Method for determining the concentration of sofosbuvir in plasma and screening for non-isotopically labeled internal standard

Abstract

The application discloses a method for determining the concentration of sofosbuvir in blood plasma and screening a non-isotope labeled internal standard, wherein anlotinib, imatinib, olaparib, osimertinib and pralsetinib are used as internal standards, quantitative analysis is performed by combining LC-MS / MS with protein precipitation pretreatment. The internal standard screening method is screened from candidate compounds by systematically evaluating four dimensions of protein precipitation compatibility, matrix effect, detection stability and extraction recovery, and an internal standard consistent with the behavior of the measured substance is screened. The application solves the problem of the lack of commercial isotope internal standards for sofosbuvir, and provides a reliable and efficient detection and internal standard screening scheme.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a method for determining the concentration of surufatinib in plasma, and a method for screening non-isotope labeled internal standards therein. Background Technology

[0002] Surufatinib is an oral tyrosine kinase inhibitor that selectively targets VEGFR, FGFR1, and CSF1R kinases for the treatment of locally advanced or metastatic, well-differentiated pancreatic neuroendocrine tumors. However, the pharmacokinetic characteristics of surufatinib exhibit significant inter-individual variability, with inter-individual coefficients of variation for peak concentration and area under the curve reaching 54.6% and 50.4%, respectively. Patients receiving a standard fixed dose (300 mg) may experience poor efficacy due to insufficient drug exposure or adverse drug reactions due to excessive exposure.

[0003] Therapeutic drug monitoring is crucial for achieving personalized dosing and balancing efficacy and toxicity, with its core being the accurate and reliable quantitative detection of drug concentrations in human plasma. Liquid chromatography-tandem mass spectrometry (LC-MS / MS), due to its excellent sensitivity, specificity, and analytical throughput, is considered the gold standard for such detection. In quantitative mass spectrometry analysis, internal standards are typically introduced to compensate for systematic and random errors during the analysis process, thereby obtaining reliable results. Isotope-labeled internal standards are widely considered the optimal choice because they share the same physicochemical properties as the analyte. However, their application is often limited by factors such as high cost, poor commercial availability, or difficulty in synthesis. Surufatinib falls into this category, as there is currently no commercially available isotope-labeled internal standard. Therefore, there is an urgent need to select a suitable, stable, and cost-effective non-isotope-labeled internal standard as a practical alternative.

[0004] In existing technologies, researchers have attempted to use non-isotopically labeled internal standards for the quantitative analysis of surufatinib and related tyrosine kinase inhibitors. For example, in a phase I study, HM5003491 was used as an internal standard for surufatinib, but the structure of this compound is unclear and it is not commercially available. One study established an ultra-high performance liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in rat plasma using carbamazepine as an internal standard. In a study on the in vivo interaction between surufatinib and myricetin, lenvatinib was used as an internal standard. In studies of other tyrosine kinase inhibitors, researchers have used non-isotopically labeled internal standards, including erlotinib, carbamazepine, propranolol, and voriconazole.

[0005] However, none of the aforementioned studies systematically screened internal standards or provided a theoretical basis for selecting these substances. Regarding the selection of non-isotope-labeled internal standards, existing literature proposes some general criteria, including: the internal standard's chemical structure and functional groups should be similar to the analyte; the internal standard should have sufficient purity; the internal standard should remain stable under different solvents and conditions; the internal standard should not be present in the analyte sample; the internal standard should be co-eluted with multiple analytes; and the internal standard should be inexpensive, readily available, and / or synthesized. Selecting a suitable internal standard is usually a time-consuming process. Some researchers have attempted rapid screening using internal standard mixtures, employing a nine-component internal standard mixture screening strategy. Others have prepared mixed internal standard solutions composed of five commonly used analogs, with linearity as the primary evaluation metric. However, existing screening methods have relatively singular evaluation dimensions, lacking a systematic consideration of multiple dimensions such as matrix influence, detection stability, extraction recovery, and chromatographic resolution.

