Method for determining NBED concentration in plasma by high performance liquid chromatography-mass spectrometry

Abstract

The invention provides a method for determining NBED concentration in plasma by high performance liquid chromatography-mass spectrometry, which comprises the following steps: collecting a whole blood sample containing an NBED drug, centrifuging the whole blood sample at 6000 rpm for 5 min to obtain plasma, adding an internal standard working solution into the plasma, uniformly mixing, centrifuging at 4 DEG C at 14000 rpm for 10 min, taking supernate, blow-drying at 60 DEG C by using N2, re-dissolving by using a mobile phase with an initial proportion, and determining the concentration of NBED in the plasma. After vortex uniform mixing, centrifuging to obtain a sample solution containing the NBED medicine; injecting the sample solution containing the NBED into a high performance liquid chromatography-tandem mass spectrometer to obtain a mass spectrum; and carrying out quantitative analysis on the NBED by adopting an internal standard method according to the mass spectrum. According to the method, the concentration of the NBED in the plasma is determined by a liquid chromatography-mass spectrometry method, so that when the NBED is detected, the sensitivity is high, the analysis speed is high, a basis is provided for preclinical safety research and clinical pharmacokinetic research of the NBED, and the method can be used for supporting non-clinical and clinical research work of brand-new drug molecules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for determining the concentration of NBED in plasma using high performance liquid chromatography-mass spectrometry. Background Technology

[0002] With the rapid development of industrialization and urbanization, my country's heavy metal pollution problem is becoming increasingly serious. If heavy metals enter the human body, they may accumulate and threaten human health. Among the treatment options, excretion-promoting therapy is the main approach.

[0003] To date, the FDA has only approved three drugs—Ca / Na-DTPA, Prussian blue, and potassium iodide—for treating radionuclide poisoning in response to radionuclide poisoning. Others are still in the stages of structural optimization, efficacy confirmation, and preclinical research. Currently, the clinical treatment for uranium contamination involves intravenous injection of 1.4% sodium bicarbonate until the urine pH reaches 8.0-9.05, continuing for three days. However, low doses are not very effective, and excessive clinical dosage can easily cause acid-base imbalances and electrolyte disturbances.

[0004] NBED is a heavy metal excretion-promoting drug with independent intellectual property rights developed by the China Institute of Radiation Protection (patent number: CN112358422B). Its chemical structure is as follows: Its chemical name is N1,N2-bis(2,3-dihydroxy-4,6-disulfonic acid benzyl)ethylenediamine, and its molecular formula is C 16 H 16 N2Na4O 16 S4, preclinical studies have shown that NBED is a low-toxicity compound that can form five-membered ring chelates with actinides through hydrogen bonds, promoting the excretion of actinides in the body. In addition, NBED also has a good excretion-promoting effect on heavy metals such as copper and lead, and is very promising to show a good heavy metal excretion-promoting effect in clinical studies.

[0005] In therapeutic drug monitoring and clinical research, it is necessary to measure the blood drug concentration of subjects or patients to study their pharmacokinetic behavior. Due to the innovative structure of NBED, there are currently no biological sample detection and analysis methods for NBED both domestically and internationally. Therefore, there is an urgent need to develop a rapid, specific, highly sensitive and stable quantitative analysis method to quantitatively measure NBED for accurate analysis of large batches of samples in clinical research of NBED-related drugs.

[0006] The above problems urgently need to be addressed. Summary of the Invention

[0007] The method for determining the concentration of NBED in plasma using high-performance liquid chromatography-mass spectrometry aims to solve the technical problems existing in the prior art.

[0008] The present invention adopts the following technical solution: This invention provides a method for determining the concentration of NBED in plasma using high performance liquid chromatography-mass spectrometry, comprising the following steps: Whole blood samples containing NBED were collected. The whole blood samples were centrifuged at 6000 rpm for 5 min to obtain plasma. Internal standard working solution was added to the plasma and mixed well. The plasma was then centrifuged at 14000 rpm for 10 min at 4 °C. The supernatant was dried with N2 at 60 °C. The plasma was reconstituted with the mobile phase of the initial ratio. After vortexing and centrifugation, the sample solution containing NBED was obtained. The sample solution containing NBED was injected into a high-performance liquid chromatography-tandem mass spectrometry instrument for chromatographic and mass spectrometric detection to obtain a mass spectrum; Quantitative analysis of NBED was performed using the internal standard method based on the mass spectrum.

