Kit and method for detecting concentration of Venoclarias

Abstract

The invention provides a kit for detecting the concentration of Venoclarias and a Venoclarias concentration detection method, and belongs to the technical field of in-vitro diagnos.The kit comprises a calibrator, an internal standard mother solution, a quality control product, an internal standard-sample releasing agent, a sample dilution treating fluid and a mobile phase. And the detection of low-concentration Venokacrah can be realized. The kit provided by the invention also has the advantages of high sensitivity, strong anti-interference capability and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a kit for detecting the concentration of venetoclax and a method for detecting the concentration of venetoclax. Background Art

[0002] Venetal, an oral, selective B-cell lymphoma-2 (BCL-2) inhibitor, has demonstrated remarkable therapeutic potential and clinical value in the treatment of various hematologic malignancies. In chronic lymphocytic leukemia (CLL), overexpression of BCL-2 leads to impaired apoptosis and accumulation of mature leukemic lymphocytes. In non-Hodgkin's lymphoma (NHL), overexpression of BCL-2 promotes cell survival and enhances resistance to cancer therapies. Venetal binds to BCL-2 with high specificity and inhibits its overexpression, thereby suppressing the release of pro-apoptotic proteins from anti-apoptotic proteins and triggering cell death. Venetal kills tumor cells primarily by inducing intrinsic apoptosis. In vitro studies have shown that venetoclax is primarily metabolized by CYP3A4 and is a substrate of the P-glycoprotein efflux pump, exhibiting strong drug interactions with drugs such as ketoconazole, posaconazole, and rifampicin. Long-term, high-dose use of venetoclax can lead to life-threatening tumor lysis syndrome and common adverse reactions, including neutropenia and thrombocytopenia. Furthermore, the relative bioavailability of venetoclax is nonlinear across patients, exhibiting significant individual variability. Therefore, in clinical practice, therapeutic drug monitoring (TDM) is necessary for patients taking venetoclax. This allows for dosage adjustments based on monitored concentrations to ensure effective exposure in the patient's body, while also reducing the incidence of adverse drug reactions and improving the safety and efficacy of clinical medication.

[0003] Currently, there are only a few methods for the quantitative determination of venetoclax in biological fluids, primarily including fluid scintillation counting (LSC), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, these methods typically require multiple sample preparation steps, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE), as well as lengthy processing times. Therefore, given the low concentrations of analytes, the presence of numerous interfering substances, the small sample size, and the difficulty in obtaining clinical samples, it is imperative to develop detection methods with higher sensitivity, specificity, and rapid analysis speeds. Furthermore, most clinical mass spectrometry-based testing methods are laboratory-developed methods (LDTs), using reagents that are not part of the kit. This makes it difficult to trace the methods back to reference materials or methods, and the accuracy and reproducibility of the test results await further clinical validation. In summary, there is currently a lack of a commercially available mass spectrometry-based detection kit that is both efficient and sensitive. Summary of the Invention

[0004] The object of the present invention is to provide a kit which is highly sensitive, easy to use and can be used for detecting low-concentration venetoclax.

[0005] To achieve the above object, the present invention provides a first aspect of a kit for detecting the concentration of venetoclax, comprising: Calibrators: including several venetoclax methanol solutions with concentrations ranging from 1 to 100.0 ng / mL; Internal standard stock solution: including d8-Venetradecanoic acid methanol solution; Quality control products: including low-concentration venetoclax methanol solution, medium-concentration venetoclax methanol solution, and high-concentration venetoclax methanol solution. The venetoclax concentration range of the low-concentration venetoclax methanol solution is 1-5 ng / mL; the venetoclax concentration range of the medium-concentration venetoclax methanol solution is 10-50 ng / mL; and the venetoclax concentration range of the high-concentration venetoclax methanol solution is 60-100 ng / mL. Internal standard-sample release agent: including 5 ng / mL d8-venetoclax methanol solution; Sample dilution solution: 50% (v / v) methanol in water; Mobile phase: includes mobile phase A and mobile phase B, wherein mobile phase A is 5 mM ammonium acetate aqueous solution and mobile phase B is methanol.

[0006] The present invention provides a standardized kit that integrates calibrators, quality control products, internal standard solutions, sample treatment solutions, and a mobile phase system, effectively simplifying the process required for reagent preparation. Furthermore, the calibrators provided by the present invention cover a range of 1 to 100 ng / mL, ensuring the detectability of trace drugs.