[0006] In summary, existing technologies lack a screening method for non-isotope-labeled internal standards for surufatinib, as well as a reliable and efficient liquid chromatography-tandem mass spectrometry (LC-MS / MS) quantitative method based on such a screening method. This invention addresses this technical problem. Summary of the Invention

[0007] In view of this, the present invention provides a method for determining the concentration of surufatinib in plasma and screening non-isotope-labeled internal standards, in order to solve the technical problem that it is difficult to accurately quantify the concentration of surufatinib in plasma due to the lack of commercially available isotope-labeled internal standards in therapeutic drug monitoring; and to solve the technical problem that existing methods cannot fully assess the applicability of internal standards.

[0008] The present invention provides a liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma, comprising the following steps:

[0009] 1) Mix surufatinib with a solvent to prepare standard curve working solutions of a series of concentrations and quality control working solutions of at least two concentration levels;

[0010] 2) Prepare a mixed internal standard working solution containing a non-isotope-labeled internal standard, wherein the non-isotope-labeled internal standard is selected from at least one of anlotinib, imatinib, olaparib, osimertinib, and praltinib;

[0011] 3) Sample pretreatment:

[0012] Take blank plasma and add the corresponding concentration of the standard curve working solution or quality control working solution to obtain the corresponding standard curve mixture and quality control mixture;

[0013] Take the same volume of the plasma to be tested, the standard curve mixture, and the quality control mixture. Then, take the same volume of the mixed internal standard working solution and add it to the plasma to be tested, the standard curve mixture, and the quality control mixture. After protein precipitation reaction, centrifuge the mixture and take the supernatant from the centrifugation to obtain the sample to be tested, the standard curve sample, and the quality control sample.

[0014] 4) Liquid chromatography-tandem mass spectrometry analysis: The standard curve sample, quality control sample and test sample obtained in step 3) were analyzed by liquid chromatography-tandem mass spectrometry to obtain the peak area ratio of surufatinib to non-isotope labeled internal standard in each sample.

[0015] 5) Calculation: Establish a standard curve with the known concentration of surufatinib in the standard curve sample as the x-axis and the corresponding peak area ratio as the y-axis; substitute the peak area ratio corresponding to the quality control sample into the standard curve to calculate the concentration of surufatinib in the quality control sample; compare the calculated surufatinib concentration with the known surufatinib concentration in the quality control sample; if the relative standard deviation requirement is met, the standard curve is qualified; if the relative standard deviation requirement is not met, repeat steps 1) to 4) until a standard curve that meets the relative standard deviation requirement is obtained.

[0016] The concentration of surufatinib in plasma was calculated by substituting the peak area ratio of the sample to be tested into the standard curve that meets the relative standard deviation requirement.

[0017] Further, step 1) involves mixing surufatinib with the solvent, which includes: first dissolving surufatinib in methanol containing 5% dimethyl sulfoxide to prepare a standard stock solution with a mass concentration of 0.25 mg / mL; then diluting the standard stock solution with a 1:1 volume ratio acetonitrile / water mixture.

[0018] Furthermore, the concentrations of the standard curve working solution mentioned in step 1) include 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL, and the concentrations of the quality control working solution include 3 ng / mL, 30 ng / mL and 150 ng / mL.

[0019] Further, step 2) describes the preparation of a mixed internal standard working solution containing a non-isotope-labeled internal standard, which includes: preparing an internal standard stock solution by taking the non-isotope-labeled internal standard and using methanol containing 1% dimethyl sulfoxide, and then diluting it with a methanol / acetonitrile solution at a volume ratio of 1:1 to obtain a mixed internal standard working solution.

[0020] Furthermore, the conditions for the protein precipitation reaction in step 3) of sample pretreatment include: vortexing the test plasma, standard curve mixture, and quality control mixture after adding the mixed internal standard working solution for 8 min and 1300 rpm.

[0021] Furthermore, in step 4) liquid chromatography-tandem mass spectrometry analysis, the liquid chromatography uses gradient elution mode.

[0022] Furthermore, in step 4) liquid chromatography-tandem mass spectrometry analysis, the liquid chromatography conditions are as follows: the chromatographic column is a C18 column; mobile phase A is an aqueous solution containing formic acid and ammonium acetate, and mobile phase B is acetonitrile; the elution conditions of the gradient elution mode are as follows: 0-1.5 min, the volume percentage of mobile phase B increases from 10% to 60%; 1.5-2.0 min, the volume percentage of mobile phase B increases from 60% to 98%; 2.0-3.0 min, maintain 98% B; 3.0-3.5 min, the volume percentage of mobile phase B decreases from 98% to 10%; 3.5-4.0 min, maintain 10% B.