[0009] In one possible implementation, the volume ratio of plasma to internal standard working solution is 1:(2-5).

[0010] In one possible implementation, the internal standard working solution is a methanol-acetonitrile mixture with an added internal standard, wherein the concentration of the internal standard in the internal standard working solution is 8.00 ng / mL.

[0011] In one possible implementation, the internal standard is any one of lamivudine, tolbutamide, and lidocaine; In one possible implementation, the volume ratio of methanol to acetonitrile is 1:(1-3).

[0012] In one possible implementation, the chromatographic detection conditions are as follows: mobile phase A is a mixed solution of ammonia and water, and mobile phase B is a mixed solution of acetonitrile or methanol and ammonia, with gradient elution. The gradient elution program is as follows: within 0-3 min, 10% mobile phase B gradually changes to 20% mobile phase B; within 3-8 min, 20% mobile phase B gradually changes to 90% mobile phase B; within 8-10 min, the proportion of mobile phase B is maintained at 90%; within 10.1-12 min, the proportion of mobile phase B is maintained at 10%; column temperature: 40℃; sample loading volume: 10 μl; flow rate: 0.4 ml / min; injector temperature: 4℃.

[0013] In one possible implementation, the chromatographic column is any one of Ultimate® AQ-C18, ACQUITY UPLC BEHAmide, Ultimate® HILIC Amide, ACQUITY BEH C18, or Zorbax Eclipse Plus C18 (T3).

[0014] In one possible implementation, the initial ratio of mobile phase A to mobile phase B is 9:1, with the volume fraction of ammonia in mobile phase A being 0.01-0.05% and the volume fraction of ammonia in mobile phase B being 0.01-0.05%.

[0015] In one possible implementation, the mass spectrometry detection conditions are as follows: electrospray ionization (ESI) source, negative ion detection method, multiple reaction monitoring (MRM) mode, ion injection voltage -4500 V; ion source temperature 400-500 °C; gas 1 (GS1) 65 psi; gas 2 (GS2) 50 psi; curtain gas (Cur) 40 psi; collision gas (CAD) 9 psi; residence time 50 ms; mass spectrometry acquisition time 10.00 min; Q1 / Q3 resolution Unit / Unit.

[0016] In one possible implementation, the quantitative ion pair of the NBED has an m / z of 689.4→609.6, a collision energy (CE) of -13 eV, and a declustering voltage (DP) of -50 V; the analytical ion pair of the internal standard has an m / z of 228.0→133.9, a collision energy (CE) of -20 eV, and a declustering voltage (DP) of -57 V.

[0017] The technical solution adopted in this invention can achieve the following beneficial effects: This invention uses liquid chromatography-mass spectrometry to determine the concentration of NBED in plasma, which provides high sensitivity and fast analysis speed for NBED detection, providing a basis for preclinical safety studies and clinical pharmacokinetic studies of NBED, and can be used to support non-clinical and clinical research on this novel drug molecule. Attached Figure Description