[0007] Preferably, the calibrator includes seven concentration gradients, specifically 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

[0008] Preferably, the internal standard mother solution is a 10× internal standard solution, and the concentration of d8-venetoclax in the 10× internal standard solution is 50 ng / mL.

[0009] The second aspect of the present invention provides a method for detecting the concentration of venetoclax, which uses the kit described in the first aspect to detect the concentration of venetoclax.

[0010] Preferably, the above detection method comprises the following steps: S1: Preparation of internal standard-sample release agent: dilute the internal standard d8-venetoclax stock solution with the sample dilution solution to prepare an internal standard-sample release agent with a d8-venetoclax concentration of 5 ng / mL; S2: Sample pretreatment: Take the sample to be tested, add the internal standard-sample release agent prepared in step S1 at a volume twice that of the sample to be tested, vortex mix, and centrifuge at 4°C for 10 minutes; S3: Transfer the supernatant to a clean centrifuge tube and centrifuge again at 4°C. Take the final supernatant as the test solution and perform HPLC-MS / MS detection. S4: Compare the results of HPLC-MS / MS detection with the standard curve and calculate the concentration of venetoclax in the sample.

[0011] In step S1, the present invention uses 50% (v / v) methanol aqueous solution as the sample dilution treatment solution, which has a low matrix effect and can quickly remove macromolecular substances in the sample without the need for complex operations such as extraction, and has the advantage of a simple treatment method.

[0012] In step S3, the present invention uses optimized chromatographic conditions and mass spectrometry conditions to achieve the detection of low-concentration venetoclax.

[0013] Preferably, the liquid chromatography conditions for HPLC-MS / MS detection are: column temperature 45°C; flow rate 0.55 mL / min; injection volume 2 μL, mobile phase A is the mobile phase A described in the first aspect, mobile phase B is the mobile phase B described in the first aspect, and the gradient elution program is: 0-1min: 95% mobile phase B, the balance is mobile phase A; 1-1.5 min: 95%-100% mobile phase B, the balance is mobile phase A; 1.5-2 min: 100% mobile phase B, the balance is mobile phase A; 2-2.1 min: 100%-70% mobile phase B, the balance mobile phase A; 2.1~3.5min: 70% mobile phase B, the balance mobile phase A.

[0014] Preferably, the mass spectrometry conditions for HPLC-MS / MS detection are: electrospray ion source (ESI)-positive ion ionization-multiple reaction ion monitoring (MRM) for scanning detection, the Venetoclax ion pair is 868.2→321, the d-8 Venetoclax ion pair is 876.2→329.0, the ion source voltage is 4500V; the interface temperature is 300°C; the collision gas is argon; the collision gas pressure is 230kPa; the heating gas is air; the nebulizing gas and the drying hot gas are both nitrogen; the heating gas flow rate is 10L / min; the nebulizing gas flow rate is 3L / min; the drying gas flow rate is 10L / min; the collision energy is 37V, and the scanning time is 50ms.

[0015] Preferably, the sample is selected from any one or more of blood, plasma, cerebrospinal fluid, urine, venetoclax solution, and lymph fluid.

[0016] Preferably, the concentration of venetoclax in the sample is 0.1-10 ng / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a kit for detecting the concentration of venetoclax based on a protein precipitation method combined with HPLC-MS / MS. The kit is suitable for detecting low concentrations of venetoclax of 0.1 to 10 ng / mL and has the advantage of high sensitivity. 2. The detection kit provided by the present invention does not require complicated extraction, purification, drying and re-dissolution steps. The analysis of a single sample can be completed within 3.5 minutes, which greatly shortens the sample detection time and is easy to use.

[0018] 3. The kit provided by the present invention also has the advantages of good stability and high accuracy. At the same time, the kit provided by the present invention provides a standardized detection method for venetoclax detection, which can promote the mutual recognition of venetoclax detection results among different medical institutions and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The plotting result of the Venetoclax standard curve in Example 2 of the present invention is shown below: Figure 2 This is the experimental result of the verification of the anti-interference ability of Venetoclax in Example 4 of the present invention; Figure 3 This is the experimental result of sensitivity detection in Example 5 of the present invention. DETAILED DESCRIPTION

[0020] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.