[0023] This invention also discloses a method for screening non-isotope labeled internal standards described in the above methods, comprising the following steps:

[0024] (a) Provide a set of internal standard candidates for quantifying the analyte;

[0025] (b) The internal standard candidates shall be evaluated in the following four aspects:

[0026] Protein precipitation method compatibility assessment: Based on chromatographic resolution and peak asymmetry, the internal standard candidate and the analyte are evaluated to determine whether they are compatible with the same protein precipitation method.

[0027] Matrix influence assessment: Based on the coefficients of variation of matrix factor and internal standard normalized matrix factor, assess whether the internal standard candidate and the analyte are similarly affected by the matrix;

[0028] Stability assessment: The response stability of the internal standard candidate in the liquid chromatography-tandem mass spectrometry analysis process and its reliability for establishing a standard curve were evaluated based on the response variation coefficient and linear correlation coefficient.

[0029] Extraction recovery rate evaluation: The extraction recovery rates of the internal standard candidate and the analyte, and the difference between the two, are used as evaluation criteria to assess whether the extraction efficiency of the internal standard candidate and the analyte is consistent under the same pretreatment conditions.

[0030] (c) From the candidates evaluated in step (b), internal standard candidates with consistent co-elution behavior with the analyte are selected as non-isotope labeled internal standards suitable for the determination of the analyte, using chromatographic resolution as the screening index.

[0031] Furthermore, the analyte in step (a) is surufatinib, and the internal standard candidates include anlotinib, crizotinib, gefitinib, imatinib, lenvatinib, olaparib, osimertinib, praltinib, regorafenib, zanubrutinib, carbamazepine, dexamethasone, gabapentin, nematradine, and ritonavir.

[0032] The beneficial effects of this invention are:

[0033] 1. This invention proposes for the first time a multi-dimensional non-isotope labeled internal standard screening strategy that includes pretreatment compatibility, matrix effect, analytical stability, extraction recovery rate and chromatographic behavior. It overcomes the shortcomings of arbitrary internal standard selection and single evaluation in the prior art and provides universal methodological guidance for the selection of internal standards in similar situations.

[0034] 2. This invention, through its proposed method for screening non-isotope-labeled internal standards, successfully identified five suitable non-isotope-labeled internal standards for surufatinib quantification from 15 candidates: anlotinib, imatinib, olaparib, osimertinib, and pralatinib. Pralatinib was the optimal choice. Although olaparib is not a tyrosine kinase inhibitor, after systematic screening and validation, it meets the basic requirements for use as a surufatinib internal standard; when the other four preferred internal standards are unavailable, olaparib can be used as an alternative internal standard. This invention provides five interchangeable non-isotope-labeled internal standards, offering a reliable and readily available practical internal standard solution for TDM of surufatinib.

[0035] 3. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma, established based on a screened non-isotope labeled internal standard, has been verified to have good selectivity, accuracy (bias: -11.74% ~ 8.60%), precision (CV: 1.06% ~ 9.36%), excellent linearity (r² > 0.99), and good stability under different conditions, fully meeting the requirements of the guidelines for biological sample analysis.

[0036] 4. This invention validates five interchangeable qualified internal standards. In practical applications, when the preferred internal standard, pralatinib, is unavailable, it can be seamlessly replaced with other validated internal standards without requiring a complete re-validation of the methodology, significantly enhancing the practicality of the method and the operational flexibility of the laboratory.

[0037] 5. This invention directly solves the technical bottleneck of surufatinib's inaccurate quantification due to the lack of commercially available isotope internal standards, and provides key and reliable analytical tools (five interchangeable non-isotope labeled internal standards) and methods for TDM and personalized treatment of surufatinib. Attached Figure Description

[0038] Figure 1 This is a diagram showing the results of the matrix impact assessment.

