Method for determining mosapride concentration in human plasma by liquid chromatography-tandem mass spectrometry

Abstract

This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the concentration of mosapride in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Mosapride-d5 is used as an internal standard; the stationary phase of the LC column has an average particle size of 1-5 μm and an average pore size of 80-150 Å; the stationary phase comprises C18 chemical bonds; the mobile phase comprises an aqueous solution of 0.1-0.5 wt% formic acid and acetonitrile; the elution program of the LC includes: 0-0.2 min, with an acetonitrile volume content of 20-30% in the mobile phase; 0.2-0.8 min, with an acetonitrile volume content of 70-90% in the mobile phase; after 0.8 min, with an acetonitrile volume content of 20-30% in the mobile phase; the flow rate of the mobile phase is 0.6-1 mL / min; and the elution time is ≤2 min.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the concentration of mosapride in human plasma using liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Mosapride citrate, a potent and selective 5-HT4 receptor agonist, stimulates the release of acetylcholine from nerve endings by exciting 5-HT4 receptors in the myenteric plexus, effectively enhancing gastrointestinal motility. It exhibits no side effects such as extrapyramidal syndrome and has a safety profile superior to cisapride and other similar drugs, making it widely used in the clinical treatment of gastrointestinal diseases such as chronic gastritis and functional dyspepsia. Related substances (impurities) in drugs can affect drug stability, reduce efficacy, and even cause toxic side effects; their control is crucial for ensuring drug quality and medication safety. Impurities in mosapride citrate mainly originate from impure raw materials, incomplete reactions, residual intermediate products, and residual solvents and catalysts. Therefore, establishing efficient and accurate methods for detecting related substances is essential for its quality control. Currently, chromatography is the mainstream technique for detecting related substances in drugs, with high-performance liquid chromatography (HPLC) being widely used due to its high separation efficiency and specificity.

[0003] Chinese invention patent application CN105301118B discloses an HPLC method for the detection of related substances in mosapride citrate, employing an octadecylsilane-bonded silica column and gradient elution with a triethylamine-containing sodium dihydrogen phosphate solution and acetonitrile as the mobile phase. However, this method has a long detection cycle (gradient elution up to 100 min), which is not conducive to the efficient detection of batch samples. Therefore, existing detection methods cannot meet the needs of comprehensive, accurate, and efficient detection of related substances in the research and development and production of mosapride citrate. Developing a detection method with more comprehensive impurity detection, higher sensitivity, and stronger robustness is of significant practical importance. Summary of the Invention

[0004] This invention provides a method for determining mosapride in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS / MS), comprising the following steps: preparing standard curve samples, quality control samples, test samples, blank samples, zero-concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard; performing detection of the standard curve samples, quality control samples, test samples, blank samples, zero-concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard using LC-MS / MS; wherein mosapride-d5 is used as an internal standard; the stationary phase of the chromatographic column used in the LC-MS / MS has an average particle size of 1-5 μm and an average pore size of 8 μm. The liquid chromatography phase is 0-150 Å; the stationary phase comprises C18 chemical bonds; the mobile phase comprises an aqueous solution of 0.1-0.5 wt% formic acid and acetonitrile; the elution program of the liquid chromatography comprises: 20-30% acetonitrile by volume in the mobile phase for 0-0.2 min; 70-90% acetonitrile by volume in the mobile phase for 0.2-0.8 min; and 20-30% acetonitrile by volume in the mobile phase after 0.8 min; the flow rate of the mobile phase is 0.6-1 mL / min; and the elution time is ≤2 min.

