90 2.50 10 90 3.00 55 45 5.50 55 45

[0080] Mass spectrometry conditions:

[0081] The electrospray positive ion mode was used, the ion source parameters were shown in Table 4 below, and the compound parameters were shown in Table 5 below.

[0082] Table 4 Ion source parameters

[0083] Ion source CAD(psi) CUR(psi) Gas1(psi) Gas2(psi) TEM(℃) IS(V) ESI 4 25 40 40 450 5500

[0084] Table 5 Compound parameters

[0085]

[0086] According to calculation, in this embodiment, the concentration of lumepirone tosylate in the plasma sample of SD rats measured was 12.5 ng / mL.

[0087] Example 2 A method for detecting the concentration of lumepirone tosylate in plasma

[0088] The details are as follows:

[0089] The detection method is basically the same as that in Example 1, except for the following three aspects:

[0090] During the dissolution and volume determination in step (1), the organic solvent used is a methanol aqueous solution with a volume fraction of 45%.

[0091] Take 285 μL of SD rat plasma sample into a centrifuge tube, add 15 μL of internal standard working solution and 300 μL of acetonitrile, vortex at 1200 rpm for 5 min, centrifuge at 4000 g for 5 min, and take the supernatant to obtain the test solution.

[0092] The mobile phase in the liquid chromatography conditions in step (2) is: mobile phase A is a mixed aqueous solution of 0.05% formic acid and 20 mM ammonium formate, mobile phase B is acetonitrile, and the flow rate is 0.3 mL / min; the spray temperature in the mass spectrometry conditions is 500°C.

[0093] A methodological investigation was conducted on the determination method, and the results showed that the accuracy, precision, matrix effect, stability, etc. of the determination of lumepirone tosylate in heparin / EDTA anticoagulated SD rat plasma in the range of 1-400 ng / mL met the relevant requirements of methodological validation, and the method had good system adaptability, specificity and reproducibility.

[0094] Example 3 A method for detecting the concentration of lumepirone tosylate in plasma

[0095] The details are as follows:

[0096] The detection method is basically the same as that in Example 1, except for the following three aspects:

[0097] During the dissolution and volume adjustment in step (1), the reagent used is a methanol aqueous solution with a volume concentration of 45%.

[0098] Take 100 μL of SD rat plasma sample into a centrifuge tube, add 20 μL of internal standard working solution and 300 μL of acetonitrile, vortex at 1200 rpm for 5 min, centrifuge at 4000 g for 5 min, and take the supernatant to obtain the test solution.

[0099] The mobile phase in the liquid chromatography conditions of step (2) is: mobile phase A is a mixed aqueous solution of 0.15% formic acid and 5 mM ammonium formate, mobile phase B is acetonitrile, and the flow rate is 0.6 mL / min; the spray temperature in the mass spectrometry conditions is 600°C.

[0100] A methodological investigation was conducted on the determination method, and the results showed that the accuracy, precision, matrix effect, stability, etc. of the determination of lumepirone tosylate in heparin / EDTA anticoagulated SD rat plasma in the range of 1-400 ng / mL met the relevant requirements of methodological validation, and the method had good system adaptability, specificity and reproducibility.

[0101] Comparative Example 1

[0102] Comparative Example 1 is based on Example 1, except that in step (2), the mobile phase A in the liquid chromatography conditions is replaced by a mixed aqueous solution of 0.1% formic acid and 10 mM ammonium formate with a 0.1% formic acid aqueous solution, and the mobile phase B is replaced by 0.1% methanol; except for the above differences, the other parameters are the same as those in Example 1.

[0103] The results are as follows Figure 1 As shown, after changing the mobile phase, the peak shape is poor, while there is no obvious tailing phenomenon under the chromatographic conditions of Example 1.

[0104] Comparative Example 2

[0105] Comparative Example 2 is based on Example 1, and the running time of the liquid chromatography conditions in step (2) is shortened to 4.5 min, specifically:

[0106]

[0107]

[0108] The results are as follows Figure 2As shown, after changing the gradient, the retention time of the main peak keeps moving forward, while the gradient conditions in Example 1 do not have this phenomenon.

[0109] Comparative Example 3

[0110] Comparative Example 3 is based on Example 1, but in step (2), the gradient elution procedure of the liquid chromatography conditions is changed, specifically:

[0111] Time / min A％ B％ 0.01 45 55 0.50 45 55 2.00 25 75 2.01 10 90 250 10 90 300 45 55 550 45 55

[0112] The results are as follows Figure 3 As shown, after changing the gradient, the retention time of the main peak shifts, and the peak separation between the main peak and the degradation product is poor, while the gradient conditions in Example 1 do not have this phenomenon.