[0018] Figure 1 The mass spectrum of the double-blank plasma sample obtained in Example 1 of the present invention; Figure 2 The mass spectrum of the sample without internal standard (ULOQ Without IS) obtained in Example 1 of this invention; Figure 3 This is a representative physical spectrum of the zero-concentration sample obtained in Example 1 of the present invention; Figure 4 The graph shows the blood drug concentration and time curves of Beagle dogs after gavage administration of NBED at a dose of 50 mg / kg, as provided in Example 1 of this invention. Figure 5 This is a mass spectrum provided in Embodiment 1 of the present invention; Figure 6 This is the mass spectrum provided in Embodiment 3 of the present invention; Figure 7 This is the mass spectrum provided in Embodiment 6 of the present invention; Figure 8 This is the mass spectrum provided in Embodiment 7 of the present invention; Figure 9 This is the mass spectrum provided in Embodiment 8 of the present invention; Figure 10 This is the mass spectrum provided in Embodiment 9 of the present invention; Figure 11 This is a mass spectrum provided in Embodiment 10 of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0020] Example 1 1. Preparation of solutions and samples Preparation of the chelating agent NBED (N1,N2-bis(2,3-dihydroxy-4,6-disulfonic acid benzyl)ethylenediamine): In a 200 ml round-bottom flask, 1.9 g (10 mmol) of catechol-3-sulfonic acid was dissolved in 40 ml of methanol at room temperature. Then, 2 ml (30 mmol) of ethylenediamine was added, and the mixture was stirred for 40 minutes. Then, 1.5 ml of formaldehyde aqueous solution (40 mmol of formaldehyde) was slowly added, and the mixture was stirred at room temperature for another 1.2 hours. The reaction system was slowly heated to reflux and refluxed for 15 hours. The reaction was stopped, and the mixture was allowed to cool naturally to room temperature. The reaction system was concentrated until solvent-free, and 50 ml of ethyl acetate and 50 ml of water were added. After mixing and allowing the mixture to stand for separation, the upper ethyl acetate layer was collected. 10 g of anhydrous sodium sulfate was added, and the mixture was concentrated and dried to obtain the intermediate N1,N2-bis(2,3-dihydroxy-4-sulfonic acid benzyl)ethylenediamine, with a yield of 37%. The intermediate N1,N2-bis(2,3-dihydroxy-4-sulfonylbenzyl)ethylenediamine was added in batches to 96% (m / m) concentrated sulfuric acid (mass ratio of 1:10), stirred at 50°C for 12 hours, extracted with 250 ml of diethyl ether for 5-15 min, the ether layer was collected, evaporated and dried to obtain NBED with a yield of 25% and a purity of 95%.

[0021] Standard series samples: Weigh the prepared NBED, dissolve and dilute with pure water to prepare an NBED stock solution with a concentration of approximately 1.0 mg / mL. Pipette 250 μL of the NBED stock solution and serially dilute it with plasma precipitation supernatant (heparin-anticoagulated whole blood and methanol-acetonitrile mixture at a ratio of 1:3, vortex for at least 3 min, and centrifuge at 4 ℃ and 14000 rpm for 10 min to obtain plasma precipitation supernatant) to prepare standard curve working solutions with concentrations of 125, 250, 500, 1250, 2500, 5000, 12500, and 25000 ng / mL. Take different concentrations of standard curve working solutions and dilute them with blank plasma to prepare a standard series of samples with concentrations of 5.00, 10.0, 20.0, 50.0, 100, 200, 500, and 1000 ng / mL.

[0022] Quality control (QC) samples: Take 200 μL of NBED stock solution with a concentration of 1.0 mg / mL, and further dilute it with plasma precipitation supernatant (whole blood and methanol-acetonitrile mixed solution with a volume ratio of 1:3) to prepare four quality control working solutions with concentrations of 125, 375, 3750, and 20000 ng / mL.

[0023] Four quality control working solutions with concentrations of 125, 375, 3750, and 20000 ng / mL were taken and diluted with blank plasma to obtain quality control plasma sample solutions with a lower limit of quantitation of 5.00 ng / mL, a low quality control (LQC) concentration of 15.0 ng / mL, a medium quality control (MQC) concentration of 150 ng / mL, and a high quality control (HQC) concentration of 800 ng / mL.

[0024] Internal standard working solution: Weigh lamivudine reference standard (purchased from RXA, purity 99.70%, batch number 21-L0232-07, stored in a light-proof, sealed container at 2~8 ℃), dissolve and dilute with methanol to prepare an internal standard stock solution with a concentration of 10 μg / mL; pipette the 10 μg / mL internal standard stock solution and dilute with a methanol-acetonitrile mixed solution (where the volume ratio of methanol to acetonitrile is 1:1) to obtain an internal standard working solution with a lamivudine concentration of 8.00 ng / mL.

[0025] 2. Pretreatment of plasma samples Whole blood samples were collected from Beagle dogs and anticoagulated with EDTA-K2. The whole blood samples were centrifuged at 6000 rpm for 5 min in a high-speed refrigerated centrifuge (Eppendorf, model: Centrifuge 5424R). 250 μl of plasma was taken and 750 μl of a methanol-acetonitrile mixture containing 8.00 ng / mL lamivudine was added. The mixture was mixed for at least 10 s to precipitate proteins. The mixture was centrifuged at 14000 rpm for 10 min at 4 °C. 180 μl of the supernatant was placed in a nitrogen blower (Shanghai Xiwei Medical Technology Co., Ltd., model: Arica V96) and dried under N2 at 60 °C. The mixture was reconstituted with 80 μl of the initial mobile phase and vortexed for at least 10 s in a Vortex (Vortex QL-901). The mixture was then centrifuged at 14000 rpm for 10 min at 4 °C.