[0005] Currently, the detection time for mosapride is relatively long. This invention sets up standard curve samples, quality control samples, blank samples, zero-concentration samples, and samples without internal standards for the upper limit of quantitation. These samples respectively achieve quantitative calibration, detection accuracy quality control, matrix interference screening, internal standard validity verification, and upper limit of quantitation accuracy verification. By avoiding systematic errors and matrix interference errors in the detection process at the sample level, the detection process can be streamlined without additional verification and rework steps, significantly improving the efficiency of the detection process. At the same time, the chromatographic column uses a C18 chemically bonded stationary phase, which can form hydrophobic interactions with the lipophilic groups in the mosapride molecule, achieving rapid preliminary separation of mosapride from strongly polar endogenous impurities in plasma. The mobile phase uses an aqueous solution of 0.1~0.5wt% formic acid + acetonitrile. Formic acid can protonate the nitrogen-containing groups in the mosapride molecule, which not only improves the chromatographic retention of mosapride on the C18 column, but also enhances its ionization efficiency in the subsequent positive ion mode of mass spectrometry. Acetonitrile, as a strong eluent, can quickly break the interaction between mosapride and the stationary phase to achieve rapid elution.

[0006] Optionally, the stationary phase of the column used in the liquid chromatography has an average particle size of 3 μm and an average pore size of 100 Å.

[0007] The 3μm average particle size and 100Å average pore size parameters specified in this invention are highly compatible with the molecular size and physicochemical properties of mosapride, forming a synergistic effect to improve precision: the 100Å average pore size matches the molecular dynamic diameter of mosapride, allowing mosapride molecules to smoothly enter the stationary phase channels and achieve sufficient and uniform interaction with the C18 bonded phase, avoiding retention time fluctuations and peak broadening problems caused by excessively large / small pore sizes; the small 3μm particle size significantly increases the specific surface area of ​​the chromatographic column stationary phase, improving column efficiency and making the mass transfer rate of mosapride in the chromatographic column faster and more uniform, greatly improving the separation degree from impurities in plasma and reducing the interference of impurity peaks on the peak shape of mosapride; the compatible particle size and pore size make the hydrophobic interaction between the C18 bonded phase and mosapride more stable, and the consistency of the chromatographic peak shape and retention time of mosapride is significantly improved during multiple repeated injections, reducing the dispersion of detection results from the separation level, and ultimately achieving a significant improvement in detection precision.

[0008] Optionally, the elution time is ≤1.5 min.

[0009] Optionally, the elution program of the liquid chromatography includes: 0-0.2 min, the volume content of acetonitrile in the mobile phase is 25%; 0.2-0.8 min, the volume content of acetonitrile in the mobile phase is 80%; 0.8-1.5 min, the volume content of acetonitrile in the mobile phase is 25%; and the flow rate of the mobile phase is 0.6-0.8 mL / min.

[0010] The gradient elution program of this invention is tailored to the adsorption-desorption kinetics of mosapride. Combined with an optimal flow rate of 0.6–0.8 mL / min, each elution stage is precisely coordinated with the flow rate, achieving ultra-fast detection while ensuring separation efficiency: 0–0.2 min, acetonitrile volume content 25%: The low-proportion organic mobile phase system allows mosapride to be fully retained on the C18 column, while simultaneously allowing highly polar endogenous impurities in the plasma to rapidly elute with the mobile phase, achieving rapid separation of mosapride from these impurities and laying the foundation for subsequent rapid elution; 0.2–0.8 min, acetonitrile volume content surges to 80%: A high proportion of strong eluent rapidly breaks the hydrophobic interaction between mosapride and the C18 column, allowing mosapride to rapidly desorb from the stationary phase and elute with the mobile phase, avoiding… The extended detection time due to slow elution is addressed in this stage, which is the core elution stage of mosapride, where rapid desorption is achieved to suit its lipophilic properties. The acetonitrile volume content drops to 25% within 0.8–1.5 min, rapidly restoring the column to its initial equilibrium state. This eliminates the need for additional long equilibration steps, preparing the column for the next injection and significantly shortening the interval between single detections. Simultaneously, a flow rate of 0.6–0.8 mL / min strikes a balance between separation efficiency and elution speed. Excessive flow rate reduces the separation of mosapride from weakly polar impurities, while excessively slow flow rate prolongs mass transfer and elution times at each stage. At this flow rate, mosapride achieves optimal mass transfer and elution in the column, synergistically complementing the gradient elution stage switching, ultimately further reducing the single detection time to 1.5 min.