[0113] Effect Experiment

[0114] This experiment is a methodological investigation of the method for determining the concentration of lumepirone tosylate in plasma described in Example 1. Specifically, it includes the following contents:

[0115] 1. Preparation of different samples

[0116] Preparation of standard curve linear samples: Take 285 μL of SD rat blank plasma, add 15 μL of different concentrations of lumepirone tosylate standard curve working solution, vortex for 30 s, mix well, and prepare lumepirone tosylate standard curve samples with concentrations of 1.000, 2.000, 10.000, 40.000, 100.000, 300.000 and 400.000 ng / mL, respectively.

[0117] Preparation of quality control samples: Take 285 μL of blank plasma of SD rats, add 15 μL of lumepirone tosylate standard working solution of different concentrations, vortex for 30 s, mix well, and prepare samples of lumepirone tosylate LQC, MQC and HQC with concentrations of 3.000, 30.000, and 300.000 ng / mL, respectively.

[0118] Sample preparation for matrix effect investigation:

[0119] Solution sample (Sol): Take 285 μL of ultrapure water into a centrifuge tube, add 15 μL of lumepirone tosylate quality control solution respectively, to obtain 3, 30 and 300 ng / mL sample solutions, and the rest of the operation is the same as the "Preparation of lumepirone tosylate standard curve working solution" in Example 1.

[0120] Plasma matrix sample (SAE): Take 285 μL of plasma into a centrifuge tube, replace the quality control working solution and internal standard solution with 15 μL of 50% methanol water, add 300 μL of acetonitrile, vortex mix at 1200 rpm for 5 minutes, centrifuge at 4000g for 5 minutes, and take the supernatant.

[0121] Plasma stability test samples: Take an appropriate amount of lumepirone tosylate stock solution into a centrifuge tube, dilute it to a certain concentration with 50% methanol, and then dilute it with blank SD rat plasma to obtain simulated plasma samples with concentrations of 3, 30 and 300 ng / mL, respectively. Place them at room temperature, 4°C and -20°C refrigerator for a long time for stability study.

[0122] 2. Methodological Investigation

[0123] (1) Specificity investigation

[0124] Under the established detection conditions and sample processing conditions, endogenous substances had little interference with the determination. The retention time of lumepirone tosylate was 3.39 min, and the retention time of the internal standard lumepirone ditosylate-d4 was 3.40 min. 285 μL of blank plasma from 6 single-source SD rats was taken respectively, and the sample was injected according to the operation under "Step (1) Preparation of experimental solution" in Example 1. The obtained product ion mass spectra of lumepirone tosylate and its internal standard were as shown in the figure. Figure 4 As shown, A represents the chromatogram of lumepirone tosylate, and B represents the chromatogram of lumepirone ditosylate-d4; a certain concentration of working solution was added to blank plasma, and the same method was used to obtain the blank SD rat plasma. The chromatogram after adding LLQC concentration of lumepirone tosylate and internal standard treatment was as shown in Figure 5 As shown, I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4; the chromatogram of blank SD rat plasma after adding STD8 concentration of lumepirone tosylate and internal standard treatment is shown in Figure 6 As shown, I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4. Plasma samples were taken from SD rats 2 hours after oral administration, and the operation was performed according to the "Step (1) Preparation of Experimental Solution" in Example 1 to obtain the chromatogram of plasma samples from SD rats after oral administration of lumepirone tosylate as shown in FIG. Figure 7 I represents the chromatogram of lumeperone tosylate, and II represents the chromatogram of lumeperone ditosylate-d4; plasma samples of SD rats were taken 2 hours after injection, and the operation under "Step (1) Preparation of experimental solution" in Example 1 was performed to obtain the chromatogram of plasma samples of SD rats after injection of lumeperone tosylate as shown in the figure. Figure 8 Described, I represents the chromatogram of lumepirone tosylate, and II represents the chromatogram of lumepirone ditosylate-d4.

[0125] (2) System adaptability investigation

[0126] Each analytical batch was first investigated for this project. The LLQC sample was injected 6 times, and the RSD of the internal standard peak area was less than 5%, and the difference in internal standard retention time was less than 0.3min. The results showed that the determination method had good specificity.

[0127] Table 6 System suitability investigation results

[0128]

[0129] (3) System Residue Investigation

[0130] After running STD8, the LLQC samples and blank samples were run separately, each with three injections. The results are shown in Table 7.