[0026] 3. Chromatographic and mass spectrometric conditions Each sample was analyzed using a Triple Quad 5500 (AB SCIEX) liquid chromatography-tandem mass spectrometry system and an LC-20AD high-performance liquid chromatograph (Shimadzu Corporation, Japan). The chromatographic conditions were: Ultimate® AQ-C18 column (2.1 × 50 mm, 3 μm), column temperature 40 °C, injection volume 10 µL, and injector temperature 4 °C. The temperature was ℃. Mobile phase A was 0.05% ammonia-water, and mobile phase B was 0.05% ammonia-acetonitrile. The gradient elution program was as follows: 0.00 min, 90% A, 10% B, flow rate 0.4 mL / min; 3.00 min, 80% A, 20% B, flow rate 0.4 mL / min; 8.00 min, 10% A, 90% B, flow rate 0.4 mL / min; 10.00 min, 10% A, 90% B, flow rate 0.4 mL / min; 10.01 min, 90% A, 10% B, flow rate 0.4 mL / min; 12.00 min, 90% A, 10% B, flow rate 0.4 mL / min. The autosampler was cleaned by washing the needle before and after sample aspiration, 2000 µL each time, immersing for 10 seconds, with a washing solution of methanol:water (1:1, v / v).

[0027] Mass spectrometry detection conditions: Electrospray ionization (ESI) source was used, detection mode was negative ion mode; scanning mode was multiple ion reaction monitoring (MRM); quantitative ion pair of NBED: 689.4→609.6; quantitative ion of lamivudine (internal standard): m / z 228.0→133.9; ion ejection voltage: -4500 V; ion source temperature: 500 ℃; gas 1 (GS1): 65 psi; gas 2 (GS2): 50 psi; curtain gas (Cur): 40 psi; collision gas (CAD): 9 psi; ion source: ESI; mass spectrometry acquisition time: 10.00 min; Q1 / Q3 resolution: Unit / Unit.

[0028] Table 1. Mass spectrometry detection conditions for analytes and internal standards.

[0029] 4. Selective investigation By preparing different types of samples—double blank plasma (DB) samples, upper limit of quantitation (ULOQ) samples without internal standards (ULOQ Without IS) and zero concentration samples—and comparing the detection results, the selectivity of the method was evaluated based on specific acceptance criteria to determine the method's performance in excluding matrix interference.

[0030] Plasma blank matrix was collected from six different individuals of Beagle dogs. One double-blank (DB) plasma sample and one zero-concentration (Zero) sample were prepared from each source matrix, for a total of 12 samples. In addition, plasma blank matrix was collected from three other different individuals of Beagle dogs. One upper limit of quantitation (ULOQ) sample without internal standard (IS) was prepared from each source blank plasma sample, for a total of 3 samples.

[0031] Acceptance Criteria: In blank samples, the peak area of ​​interfering peaks at the retention time of the analyte should not exceed 20.0% of the average peak area of ​​the analyte in LLOQ samples; at the internal standard retention time, the peak area of ​​interfering peaks should not exceed 5.0% of the average internal standard area in all standard curve samples and quality control samples. In an individual normal blank matrix, at least 80% of the normal blank matrix should meet the above criteria. In upper limit of quantitation (ULOQ Without IS) samples, the peak area of ​​the internal standard retention time should not exceed 5.0% of the average internal standard area in all standard curve samples and quality control samples. In zero concentration (Zero) samples, the peak area of ​​the analyte retention time should not exceed 20.0% of the average peak area of ​​LLOQ samples in the standard curve that meets the acceptance criteria. In an individual normal blank matrix, at least 80% of the normal blank matrix should meet the above criteria.