[0011] The concentration gradient range of the standard curve samples is 0.5-100 ng / mL.

[0012] Optionally, the concentration gradient of the standard curve samples is 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL and 100 ng / mL.

[0013] The ionization modes of the mass spectrometer are: electrospray ionization source, positive ion mode, and multiple reaction monitoring.

[0014] The ion source parameters of the mass spectrometer include the following parameters: collision gas 8.00 psi, curtain gas 35.00 psi, first ion source gas 35.00 psi, second ion source gas 60.00 psi, ion source spray voltage 5500.00 V, and ion source temperature 550.00 °C.

[0015] The reaction ion parameters of mosapride in the mass spectrometer were as follows: monitored ion pair 422.3 / 198.2, declustering voltage 105.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 30.00eV, and residence time 200.00msec.

[0016] The reaction ion parameters of mosapride-d5 in the mass spectrometer were as follows: monitored ion pair 427.2 / 203.1, declustering voltage 105.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 31.00eV, and residence time 200.00msec.

[0017] Mosapride is a nitrogen-containing compound that readily undergoes protonation ionization in positive ion mode. All mass spectrometry parameters defined in this invention are adapted to the ionization and fragmentation characteristics of mosapride, and the parameters work synergistically to achieve high sensitivity and high specificity at the ion level, effectively avoiding interference from complex endogenous substances in plasma. The selection of characteristic ion pairs achieves high specificity: after ionization by an electrospray positive ion source, mosapride readily forms a characteristic precursor ion with m / z 422.3, which, after collisional fragmentation, can only produce an m / z of 198. 2. Characteristic daughter ions; the internal standard mosapride-d5, due to isotopic labeling, ionizes to form a parent ion with m / z 427.2, which fragments to produce daughter ions with m / z 203.1. This ion pair is a unique characteristic ion pair for mosapride and its intermediate standard. Other endogenous substances in plasma do not exhibit corresponding ionization or fragmentation behavior and cannot generate a response signal from this ion pair, thus eliminating interference from endogenous substances at the detection source and achieving extremely high detection specificity; the synergistic enhancement of detection sensitivity by the ion source and reaction ion parameters: limited A fixed declustering voltage of 105.00 V efficiently dissociates mosapride precursor ions from solvent clusters, improving the effective concentration and transport efficiency of the precursor ions. Collision energies of 30.00 eV (mosapride) and 31.00 eV (mosapride-d5) represent the optimal fragmentation energies for both, enabling efficient and directional fragmentation of the precursor ions into characteristic daughter ions and enhancing the response intensity of the daughter ions. An inlet and outlet voltage of 10.00 V and a residence time of 200.00 msec further optimize ion transport in the mass spectrometer. Efficiency and signal acquisition intensity: The parameters are synergistically adapted to the ionization and fragmentation characteristics of mosapride, significantly improving the signal intensity of ion detection and achieving high-sensitivity detection of low concentrations of mosapride in plasma. Matching of internal standard and analyte mass spectrometry parameters: The mass spectrometry parameters of mosapride-d5 are highly consistent with those of mosapride, with only the collision energy fine-tuned to match its isotopic fragmentation characteristics. This ensures accurate calibration of mosapride by the internal standard, further reducing interference from matrix effects and injection errors, and improving the accuracy of detection results.

[0018] Beneficial effects 1. This invention, by setting standard curve samples, quality control samples, test samples, blank samples, zero concentration samples, and samples without internal standard at the upper limit of quantitation, combined with specific chromatographic columns and liquid chromatography elution programs, can shorten the detection time of mosapride in human plasma to less than 2 minutes.