[0131] Table 7 System residue investigation results

[0132]

[0133] The results showed that lumepirone tosylate and lumepirone ditosylate-d4 in the STD8 sample had no interference with the rat blank sample. Therefore, under this method, the quantitative detection of lumepirone tosylate and lumepirone ditosylate-d4 was not affected by the residual analyte and internal standard.

[0134] Standard curve and linear range

[0135] Take 285 μL of blank SD rat plasma and place it in a centrifuge tube. According to the operation under "Step (1) Preparation of experimental solution" in Example 1, prepare a standard curve plasma linear sample, with the concentration of lumepirone tosylate as the abscissa (X) and the peak area ratio of lumepirone tosylate to the internal standard as the ordinate (Y). Use the weighted least squares method to perform linear regression to obtain the standard curve for the determination of lumepirone tosylate in SD rat plasma as shown in the figure below. Fig. 9 As shown; the standard curve data and correlation coefficient results of three analytical batches of method validation of lumepirone tosylate in SD rat plasma are shown in Table 8.

[0136] Table 8 Statistical table of standard curve data and correlation coefficient results of 3 analytical batches of lumepirone tosylate in rat plasma method validation

[0137]

[0138]

[0139] The results showed that the linear range of the concentration determination method of lumepirone tosylate in SD rat plasma was 1-400 ng / mL, and the linear relationship was good within the linear range.

[0140] (4) Precision and accuracy

[0141] Take 285 μL of blank SD rat plasma, place it in a centrifuge tube, add 15 μL of different concentrations of lumepirone tosylate working reference substance, and the rest of the operation is the same as that in Example 1 under "Step (1) Preparation of experimental solution", prepare LLQC, LQC, MQC and HQC samples with concentrations of 1.000, 3.000, 30.000, and 300.000 ng / mL, respectively, prepare 6 samples in parallel for each concentration, measure 3 batches, and bring into the standard curve to calculate the measured concentration. The accuracy and precision results of the determination method of lumepirone tosylate in SD rat plasma are shown in Table 9.

[0142] Table 9 Statistical table of accuracy and precision of the determination method of lumepirone tosylate in SD rat plasma

[0143]

[0144] The results in Table 9 show that the average accuracy of LLQC is 106.67%-110.00%, the intra-day precision (RSD) is 2.63%-2.87%, and the inter-day precision (RSD) is 1.56%; the accuracy of low, medium and high concentration QC is 98%-105%, the intra-day precision (RSD) is 1.74%-4.85%, and the inter-day precision (RSD) is 0.31%-1.74%, which meets the requirements.

[0145] (5) Investigation of matrix effects

[0146] Low, medium and high concentrations of SAE and Sol samples (each concentration was run in parallel for at least 5 times) were injected and analyzed, and the chromatographic peak area of ​​the SAE sample (ASAE) of the same concentration was compared with the chromatographic peak area of ​​the Sol sample (ASol) to obtain the matrix factor (ME). The matrix factor of the internal standard was calculated in the same way. The statistics of the matrix effect investigation of the determination method of lumepirone tosylate in SD rat plasma are shown in Table 10.

[0147] Table 10 Matrix effect investigation results

[0148]

[0149] From the results in Table 10, it can be seen that the RSD after normalization of ME is in the range of 4.81%-6.56%, and the matrix effect is acceptable.

[0150] (6) Stability investigation

[0151] In this embodiment, the stability mainly examines the stability of drug-containing plasma and the stability of plasma samples after treatment. The stability of plasma samples after treatment refers to the stability of plasma samples during placement in the injector.

[0152] Drug-containing plasma stability

[0153] Freshly prepared simulated plasma samples (3, 30 and 300 ng / mL) were placed at room temperature (0 h, 4 h), placed at -20°C (7 days, 10 days), and frozen and thawed at -20°C (1 time, 3 times), respectively, and then prepared and measured according to the "Step (1) Preparation of Experimental Solution" in Example 1, and the concentration and accuracy were calculated using the standard curve of the same batch. Three points were run in parallel for each concentration. The stability test results are detailed in Tables 11-13.

[0154] Table 11Study on the stability of SD rat plasma at room temperature

[0155]

[0156] Table 12 Study on the stability of SD rat plasma after repeated freezing and thawing

[0157]

[0158] Table 13 Study on the long-term cryopreservation stability of SD rat plasma

[0159]

[0160]

[0161] From the data results in Tables 11-13, it can be seen that drug-containing plasma is stable when placed at room temperature for 4 hours, repeatedly frozen and thawed 3 times, or frozen for 20 days.