[0032] Fifteen samples were placed in a liquid chromatography-tandem mass spectrometry system (Triple Quad 5500 (AB SCIEX)) and an LC-20AD high performance liquid chromatograph (Shimadzu Corporation, Japan) for detection. The peak area data of the analyte and internal standard at their respective retention times were recorded. Table 2 is a table of selectivity evaluation of the internal standard. Mass chromatograms of six double blank plasma samples (DB) from different sources were obtained. Figure 1 The mass spectrum of the double-blank plasma sample obtained in Example 1 of the present invention; Figure 2 The mass spectrum of the sample without internal standard (ULOQ Without IS) obtained in Example 1 of this invention; Figure 3 This is a representative mass spectra of the zero-concentration sample obtained in Example 1 of the present invention; as shown... Figure 1 Based on Table 2, no endogenous interference peaks from internal standards were found in the plasma of Beagle dogs. Figure 2 Table 2 shows that NBED does not interfere with the accurate determination of the internal standard lamivudine. Figure 3 Table 2 shows that the internal standard lamivudine does not interfere with the accurate determination of NBED. Therefore, under the experimental conditions, the plasma matrix does not interfere with the separation and determination of the analyte and the internal standard. NBED and lamivudine do not affect each other's detection results, and the selectivity meets the requirements.

[0033] Table 2 Selective Examination Table

[0034] 5. Standard curve and detection limit investigation Two sets of standard samples with eight concentration points (5.00, 10.0, 20.0, 50.0, 100, 200, 500, 1000 ng / mL) were prepared and placed at the beginning and end of each analytical batch. Each analytical batch included one double blank sample (DB) and one zero-concentration sample (Zero) to ensure that the blank matrix and added reagents did not interfere with the detection of the compounds. Measurements were performed according to the analytical method, and the signal response and noise response values ​​at each concentration were recorded.

[0035] Calculate the signal-to-noise ratio (S / N = analyte signal response value / noise response value) at different concentrations.

[0036] Plot a signal-to-noise ratio (SNR) versus concentration curve with concentration as the x-axis and the ratio of NBED to lamivudine mass spectrometry peak areas as the y-axis. Use a weighted least squares method (e.g., 1 / X). 2A weighted linear regression was performed, and the typical linear regression equation for the standard curve of the NBED to be tested was: y = -0.0361 + 0.0215x. The lowest point of the linear range of the standard curve was taken as the lower limit of quantitation. The lower limit of quantitation was found when the concentration of the NBED to be tested in the sample was 5 ng / mL, the signal-to-noise ratio was 17.8, and the concentration corresponding to a signal-to-noise ratio of 3:1 was identified as the limit of detection.

[0037] 6. Precision and accuracy assessment The specific data on the precision and accuracy of detecting the concentration of NBED in different batches of plasma are shown in Table 3. The intra-batch precision of each concentration level is ≤15%, and the lower limit of quantitation is ≤20%. The intra-batch accuracy deviation of each concentration level does not exceed ±15% of the theoretical value, and the lower limit of quantitation does not exceed ±20% of the theoretical value, all of which meet the acceptance criteria. This indicates that the method is reliable and reproducible for the determination of the compound NBED.

[0038] Table 3 Precision and accuracy data (n=6)

[0039] 7. Matrix effect Interference from endogenous substances or other components in biological samples (such as plasma, urine, etc.) with analyte determination may lead to deviations in analytical results. The matrix effect is assessed by comparing the response value of the analyte in pure solution with the response value of the same amount of analyte added to a blank matrix after sample extraction.

[0040] Quality control (QC) samples were prepared by adding quality control working solution to six batches of blank matrix from different donors. The results were evaluated by analyzing at least three replicates of LQC and HQC samples, each replicate prepared using at least six different sources / batches of matrix. The average accuracy deviations (%) of NBED in LQC and HQC were -1.5% to 11.5% and -0.8% to 9.7%, respectively, and the precisions were 0.9% to 5.0% and 0.8% to 3.8%, respectively. These meet acceptable standards (for each source / batch of matrix, the average accuracy deviation should be within ±15%, and the precision should not exceed 15%), indicating that when using the protein precipitation method of this invention for plasma sample pretreatment, the plasma matrix effect does not interfere with the accuracy of analyte concentration or content detection.

[0041] 8. Stability test Room temperature stability study: Plasma samples of each LQC (15.0 ng / mL) and HQC (800 ng / mL) were stored at room temperature for 6 hours before being sampled and analyzed.