[0019] 2. By limiting the average particle size of the stationary phase used in liquid chromatography to 3 μm and the average pore size to 100 Å, the precision of detection can be improved.

[0020] 3. The elution program for liquid chromatography is defined as follows: 0-0.2 min, acetonitrile volume content in the mobile phase is 25%; 0.2-0.8 min, acetonitrile volume content in the mobile phase is 80%; 0.8-1.5 min, acetonitrile volume content in the mobile phase is 25%; and the flow rate of the mobile phase is 0.6-0.8 mL / min. This allows for a one-step reduction of the detection time to 1.5 min.

[0021] 4. By limiting parameters such as ion pairs, collision energy, and declustering voltage in mass spectrometry, detection sensitivity and specificity can be improved, while reducing interference from endogenous substances.

[0022] 5. The detection method of this invention meets the clinical requirements for precision, accuracy, and durability. Attached Figure Description

[0023] Figure 1 The conditions for liquid chromatography in Example 1 are as follows.

[0024] Figure 2 The conditions for mass spectrometry in Example 1 are (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0025] Figure 3 The results of precision, accuracy and recovery tests for Example 1 are shown.

[0026] Figure 4 The results are from the selective test in Example 1.

[0027] Figure 5 The results are from the durability test of Example 1.

[0028] Figure 6 This is the scanning spectrum of mosapride ions in Example 1.

[0029] Figure 7 This is the scanning spectrum of the mosapride-d5 ion in Example 1.

[0030] Figure 8 The standard curve is obtained from the standard curve sample of Example 1. Detailed Implementation

[0031] Example 1 A method for determining the concentration of mosapride in human plasma by liquid chromatography-tandem mass spectrometry comprises the following steps: Mosapride standard (mosapride citrate, purchased from the National Institutes for Food and Drug Control, batch number: 100656-201903) and mosapride-d5 standard (purchased from TLC, batch number: 2079-017A3) were prepared with dimethyl sulfoxide to prepare 1.00 mg / mL mosapride stock solution and mosapride-d5 stock solution, respectively. The above mosapride stock solutions were diluted with 50% (v / v) acetonitrile aqueous solution to prepare standard curve sample working solution and quality control sample working solution. The above mosapride-d5 stock solution was diluted with 50% (v / v) acetonitrile aqueous solution to prepare 100 ng / mL internal standard working solution.

[0032] The concentrations of mosapride in the standard curve solutions were 0.5 ng / mL (lower limit of quantitation, LLOQ), 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, and 100 ng / mL (upper limit of quantitation, ULOQ).

[0033] The mosapride concentrations in the quality control solutions were 0.5 ng / mL (lowest quality control concentration, LLOQQC), 1.5 ng / mL (low quality control concentration, LQC), 7.5 ng / mL (medium quality control concentration, GMQC), 30 ng / mL (medium quality control concentration, MQC), and 75 ng / mL (high quality control concentration, HQC).

[0034] The blank matrix was derived from blank whole blood, individual human plasma, or high-lipid plasma with K2EDTA as an anticoagulant from Kangbo Hospital in Hangzhou, Zhejiang Province, and Liling Traditional Chinese Medicine Hospital in Hunan Province, as well as laboratory-prepared hemolyzed plasma.