[0039] Figure 2 Chromatographic peaks of surufatinib with five internal standards (anlotinib, imatinib, olaparib, osimertinib, and pralatinib).

[0040] Figure 3 This is a graph showing the residual effect.

[0041] Figure 4 The distribution of the actual sample concentration is shown in the graph (N=15). Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] Example 1: The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma in this example includes the following steps:

[0044] 1) Mix surufatinib with a solvent to prepare standard curve working solutions of a series of concentrations and quality control working solutions at at least two concentration levels. This step specifically includes:

[0045] First, weigh out surufatinib standard and dissolve it in methanol containing 5% dimethyl sulfoxide to prepare a standard stock solution with a mass concentration of 0.25 mg / mL. Then, dilute the standard stock solution with a 1:1 volume ratio acetonitrile / water mixture to prepare standard curve working solutions with concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. Dilute the standard stock solution with a 1:1 volume ratio acetonitrile / water mixture to prepare quality control working solutions with three concentration levels: 3 ng / mL, 30 ng / mL, and 150 ng / mL.

[0046] 2) Prepare a mixed internal standard working solution containing a non-isotope-labeled internal standard, wherein the non-isotope-labeled internal standard is selected from at least one of anlotinib, imatinib, olaparib, osimertinib, and praltinib. This step includes:

[0047] Non-isotope-labeled internal standards were prepared into internal standard stock solutions with a mass concentration of 1 mg / mL using methanol containing 1% dimethyl sulfoxide, and then diluted with a methanol / acetonitrile solution with a volume ratio of 1:1 to prepare a mixed internal standard solution with a concentration of 150 ng / mL.

[0048] 3) Sample pretreatment:

[0049] Blank plasma was taken and the corresponding concentration of the standard curve working solution or quality control working solution was added to it to obtain the corresponding standard curve mixture and quality control mixture.

[0050] Take equal volumes (100 μL) of the test plasma, standard curve mixture, and quality control mixture. Then, add equal volumes (300 μL) of the mixed internal standard working solution to the same volume of the test plasma, standard curve mixture, and quality control mixture. After protein precipitation, centrifuge and collect the supernatant (200 μL) to obtain the test sample, standard curve sample, and quality control sample. The protein precipitation reaction conditions in this step include: vortexing the test plasma, standard curve mixture, and quality control mixture after adding the mixed internal standard working solution for 8 min at an oscillation intensity of 1300 rpm. The centrifugation conditions include: centrifugation at 14000 g for 10 min.

[0051] 4) Liquid chromatography-tandem mass spectrometry analysis: The standard curve sample, quality control sample and test sample obtained in step 3) were analyzed by liquid chromatography-tandem mass spectrometry to obtain the peak area ratio of surufatinib to non-isotope labeled internal standard in each sample.

[0052] The liquid chromatography conditions used in this step are as follows:

[0053] The chromatographic column was a C18 column; mobile phase A was an aqueous solution of 2 mmol / L ammonium acetate and 0.1% formic acid, and mobile phase B was acetonitrile; gradient elution was used, with the following elution conditions: 0–1.5 min, the volume percentage of mobile phase B increased from 10% to 60%; 1.5–2.0 min, mobile phase B increased from 60% to 98%; 2.0–3.0 min, maintaining 98% B; 3.0–3.5 min, mobile phase B decreased from 98% to 10%; 3.5–4.0 min, maintaining 10% B. The flow rate was 0.4 mL / min; the injector temperature was 15 ℃; the column oven temperature was 40 ℃; and the injection volume was 1 μL.

[0054] Mass spectrometry conditions in this step:

[0055] The ion source was an electrospray ionization source, and mass monitoring was performed in positive ion mode. The acquisition mode was multiple reaction monitoring. Mass spectrometry parameters: ion spray voltage 5500 V, ion source temperature 550 ℃, inlet potential 10 V, collision gas 8.0 psi, curtain gas 20 psi, and ion sources Gas1 and Gas2 both 55 psi.