[0042] -60 to -90℃ 4-cycle freeze-thaw: The stability of LQC (15.0 ng / mL) and HQC (800 ng / mL) quality control samples after 4 freeze-thaw cycles was investigated at -60℃ to -90℃. Each sample was frozen for at least 12 hours (at least 24 hours for the first freeze), thawed naturally at room temperature, and analyzed after at least four freeze-thaw cycles (n≥3 for each concentration level).

[0043] Whole blood stability: Before preparing whole blood stability samples, heparin sodium anticoagulated whole blood should be incubated at 37 °C for 10–15 min. Add NBED solution to whole blood to prepare low-concentration and high-concentration whole blood samples containing 15.0 ng / mL and 800 ng / mL NBED (the proportion of organic solvent should be as low as possible, not exceeding 5% of the total volume), and gently invert to mix to avoid hemolysis. Incubate the prepared whole blood stability samples at 37 °C for 10 min to ensure that the distribution of the analyte in plasma and blood cells reaches equilibrium. Then gently invert to mix and quickly divide into two parts (to avoid uneven concentrations caused by blood cell sedimentation). Centrifuge one part immediately to collect plasma, and then refrigerate as the 0-hour sample. Store the other part of the whole blood stability sample on ice for at least 4 hours before centrifuging to collect plasma as the whole blood stability sample. Take both plasma samples from the refrigerator at the same time, and extract and analyze them together with the standard curve sample, quality control sample, and blank sample, with n≥3 for each low-concentration and high-concentration level.

[0044] After treatment, the samples were refrigerated at 2–8°C for 24 hours: After successful injection of an analytical batch, LQC (15.0 ng / mL) and HQC (800 ng / mL) quality control samples (n≥3) used for stability assessment were placed in a refrigerator for 24 hours before injection. Freshly prepared standard curve samples were used for quantitative analysis during injection to assess the stability of the treated samples. Stability time interval: The time when the sample was placed into the autosampler was designated as time 0. The duration of stability of the treated samples was calculated using the time difference between time 0 and the completion of injection of the last stable sample in the autosampler; that is, the treated samples were stable within this time period.

[0045] The results of the stability test of the NBED in plasma samples (n=3) are shown in Table 4. The results show that the NBED was stable under the test conditions.

[0046] Table 4. Stability test data (n=3)

[0047] 9. In vivo pharmacokinetic studies Six healthy, general-grade Beagleeagle dogs were administered NBED (non-nitrogenous epidural) via gavage at a dose of 50 mg / kg. Whole blood samples were collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 4 h, 8 h, 12 h, and 24 h. After EDTA-K2 anticoagulation, the whole blood samples were centrifuged at 6000 rpm for 5 min in an Eppendorf Centrifuge 5424R high-speed refrigerated centrifuge. 250 μl of plasma was collected and mixed with 750 μl of a methanol-acetonitrile mixture containing 8.00 ng / mL lamivudine for at least 10 s to precipitate proteins. The mixture was then centrifuged at 14000 rpm for 10 min at 4 °C. 180 μl of the supernatant was collected and dried under nitrogen (N2) in a nitrogen blower (Shanghai Xiwei Medical Technology Co., Ltd., model: Arica V96) at 60 °C. The supernatant was reconstituted with 80 μl of the initial mobile phase and then vortexed. Vortex mix (QL-901) for at least 10 seconds, then centrifuge at 14,000 rpm for 10 minutes at 4°C.

[0048] Each sample was analyzed using a Triple Quad 5500 (AB SCIEX) liquid chromatography-tandem mass spectrometry system and an LC-20AD high-performance liquid chromatograph (Shimadzu Corporation, Japan). Figure 4 The graph shows the blood drug concentration and time curves of Beagle dogs after being administered NBED by gavage at a dose of 50 mg / kg, as provided in Embodiment 1 of the present invention. Figure 5 This is a mass spectrum provided in Embodiment 1 of the present invention; as shown below. Figure 4 As shown, this detection method has a high sensitivity that can fully depict pharmacokinetic characteristics, a wide linear range (5.00~1000 ng / mL), and the selection of the linear range is close to the concentration level of the actual sample. The established method has been successfully applied to detect the concentration of NBED in plasma. It also has a wide range of applications, and can be used for the detection of drug concentrations in the plasma of humans, rats, mice, beagles, Bama miniature pigs, and other animals.