[0162] Stability of plasma samples after treatment

[0163] The low, medium and high QC samples were processed and placed in the automatic sampler of the liquid chromatograph for 1 day (about 24 hours) before being re-measured. The standard curve of the current batch was substituted to calculate the measured concentration and accuracy. The statistical results of the automatic sampler stability investigation after processing of lumepirone tosylate in SD rat plasma are shown in Table 14.

[0164] Table 14 Study on the stability of lumepirone tosylate in SD rat plasma after treatment with autosampler

[0165]

[0166] The data results in Table 14 show that the sample is stable after being placed in the autosampler for 1 day after processing.

[0167] (7) Reproducibility study

[0168] A total of 4 plasma samples at 4 time points (30 min, 1 h, 2 h, and 4 h) were selected for reanalysis (accounting for approximately 20% of the total number of samples). The statistical results of the sample reanalysis (ISR) are shown in Table 15.

[0169] Table 15 Statistics of sample reanalysis (ISR) results

[0170]

[0171]

[0172] The data in Table 15 show that the difference between the two analyses of 10% of the samples was less than 10%, which proves that the determination method has good reproducibility and the determination results are stable and reliable.

[0173] In summary, the data of method validation showed that the accuracy, precision, matrix effect, stability, etc. of the determination of lumepirone tosylate in heparin / EDTA anticoagulated SD rat plasma in the range of 1-400 ng / mL met the relevant requirements of method validation, and the method had good system adaptability, specificity and reproducibility. This method can be used for the determination of lumepirone tosylate in heparin sodium and EDTA anticoagulated SD rat plasma.

Claims

1. A method for detecting the concentration of lumepirone tosylate in plasma, characterized in that: The steps include: (1) Prepare experimental solutions; (2) Liquid chromatography-tandem mass spectrometry analysis: Liquid chromatography-tandem mass spectrometry was used to inject the test sample for detection, where: Chromatographic conditions: Mobile phase A is a mixed aqueous solution of 0.05-0.15% formic acid and 5-20 mM ammonium formate, mobile phase B is acetonitrile, flow rate is 0.3-0.6 mL / min, gradient elution is adopted, and the gradient elution program is as follows: Mass spectrometry conditions: electrospray positive ionization mode was used.

2. The detection method according to claim 1, characterized in that: The specific operation steps of preparing the experimental solution described in step (1) are as follows: First, prepare the required lumepirone tosylate standard working solution, internal standard working solution and quality control working solution respectively; Then add the internal standard working solution to the plasma sample, then continue to add acetonitrile to the plasma sample for vortexing, centrifuge and take the supernatant to obtain the test solution; Among them, the volume ratio of methanol and water in the needle washing solution of the liquid phase system is: 40-50:60-50.

3. The detection method according to claim 2, characterized in that: In step (1), the working solution of the standard compound and the quality control working solution are a methanol solution of lumepirone tosylate, and the working solution of the internal standard is a methanol solution of lumepirone ditosylate-d4.

4. The detection method according to claim 2, characterized in that: The concentration of lumepirone ditosylate-d4 in the preparation of the internal standard working solution described in step (1) is 50-100 ng / mL.

5. The detection method according to claim 2, characterized in that: The concentration of lumepirone tosylate in the preparation of the standard curve working solution described in step (1) is 20-8000 ng / mL.

6. The detection method according to claim 1, characterized in that: The chromatographic column packing material is C18, the specification is 3.5μm, 2.1×100mm, the automatic sampler temperature is 0-10℃, and the column temperature is 35-45℃.

7. The detection method according to claim 1, characterized in that: In the electrospray positive ion mode described in step (2), the collision voltages of lumepirone tosylate and its internal standard are 30-40 V, respectively, and lumepirone tosylate and lumepirone ditosylate-d4 are detected simultaneously.

8. The detection method according to claim 1, characterized in that: In the electrospray positive ion mode described in step (2), the quantitative parent ion of lumepirone tosylate is 394.4 m / z, and the quantitative daughter ion is 165.0; the quantitative parent ion of deuterated lumepirone tosylate is 398.4 m / z, and the quantitative daughter ion is 168.

9.

9. The detection method according to claim 1, characterized in that: The mass spectrometry conditions also include: electrospray voltage of 5000-6000 V, curtain gas pressure of 35-45 psi, nebulization pressure of 35-45 psi, and ion source temperature of 500-600° C.

10. Use of the detection method according to any one of claims 1 to 9 in detecting the concentration of lumepirone tosylate in blood.

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