[0056] 5) Calculation: Establish a standard curve with the known concentration of surufatinib in the standard curve sample as the x-axis and the corresponding peak area ratio as the y-axis. Substitute the peak area ratio of the quality control sample into the standard curve to calculate the concentration of surufatinib in the quality control sample. Compare the calculated surufatinib concentration with the known surufatinib concentration of the quality control sample. If the relative standard deviation requirement is met, the standard curve is qualified; if the relative standard deviation requirement is not met, repeat steps 1) to 4) until a standard curve that meets the relative standard deviation requirement is obtained. Substitute the peak area ratio of the test sample into the standard curve that meets the relative standard deviation requirement to calculate the concentration of surufatinib in plasma.

[0057] Example 2:

[0058] The method for screening non-isotope labeled internal standards in the method described in Example 1, as shown in this embodiment, includes the following steps:

[0059] (a) Provide a set of internal standard candidates for quantifying the analyte. The analyte in this step is surufatinib, and the internal standard candidates specifically include anlotinib, crizotinib, gefitinib, imatinib, lenvatinib, olaparib, osimertinib, praltinib, regorafenib, zanubrutinib, carbamazepine, dexamethasone, gabapentin, nematradine, and ritonavir.

[0060] (b) The internal standard candidates were used as non-isotope labeled internal standards, and the concentration of surufatinib in plasma was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) under the same chromatographic and mass spectrometric conditions as in Example 1. The mass spectrometric parameters of surufatinib and the 15 internal standard candidates are shown in Table 1.

[0061] Table 1. Detailed information on surufatinib and 15 potential internal standard candidates

[0062]

[0063] The internal standard candidates were then evaluated from the following four aspects:

[0064] Compatibility assessment of protein precipitation methods: Chromatographic resolution and peak asymmetry were used as evaluation criteria to assess whether the internal standard candidates and analytes were compatible with the same protein precipitation method. To optimize protein precipitation efficiency, key parameters such as precipitation solvent, shaking time, and shaking intensity were investigated. Eleven precipitation solvents were prepared, including pure methanol, pure acetonitrile, and mixtures of different ratios. Experimental results showed that pure methanol, pure acetonitrile, and methanol / acetonitrile (1:1 and 3:1, v / v) mixtures all achieved effective precipitation. The chromatographic resolution (R) of the 11 internal standard candidates was lowest in the methanol / acetonitrile (1:1, v / v) mixture. Therefore, the methanol / acetonitrile (1:1, v / v) mixture was selected as the final precipitation solvent. Shaking times were tested at 2, 4, 6, 8, and 10 min, and shaking intensities were tested at 1000, 1100, 1200, 1300, and 1400 rpm. Experimental results showed that the chromatographic resolution (R) values ​​of the 10 internal standard candidates were lowest when the shaking time was 8 min. Under different shaking intensities, the R values ​​of the 15 internal standard candidates remained consistent, and the coefficients of variation were all below 10%. Considering both the chromatographic resolution (R) value and the peak asymmetry (As) value, the optimal shaking conditions were determined to be a shaking time of 8 min and a shaking intensity of 1300 rpm.

[0065] Matrix Influence Assessment: Based on the coefficients of variation of matrix factors and normalized matrix factors (MFi), the similarity of matrix influence on the internal standard candidates and the analytes was assessed. Normalized matrix factors and their coefficients of variation were measured in three matrices: normal plasma, lipemic, and hemolytic. Internal standard candidates with minimal matrix influence and stable responses were screened. Experimental results showed that in the normal plasma matrix, the matrix factor (MF) values ​​of nelmatidine, regorafenib, ritonavir, and zanubrutinib were all greater than 1, indicating enhanced response; while in the lipemic and hemolytic matrices, their responses were significantly inhibited. Gabapentin showed significant enhanced response in all three matrices. The coefficients of variation of normalized matrix factors (MFi) for nelmatidine, regorafenib, ritonavir, and zanubrutinib in the three matrices were as high as 41.04%, 99.90%, 56.06%, and 45.61%, respectively. Further analysis showed that the as values ​​(As) of carbamazepine, gefitinib, nelmatvir, regorafenib, ritonavir, and zanubrutinib varied significantly in different matrices (coefficient of variation > 15%). The R values ​​of gabapentin and regorafenib were also significantly affected by the matrices, with coefficients of variation of 20.95% and 27.10%, respectively. Therefore, carbamazepine, gabapentin, gefitinib, nelmatvir, regorafenib, ritonavir, and zanubrutinib are unsuitable as internal standards for surufatinib due to their susceptibility to matric influence. Results are as follows... Figure 1 .