[0035] Dilute the quality control solution with the plasma to be tested (quality control solution: 150 ng / mL, dilution factor: 10) to obtain a 15 ng / mL test solution; In a 2.2 mL 96-well polypropylene plate, under room temperature and white light conditions, 50 μL of standard curve solution, 50 μL of quality control solution, and 50 μL of test solution were mixed with 25 μL of internal standard solution to obtain 75 μL of standard curve sample, 75 μL of quality control sample, and 75 μL of test sample, respectively. 50 μL of blank matrix was mixed with 25 μL of acetonitrile (50 wt% concentration, water as solvent) to obtain 75 μL of blank sample. Finally, 50 μL of blank matrix was mixed with 25 μL of internal standard solution to obtain a zero-concentration sample. For the sample, 50 μL of the highest concentration mosapride control solution HQC was mixed with 25 μL of acetonitrile to obtain a 75 μL sample without internal standard and an upper limit of quantitation. All the above samples were diluted with 300 μL of acetonitrile, mixed thoroughly, and centrifuged at 3220 g for 5 min at 4 °C. 100 μL of the supernatant was transferred to another 96-well polypropylene plate, 100 μL of purified water was added, and the plate was vortexed at room temperature for 3 min. The plate was then placed in an autosampler and detected by liquid chromatography-tandem mass spectrometry. Chromatographic acquisition and peak integration were performed using ABSciex Analyst software (version 1.7.2). The standard curve was obtained using regression analysis with the Watson LIMS system (version 7.6.1), with the chromatographic response ratio of the analyte to the internal standard as the ordinate, and weighted averages (W=1 / x) calculated. 2 The least squares method uses linear regression between the concentration (x) of the analyte in plasma and the response ratio (y). The resulting regression equation (y=ax+b) is the standard curve. The drug concentration of the sample is calculated from the fitted standard curve equation. Figure 8 As shown, a=0.015; b=0.000290; R 2 =0.9993.

[0036] The conditions for the liquid chromatography are as follows: Figure 1 As shown, the mass spectrometry conditions are as follows: Figure 2 As shown, the scanning spectrum of mosapride ions is as follows: Figure 6 As shown, the scanning spectrum of mosapride-d5 ions is as follows: Figure 7 As shown.

[0037] Performance testing methods and data 1. Precision and accuracy Intra-batch precision and accuracy Intra-batch precision and accuracy were assessed using quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC), with six replicates for each concentration of quality control sample.

[0038] Precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0039] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0040] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0041] Inter-batch precision and accuracy analysis Inter-batch precision and accuracy were assessed by examining at least three independent validation analysis batches (intra-batch precision and accuracy analysis batches, completed within at least two days) using freshly prepared quality control samples with a blank matrix.

[0042] The quality control samples used to calculate inter-batch precision and accuracy were derived from the quality control samples (LLOQQC, LQC, GMQC, MQC, and HQC) prepared to examine intra-batch precision and accuracy, with six replicates for each concentration level quality control sample per validation analysis batch.

[0043] Acceptance criteria: The deviation of the overall mean concentration of each quality control sample at each concentration level from its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%).

[0044] The overall coefficient of variation for the measured concentration of each quality control sample at each concentration level shall not exceed 15.0% (the coefficient of variation for LLOQQC shall not exceed 20.0%).

[0045] If an analytical batch fails to meet the acceptance criteria, three additional accuracy and precision analytical batches are tested to validate the methodology.

[0046] like Figure 3 As shown, Example 1 has high precision and accuracy.

[0047] 2. Extraction recovery rate Blank matrix from the same batch (or source) as the routine quality control samples (extracted samples, test samples) was used as blank samples. After extraction, the analyte and internal standard were added to the extract of the blank samples to prepare low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC). Six replicates of each concentration were used as reference samples.

[0048] Test samples are routine quality control samples or samples prepared using the same process, including low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC) (6 replicates for each concentration).

[0049] Analyte extraction recovery rate calculation: The peak area of ​​the analyte in each concentration of routine quality control sample (test sample) is divided by the average peak area of ​​the analyte in the reference sample of the same concentration.

[0050] Internal standard extraction recovery rate calculation: The peak area of ​​the internal standard in each routine quality control sample (test sample) is divided by the average peak area of ​​the internal standard in the reference sample.

[0051] Acceptance criteria: The overall coefficient of variation for analyte extraction does not exceed 15.0%; the coefficient of variation for internal standard extraction recovery does not exceed 15.0%.