[0049] Example 2 The implementation method of Example 2 is the same as that of Example 1, except that: The chromatographic column used was an ACQUITY UPLC BEH Amide (2.1*100mm, 1.7μm).

[0050] Example 3 The implementation method of Example 3 is the same as that of Example 1, except that: The chromatographic column used was Ultimate® HILIC Amide (2.1×100mm, 3μm).

[0051] Example 4 The implementation method of Example 4 is the same as that of Example 1, except that: The chromatographic column used was an ACQUITY BEH C18 (3.0×50mm, 1.7μm).

[0052] Example 5 The implementation method of Example 5 is the same as that of Example 1, except that: The chromatographic column used was a Zorbax Eclipse Plus C18 (T3) (4.6 × 50 mm, 1.8 μm).

[0053] Example 6 The implementation method of Example 6 is the same as that of Example 1, except that: Mobile phase A is 0.05% ammonia-water, and mobile phase B is 0.05% ammonia-methanol.

[0054] Example 7 The implementation method of Example 7 is the same as that of Example 1, except that: The chromatographic conditions for isocratic elution were as follows: Ultimate® AQ-C18 column (2.1 × 50 mm, 3 μm), column temperature 40 ℃, injection volume 10 µL, injector temperature 4 ℃, mobile phase A was 0.05% ammonia-water, mobile phase B was 0.05% ammonia-acetonitrile, 80% A, 20% B. The autosampler was cleaned by washing the needle before and after sample aspiration, 2000 µL each time, immersing for 10 seconds, with a washing buffer of methanol:water (1:1, v / v).

[0055] Example 8 The implementation method of Example 8 is the same as that of Example 1, except that: The mass spectrometry conditions were as follows: electrospray ionization (ESI) source, negative ion mode detection, multiple ion reaction monitoring (MRM) scanning mode, ion ejection voltage -4000 V, ion source temperature 400℃, gas 1 (GS1) 65 psi, gas 2 (GS2) 50 psi, curtain gas (Cur) 30 psi, collision gas 7 p.si, ESI source, mass spectrometry acquisition time 10.00 min, Q1 / Q3 resolution Unit / Unit.

[0056] Example 9 The implementation method of Example 9 is the same as that of Example 1, except that: The internal standard was tolbutamide.

[0057] Example 10 The implementation method of Example 10 is the same as that of Example 1, except that: The internal standard was lidocaine.

[0058] Example 11 A 1:1 volume ratio of methanol to acetonitrile was used as a protein precipitant to prepare plasma samples.

[0059] Example 12 The volume ratio of methanol to acetonitrile is 1:3.

[0060] Examples 1-6 used different chromatographic columns for detection. Figure 6 The mass spectrum provided in Example 3; by Figure 6 It can be seen that the chromatographic peak broadening indicates poor sensitivity. The results show that the Ultimate AQ-C18 (2.1 × 50 mm, 3 μm) chromatographic column of Example 1 has good peak shape, appropriate retention time, and good detection effect.

[0061] Example 6 uses methanol as the organic phase for detection. Figure 7 The mass spectrum provided in Embodiment 6 of the present invention is derived from... Figure 7 It is known that when methanol is used as the organic phase for detection, the peak shape is more pronounced than that of acetonitrile. Example 1 provides the optimal peak shape, ensuring that the chromatographic peak is sharp and symmetrical, thereby improving the sensitivity and accuracy of the analysis.

[0062] Example 7 uses isocratic elution chromatographic conditions. Figure 8 The mass spectrum provided in Example 7 of this invention is shown below. By comparing the chromatographic conditions of isocratic and gradient elution, it was found that although isocratic elution is simple to operate, it cannot guarantee that interfering components in the plasma matrix cannot be effectively separated from NBED. In addition, after changing isocratic elution to gradient elution, the response of the analyte NBED was also improved. Therefore, in order to ensure that the specificity of the method meets the requirements and that there are no problems such as residues, the initial ratio, rate of change and final ratio of the gradient were adjusted and optimized. Finally, the gradient elution program described in Table 2 was determined, which can achieve the separation of the analyte quickly, while maintaining the sharpness and symmetry of the chromatographic peak.