[0066] Stability Assessment: The response stability of internal standard candidates in liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis and their reliability in establishing standard curves were evaluated based on the response variation coefficient and linear correlation coefficient. Three sets of standard curves and quality control samples were prepared for the following assessments: (i) the stability of the internal standard in intra-batch and inter-batch standard curves was examined; (ii) a linear equation was established with 1 / x² as the weighting factor to obtain the dominant correlation coefficient (r²); (iii) the accuracy of low, medium, and high concentration quality control samples was calculated based on the linear equation. The experimental results showed that nematimivir, regorafenib, and ritonavir exhibited large peak area response variations (coefficient of variation > 15%) within a single standard curve; the response variations of the above three analytes and zanubrutinib among the three independent standard curves all exceeded 15%, indicating poor analytical stability when used as internal standards. Except for gefitinib, regorafenib, ritonavir, and zanubrutinib, the r² of other internal standard candidates was greater than 0.99, meeting the requirements for reliable detection.

[0067] Recovery evaluation: The recovery rates of the internal standard candidate and the analyte, and the difference between their respective recoveries, were used as evaluation criteria to assess whether the extraction efficiencies of the internal standard candidate and the analyte were consistent under the same pretreatment conditions. At three quality control concentration levels, except for regorafenib, nelmatvir, and ritonavir, the recoveries of surufatinib and 12 internal standard candidates ranged from 93.77% to 112.60%. Gefitinib and osimertinib had the lowest recovery rate differences, both less than 5%; the differences for anlotinib, carbamazepine, dexamethasone, gabapentin, imatinib, lenvatinib, olaparib, and praltinib did not exceed 10%; the differences for nelmatvir and regorafenib were approximately 15% and 20%, respectively.

[0068] (c) From the candidates evaluated in step (b), chromatographic resolution was used as the screening criterion to select internal standard candidates with co-elution behavior consistent with the analyte as non-isotope-labeled internal standards suitable for the determination of the analyte. Based on the above multidimensional screening results, anlotinib, imatinib, olaparib, osimertinib, and pralatinib were suitable as internal standards for surufatinib in all evaluation dimensions. Among them, pralatinib and surufatinib showed the best co-elution effect, with the lowest chromatographic resolution between them. The results are as follows: Figure 2 .

[0069] Anlotinib, imatinib, olaparib, osimertinib, and pralatinib were used as internal standards for surufatinib to conduct a comprehensive methodological validation.

[0070] Selective Validation

[0071] Selectivity was investigated using eight blank plasma matrices from different sources (including six normal blank plasmas, one lipemic plasma, and one hemolyzed plasma). The results showed that no interference from endogenous substances was detected at the retention time of surufatinib in the blank plasma samples. For the five internal standards, the responses of endogenous substances in the blank plasma were all below 5% of their signals.

[0072] Matrix effect verification

[0073] Eight blank plasma matrices from different sources (same as selectivity validation) were used to investigate the matrix effect in low- and high-concentration quality control samples. The experimental results showed that the accuracy of the determination was within ±15% of the labeled concentration in different plasma matrices, and the precision (coefficient of variation) was less than 15%.

[0074] Standard curve and lower limit of quantitation verification

[0075] The standard curve should include at least six calibration concentration levels. The concentrations calculated from the calibration standards should be within ±15% of the labeled values, and within ±20% of the lower limit of quantitation. At least 75% of the calibration standards should meet these criteria.

[0076] The standard curve for surufatinib ranged from 1 to 200 ng / mL. Standard curves were prepared daily for three consecutive days, yielding three independent curves. The accuracy at all seven concentration levels met guideline requirements, with biases within ±10%.