[0021] It should be noted that the endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0022] As described in the background art, the current detection methods for venetoclax are mostly aimed at detecting high concentrations of venetoclax. Based on this, a specific embodiment of the present invention provides a kit for detecting venetoclax concentration based on HPLC-MS / MS, the kit comprising: Calibrators: including several venetoclax methanol solutions with concentrations ranging from 1 to 100.0 ng / mL; Internal standard stock solution: including d8-Venetradecanoic acid methanol solution; Quality control products: including low-concentration venetoclax methanol solution, medium-concentration venetoclax methanol solution, and high-concentration venetoclax methanol solution. The venetoclax concentration range of the low-concentration venetoclax methanol solution is 1-5 ng / mL; the venetoclax concentration range of the medium-concentration venetoclax methanol solution is 10-50 ng / mL; and the venetoclax concentration range of the high-concentration venetoclax methanol solution is 60-100 ng / mL. Internal standard-sample release agent: including 5 ng / mL d8-venetoclax methanol solution; Sample dilution solution: 50% (v / v) methanol in water; Mobile phase: includes mobile phase A and mobile phase B, wherein mobile phase A is 5 mM ammonium acetate aqueous solution and mobile phase B is methanol.

[0023] In the above embodiment, the calibrator preferably includes seven concentration gradients, specifically 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

[0024] The calibrators of the above-mentioned concentration gradient have been clinically verified to meet the detection requirements and show good detection sensitivity within the linear range, which can ensure that the analytical system can achieve accurate quantification within the instrument detection limit.

[0025] In the above embodiment, the internal standard mother liquor adopts d8-Venetra methanol solution. In actual operation, the internal standard mother liquor is 10× internal standard solution. When used, the internal standard working solution can be obtained by diluting it with methanol. The role of the internal standard solution is to assist in judging the efficiency of the pretreatment process and whether the instrument and detection conditions are normal while ensuring the detection sensitivity.

[0026] In the above embodiment, the quality control product is used for the verification test of the detection method, and the verification test of the detection method includes a precision test, a recovery test and a stability test.

[0027] The embodiment of the present invention also provides a method for detecting the concentration of venetoclax, which comprises the following steps: S1: Preparation of internal standard-sample release agent: dilute the internal standard d8-venetoclax stock solution with the sample dilution solution to prepare an internal standard-sample release agent with a d8-venetoclax concentration of 5 ng / mL; S2: Sample pretreatment: Take the sample to be tested, add the internal standard-sample release agent prepared in step S1 at a volume twice that of the sample to be tested, vortex mix, and centrifuge at 4°C for 10 minutes; S3: Transfer the supernatant to a clean centrifuge tube and centrifuge again at 4°C. Take the final supernatant as the test solution and perform HPLC-MS / MS detection. S4: Compare the results of HPLC-MS / MS detection with the standard curve and calculate the concentration of venetoclax in the sample.

[0028] Ultra-high performance liquid chromatography tandem mass spectrometry was used to detect the concentration of venetoclax in pretreated cerebrospinal fluid samples. First, ultra-high performance liquid chromatography was used to separate the target analyte from the interfering components in the cerebrospinal fluid matrix. Then, the mass spectrometry isotope internal standard quantification method was used, with the sample and internal standard peak area ratio As / Ais as the vertical axis (Y) and the venetoclax concentration C (ng / mL) as the horizontal axis (X). The 1 / x 2 The weight coefficient was regressed to establish a standard curve for venetoclax, thereby calculating the concentration of venetoclax in the sample. In step S2 of the above embodiment, the liquid chromatography conditions for HPLC-MS / MS detection were: column temperature 45° C.; flow rate 0.55 mL / min; injection volume 2 μL; mobile phase A was the aforementioned mobile phase A; mobile phase B was the aforementioned mobile phase B; and the gradient elution program was: 0-1min: 95% mobile phase B, the balance is mobile phase A; 1-1.5 min: 95%-100% mobile phase B, the balance is mobile phase A; 1.5-2 min: 100% mobile phase B, the balance is mobile phase A; 2-2.1 min: 100%-70% mobile phase B, the balance mobile phase A; 2.1~3.5min: 70% mobile phase B, the balance mobile phase A.