[0063] Figure 9 This is a mass spectrum provided in Example 8 of the present invention; compared with Example 1, the chromatographic peak response is worse under the mass spectrometry conditions of Example 8.

[0064] Figure 10 This is the mass spectrum provided in Embodiment 9 of the present invention; Figure 11 This is a mass spectrum provided in Example 10 of the present invention. Using tolbutamide and lidocaine as internal standards, the peaks are symmetrical and sharp; therefore, the detection effect under these conditions is comparable to that under the optimal conditions.

[0065] In summary, the detection method of this invention has undergone complete methodological validation, exhibiting high specificity, high sensitivity, precision, accuracy, and rapid analysis speed. It meets the analytical needs of non-clinical and clinical research on NBED in innovative drug molecules and fills the gap in the detection of NBED in plasma using high-performance liquid chromatography-mass spectrometry.

[0066] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A method for determining the concentration of NBED in plasma using high performance liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: Whole blood samples containing NBED were collected, and the whole blood samples were centrifuged at 6000 rpm for 5 min to obtain plasma. Internal standard working solution was added to the plasma, mixed well, and centrifuged at 14000 rpm for 10 min at 4℃. The supernatant was dried with N2 at 60℃, reconstituted with the mobile phase of the initial ratio, vortexed, and centrifuged to prepare the sample solution containing NBED. The sample solution containing NBED was injected into a high-performance liquid chromatography-tandem mass spectrometer for chromatographic and mass spectrometric detection to obtain a mass spectrum. Quantitative analysis of NBED was performed using the internal standard method based on the mass spectrum.

2. The method according to claim 1, characterized in that, The volume ratio of plasma to internal standard working solution added is 1:(2-5).

3. The method according to claim 1, characterized in that, The internal standard working solution is a methanol-acetonitrile mixed solution with an added internal standard, and the concentration of the internal standard in the internal standard working solution is 8.00 ng / mL.

4. The method according to claim 3, characterized in that, The internal standard is any one of lamivudine, tolbutamide, and lidocaine.

5. The method according to claim 3, characterized in that, The volume ratio of methanol to acetonitrile is 1:(1-3).

6. The method according to claim 1, characterized in that, The chromatographic detection conditions are as follows: mobile phase A is a mixed solution of ammonia and water, and mobile phase B is a mixed solution of acetonitrile or methanol and ammonia. Gradient elution is performed, and the gradient elution program is as follows: within 0-3 min, 10% mobile phase B gradually changes to 20% mobile phase B; within 3-8 min, 20% mobile phase B gradually changes to 90% mobile phase B; within 8-10 min, the proportion of mobile phase B is maintained at 90%; within 10.1-12 min, the proportion of mobile phase B is maintained at 10%. Column temperature: 40℃; Sample loading volume: 10μl; Flow rate: 0.4ml / min; Injector temperature: 4℃.

7. The method according to claim 1, characterized in that, The chromatographic column in the high-performance liquid chromatography is any one of Ultimate® AQ-C18, ACQUITY UPLC BEH Amide, Ultimate® HILIC Amide, ACQUITY BEH C18, or Zorbax Eclipse Plus C18 (T3).

8. The method according to claim 6, characterized in that, The initial ratio of mobile phase A to mobile phase B is 9:1, and the volume fraction of ammonia in mobile phase A is 0.01-0.05%; the volume fraction of ammonia in mobile phase B is 0.01-0.05%.

9. The method according to claim 1, characterized in that, The mass spectrometry detection conditions are as follows: an electrospray ionization source is used, the detection method is negative ion detection, the monitoring mode is multiple reaction monitoring, the ion spray voltage is -4500 V; the ion source temperature is 400-500 ℃; gas 1 (GS1) is 65 psi; gas 2 (GS2) is 50 psi; and the curtain gas (Cur) is 40 psi. Collision air (CAD) 9 psi; Residence time 50ms; mass spectrometry acquisition time 10.00 min; Q1 / Q3 resolution Unit / Unit.

10. The method according to claim 9, characterized in that, The quantitative ion pair of the NBED has an m / z range of 689.4 to 609.6, a collision energy (CE) of -13 eV, and a declustering voltage (DP) of -50 V; the analytical ion pair of the internal standard has an m / z range of 228.0 to 133.9, a collision energy (CE) of -20 eV, and a declustering voltage (DP) of -57 V.

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