[0077] Accuracy and precision verification

[0078] The method employed limit of quantitation (LOQ) and low, medium, and high concentration quality control samples, with at least five samples per concentration, and was evaluated across at least three analytical batches. Intra-batch accuracy and precision were assessed using at least five samples per analytical batch; inter-batch accuracy and precision were assessed using at least three analytical batches over at least two days. The results showed that the intra-batch bias for surufatinib ranged from -11.74% to 8.60%, with a coefficient of variation ranging from 1.06% to 6.40%; the inter-batch bias ranged from -9.49% to 6.91%, with a coefficient of variation ranging from 3.34% to 9.36%. All results met the method validation requirements. The experimental results are shown in Table 2.

[0079] Table 2 Accuracy and Precision Results

[0080]

[0081] Dilution integrity verification

[0082] Two concentration levels exceeding the upper limit of quantitation (300 ng / mL and 1000 ng / mL) were used for 5-fold, 10-fold, and 20-fold dilutions, with at least five samples tested at each dilution. The results showed that the mean bias of the diluted plasma samples ranged from −8.68% to 6.96%, and the coefficient of variation ranged from 0.77% to 6.11%, both within the 15% limit.

[0083] Residual effect verification

[0084] Residual effects were assessed by injecting blank samples after a high-concentration sample (upper limit of quantification). Experimental results showed that no significant response of surufatinib or the five internal standards was detected in blank plasma injected after the upper limit of quantification, and the residual effect was negligible.

[0085] Stability verification

[0086] The stability of the analyte in the matrix was investigated using low- and high-concentration quality control samples, while the stability of the analyte in the stock solution was investigated using the lower limit of quantitation (LVQ) and upper limit of quantitation (UPQ). Experimental results showed that surufatinib remained stable in plasma under various conditions. No significant degradation was observed after three freeze-thaw cycles or storage at −20°C for one month. It exhibited good stability at 4°C, with all biases within ±15%. Processed plasma samples were stable for 24 h in an autosampler, with low-concentration quality control biases ranging from −9.7% to 10% and high-concentration quality control biases ranging from −5.5% to 8.0%. Surufatinib stock solution was stable for one month at −20°C, with biases ranging from −7.5% to 13.67%. Whole blood stability studies demonstrated that surufatinib remained stable within 48 h, providing sufficient time for sample processing from collection.

[0087] Clinical application

[0088] The method described in Example 1 was applied to the detection of 15 clinical plasma samples. Experimental results showed that when five different internal standards were used to quantify the same sample, there was no significant difference in the measured concentration, with a coefficient of variation ranging from 2.26% to 8.96%, indicating that the quantitative results of this method are highly reliable. The average concentration of the detected samples ranged from 14.22 to 66.38 ng / mL, all falling within the established linear range (1–200 ng / mL). See the results below. Figure 4 .

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma, characterized in that, Includes the following steps: 1) Mix surufatinib with a solvent to prepare standard curve working solutions of a series of concentrations and quality control working solutions of at least two concentration levels; 2) Prepare a mixed internal standard working solution containing a non-isotope-labeled internal standard, wherein the non-isotope-labeled internal standard is selected from at least one of anlotinib, imatinib, olaparib, osimertinib, and praltinib; 3) Sample pretreatment: Take blank plasma and add the corresponding concentration of the standard curve working solution or quality control working solution to obtain the corresponding standard curve mixture and quality control mixture; Take the same volume of the plasma to be tested, the standard curve mixture, and the quality control mixture. Then, take the same volume of the mixed internal standard working solution and add it to the plasma to be tested, the standard curve mixture, and the quality control mixture. After protein precipitation reaction, centrifuge the mixture and take the supernatant from the centrifugation to obtain the sample to be tested, the standard curve sample, and the quality control sample. 4) Liquid chromatography-tandem mass spectrometry analysis: The standard curve sample, quality control sample and test sample obtained in step 3) were analyzed by liquid chromatography-tandem mass spectrometry to obtain the peak area ratio of surufatinib to non-isotope labeled internal standard in each sample. 5) Calculation: Establish a standard curve with the known concentration of surufatinib in the standard curve sample as the x-axis and the corresponding peak area ratio as the y-axis; substitute the peak area ratio corresponding to the quality control sample into the standard curve to calculate the concentration of surufatinib in the quality control sample; compare the calculated surufatinib concentration with the known surufatinib concentration in the quality control sample; if the relative standard deviation requirement is met, the standard curve is qualified; if the relative standard deviation requirement is not met, repeat steps 1) to 4) until a standard curve that meets the relative standard deviation requirement is obtained. The concentration of surufatinib in plasma was calculated by substituting the peak area ratio of the sample to be tested into the standard curve that meets the relative standard deviation requirement.

2. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 1, characterized in that: Step 1) involves mixing surufatinib with a solvent, which includes: first dissolving surufatinib in methanol containing 5% dimethyl sulfoxide to prepare a standard stock solution with a mass concentration of 0.25 mg / mL; then diluting the standard stock solution with a 1:1 volume ratio acetonitrile / water mixture.

3. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 2, characterized in that: The concentrations of the standard curve working solution mentioned in step 1) include 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL, and the concentrations of the quality control working solution include 3 ng / mL, 30 ng / mL and 150 ng / mL.

4. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 1, characterized in that: The preparation of the mixed internal standard working solution containing the non-isotope-labeled internal standard in step 2) includes: taking the non-isotope-labeled internal standard and preparing an internal standard stock solution with methanol containing 1% dimethyl sulfoxide, and then diluting it with a methanol / acetonitrile solution with a volume ratio of 1:1 to obtain the mixed internal standard working solution.

5. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 1, characterized in that: Step 3) The conditions for the protein precipitation reaction in the sample pretreatment include: vortexing the test plasma, standard curve mixture and quality control mixture after adding the mixed internal standard working solution for 8 min and 1300 rpm.

6. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 1, characterized in that: In step 4), liquid chromatography-tandem mass spectrometry analysis, gradient elution mode is used in liquid chromatography.

7. The liquid chromatography-tandem mass spectrometry method for determining the concentration of surufatinib in plasma according to claim 6, characterized in that: In step 4), the liquid chromatography-tandem mass spectrometry analysis was performed under the following conditions: the column was a C18 column; mobile phase A was an aqueous solution containing formic acid and ammonium acetate, and mobile phase B was acetonitrile; the elution conditions for the gradient elution mode were as follows: 0-1.5 min, the volume percentage of mobile phase B increased from 10% to 60%; 1.5-2.0 min, the volume percentage of mobile phase B increased from 60% to 98%; 2.0-3.0 min, the volume percentage of mobile phase B was maintained at 98%; 3.0-3.5 min, the volume percentage of mobile phase B decreased from 98% to 10%; 3.5-4.0 min, the volume percentage of mobile phase B was maintained at 10%.

8. A method for screening non-isotope-labeled internal standards in the method according to any one of claims 1-7, characterized in that, Includes the following steps: (a) Provide a set of internal standard candidates for quantifying the analyte; (b) The internal standard candidates shall be evaluated in the following four aspects: Protein precipitation method compatibility assessment: Based on chromatographic resolution and peak asymmetry, the internal standard candidate and the analyte are evaluated to determine whether they are compatible with the same protein precipitation method. Matrix influence assessment: Based on the coefficients of variation of matrix factor and internal standard normalized matrix factor, assess whether the internal standard candidate and the analyte are similarly affected by the matrix; Stability assessment: The response stability of the internal standard candidate in the liquid chromatography-tandem mass spectrometry analysis process and its reliability for establishing a standard curve were evaluated based on the response variation coefficient and linear correlation coefficient. Extraction recovery rate evaluation: The extraction recovery rates of the internal standard candidate and the analyte, and the difference between the two, are used as evaluation criteria to assess whether the extraction efficiency of the internal standard candidate and the analyte is consistent under the same pretreatment conditions. (c) From the candidates evaluated in step (b), internal standard candidates with consistent co-elution behavior with the analyte are selected as non-isotope labeled internal standards suitable for the determination of the analyte, using chromatographic resolution as the screening index.

9. A method for screening non-isotope-labeled internal standards in any one of the methods of claims 1-6, as described in claim 8, characterized in that: The analyte in step (a) is surufatinib, and the internal standard candidates include anlotinib, crizotinib, gefitinib, imatinib, lenvatinib, olaparib, osimertinib, praltinib, regorafenib, zanubrutinib, carbamazepine, dexamethasone, gabapentin, nematradine, and ritonavir.

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