[0052] If the independent extraction recovery of the analyte or internal standard does not meet the acceptance criteria, the extraction recovery of the analytical method can be evaluated by the extraction recovery after internal standard correction.

[0053] Extraction recovery rate calculation for internal standard correction: The ratio of the peak area of ​​the analyte to its internal standard in each concentration routine quality control sample (test sample) divided by the mean ratio of the peak area of ​​the analyte to its internal standard in the reference sample of the same concentration.

[0054] Acceptance criteria for extraction recovery after internal standard correction: The overall coefficient of variation of extraction recovery after internal standard correction shall not exceed 15.0%.

[0055] like Figure 3 As shown, Example 1 has a high extraction recovery rate.

[0056] 3. Selectivity Matrix selectivity (endogenous interference) Matrix selectivity was evaluated by examining blank biological matrices from at least six different individuals, a high-lipid matrix from one individual, and a hemolyzed matrix from one individual, with measurements of blank samples without internal standards and LLOQ-level samples, respectively.

[0057] High-lipid matrix: via commercially available or simulated hyperlipidemic plasma (1 ± 0.1 mg low-density lipoprotein (LDL) and 3 ± 0.3 mg triglycerides added to 1 mL of blank plasma).

[0058] Hemolysing matrix: Whole blood was frozen at -80°C for at least 30 minutes, thawed, vortexed for at least 1 minute, and then mixed with conventional blank matrix (1:49, v:v) to prepare hemolysing blank matrix.

[0059] Acceptance criteria: The response value of interfering components in the blank matrix at the analyte retention time shall not exceed 20.0% of the analyte response value of the LLOQ sample prepared with the same individual blank matrix; the response value at the internal standard retention time shall not exceed 5.0% of the internal standard response value of the LLOQ sample prepared with the same individual blank matrix.

[0060] If a blank matrix sample from a certain source (batch) does not meet the acceptance criteria, the same method will be used to evaluate the interference of three additional blank matrices from different sources (batches) on the analyte and internal standard. If a high-lipid matrix or hemolyzed matrix does not meet the acceptance criteria, the same method will be used to evaluate one additional high-lipid matrix or hemolyzed matrix from a different source.

[0061] Interference of the analyte to the internal standard Three samples containing only a single analyte and without an internal standard were prepared, processed, and analyzed in parallel to determine the upper limit of quantitation concentration.

[0062] Acceptance criteria: The average peak area of ​​the internal standard at the retention time of a sample containing only a single analyte should not exceed 5.0% of the average peak area of ​​the internal standard in the samples meeting the lower limit of quantitation of the standard curve in the same analytical batch.

[0063] Interference of internal standard with analyte Three samples containing only a single internal standard and without the analyte were prepared, processed, and analyzed in parallel. The concentration of the internal standard was the actual concentration used.

[0064] Acceptance criteria: The average peak area at the retention time of the analyte should not exceed 20.0% of the average peak area of ​​the analyte in the standard curve samples that meet the acceptance criteria in the same analytical batch.

[0065] Selectivity of analysis batch The selectivity of the analytical batch was evaluated using the first blank matrix sample and the first blank quality control sample of the analytical batch.

[0066] Acceptance criteria: The peak area of ​​the analyte in the detection channel of both blank samples does not exceed 20.0% of the average peak area of ​​the analyte in the samples with the limit of quantitation of the effective standard curve; the peak area of ​​the internal standard in the detection channel of the first blank matrix sample does not exceed 5.0% of the average peak area of ​​the internal standard in the samples with the limit of quantitation of the effective standard curve in the same analytical batch.

[0067] like Figure 4 As shown, Example 1 has a higher selectivity.

[0068] 4. Durability The precision was assessed by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, with samples injected by different analysts (Analyst A and Analyst B) or on different instruments (LC-MS / MS, two sets of instruments of the same model with the same parameters). The accuracy was assessed by calculating the deviation (Diff%) between the mean measured concentration of the quality control samples at each concentration level and their theoretical concentration.

[0069] Acceptance criteria: The deviation between the measured mean concentration of each concentration level quality control sample and its theoretical concentration should be within ±15.0% (for LLOQQC, the deviation should be within ±20.0%), and the coefficient of variation should not exceed 15.0% (for LLOQQC, the coefficient of variation should not exceed 20.0%).

[0070] For precision and accuracy analysis batches, at least 2 / 3 of the quality control samples should have a concentration deviation of no more than ±15.0% from their theoretical concentration (LLOQQC no more than ±20.0%), and at least 1 / 2 of the samples at the same concentration level should meet the above standards.

[0071] The mass spectrometer model is TripleQuad5500+; the chromatography system includes a liquid phase pump (LC-30AD), a controller (CBM-20A), a degasser (DGU-20A5R(C), a column oven (CTO-20A), and an injection system (SIL-30ACMP).

[0072] like Figure 5 As shown, the durability test results of Example 1 meet the requirements.

Claims

1. A method for determining mosapride in human plasma by liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: Prepare standard curve samples, quality control samples, test samples, blank samples, zero concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard; perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) on the standard curve samples, quality control samples, test samples, blank samples, zero concentration samples, and samples with upper limit of quantitation (UPQ) without internal standard. Mosapride-d5 is used as an internal standard; the stationary phase of the column used in the liquid chromatography has an average particle size of 1-5 μm and an average pore size of 80-150 Å; the stationary phase comprises C18 chemical bonds; the mobile phase of the liquid chromatography comprises an aqueous solution of 0.1-0.5 wt% formic acid and acetonitrile; the elution program of the liquid chromatography includes: for 0-0.2 min, the volume content of acetonitrile in the mobile phase is 20-30%; During the first 0.2-0.8 min, the volume content of acetonitrile in the mobile phase is 70-90%; after 0.8 min, the volume content of acetonitrile in the mobile phase is 20-30%; the flow rate of the mobile phase is 0.6-1 mL / min; and the elution time is ≤2 min.

2. The method according to claim 1, characterized in that, The stationary phase of the column used in the liquid chromatography has an average particle size of 3 μm and an average pore size of 100 Å.

3. The method according to claim 2, characterized in that, The elution time is ≤1.5 min.

4. The method according to claim 3, characterized in that, The elution program for the liquid chromatography includes: 0-0.2 min, the volume content of acetonitrile in the mobile phase is 25%; 0.2-0.8 min, the volume content of acetonitrile in the mobile phase is 80%; 0.8-1.5 min, the volume content of acetonitrile in the mobile phase is 25%; and the flow rate of the mobile phase is 0.6-0.8 mL / min.

5. The method according to claim 1 or 4, characterized in that, The concentration gradient range of the standard curve samples is 0.5-100 ng / mL.

6. The method according to claim 5, characterized in that, The concentration gradients of the standard curve samples were 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, and 100 ng / mL.

7. The method according to claim 1, characterized in that, The ionization modes of the mass spectrometer are: electrospray ionization source, positive ion mode, and multiple reaction monitoring.

8. The method according to claim 1, characterized in that, The ion source parameters of the mass spectrometer include the following parameters: Collision gas 8.00psi, curtain gas 35.00psi, first ion source gas 35.00psi, second ion source gas 60.00psi, ion source spray voltage 5500.00V, ion source temperature 550.00℃.

9. The method according to claim 1, characterized in that, The reaction ion parameters of mosapride in the mass spectrometer were as follows: monitored ion pair 422.3 / 198.2, declustering voltage 105.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 30.00eV, and residence time 200.00msec.

10. The method according to claim 1, characterized in that, The reaction ion parameters of mosapride-d5 in the mass spectrometer were as follows: monitored ion pair 427.2 / 203.1, declustering voltage 105.00V, inlet voltage 10.00V, outlet voltage 10.00V, collision energy 31.00eV, and residence time 200.00msec.

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