[0029] More specifically, in step S2 of the above embodiment, the mass spectrometry conditions for HPLC-MS / MS detection are: using an electrospray ion source (ESI), positive ion ionization mode, multiple reaction ion monitoring (MRM) for scanning detection, the Venetoclax ion pair is 868.2→321, the d-8 Venetoclax ion pair is 876.2→329.0, the ion source voltage is 4500V; the interface temperature is 300°C; the collision gas is argon; the collision gas pressure is 230kPa; the heating gas is air; the nebulizing gas and the drying hot gas are both nitrogen; the heating gas flow rate is 10L / min; the nebulizing gas flow rate is 3L / min; the drying gas flow rate is 10L / min; the collision energy is 37V, and the scanning time is 50ms.

[0030] More specifically, in the above embodiment, a standard curve of venetoclax can be drawn using calibrators to confirm the concentration of venetoclax in the sample.

[0031] The technical solutions of the present invention are further described below by specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional understandings are defined herein for the purpose of illustrating or facilitating reference. Such definitions herein should not be construed as representing significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters provided by the manufacturers.

[0032] Example 1 Preparation of a kit for detecting venetoclax concentration Preparation of calibrators: The calibrators for this kit are 7 concentration gradients of venetoclax standard solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL. The specific preparation process is as follows: Preparation of standard stock solution: Dissolve 1 mg of venetoclax standard in methanol and dilute to a 10 mL volumetric flask. Mix by ultrasonic oscillation to obtain a 100 μg / mL venetoclax stock solution, which is then aliquoted for later use.

[0033] Take 10 μL of the 100 μg / mL venetoclax standard solution, add 990 μL of methanol, and shake to mix to obtain a 1 μg / mL venetoclax standard solution. Take 100 μL of the 1 μg / mL venetoclax standard solution, add 900 μL of 50% methanol-water solution, and shake to mix to obtain a 100 ng / mL venetoclax standard solution.

[0034] Take 500 μL of the 100 ng / mL venetoclax standard solution, add 500 μL of 50% methanol-water solution, and shake to mix to obtain a 50 ng / mL venetoclax standard solution.

[0035] Take 400 μL of the 50 ng / mL venetoclax standard solution, add 600 μL of 50% methanol-water solution, and shake to mix to obtain a 20 ng / mL venetoclax standard solution.

[0036] Take 500 μL of the 20 ng / mL venetoclax standard solution, add 500 μL of 50% methanol-water solution, and shake to mix to obtain a 10 ng / mL venetoclax standard solution.

[0037] Take 500 μL of the 10 ng / mL venetoclax standard solution, add 500 μL of 50% methanol-water solution, and shake to mix to obtain a 5 ng / mL venetoclax standard solution.

[0038] Take 400 μL of the 5 ng / mL venetoclax standard solution, add 600 μL of 50% methanol-water solution, and shake to mix to obtain a 2 ng / mL venetoclax standard solution.

[0039] Take 500 μL of the 2 ng / mL venetoclax standard solution, add 500 μL of 50% methanol-water solution, and shake to mix to obtain a 1 ng / mL venetoclax standard solution.

[0040] Preparation of quality control products: including low-concentration venetoclax methanol solution (2 ng / mL), medium-concentration venetoclax methanol solution (20 ng / mL), and high-concentration venetoclax methanol solution (80 ng / mL). The specific preparation process is as follows: Preparation of high-concentration venetoclax methanol solution: Take 800 μL of 100 ng / mL venetoclax standard solution, add 200 μL of 50% methanol-water solution, and shake to mix to obtain 80 ng / mL high-concentration venetoclax methanol solution.

[0041] Preparation of medium-concentration venetoclax methanol solution: Take 400 μL of 50 ng / mL venetoclax standard solution, add 600 μL of 50% methanol-water solution, and shake to mix to obtain 20 ng / mL medium-concentration venetoclax methanol solution.

[0042] Preparation of low-concentration venetoclax methanol solution: Take 100 μL of 20 ng / mL venetoclax standard solution, add 900 μL of 50% methanol-water solution, and shake to mix to obtain a 2 ng / mL low-concentration venetoclax methanol solution.

[0043] Preparation of internal standard stock solution: The internal standard solution uses the isotope labeled d8-venetoclax of the analyte venetoclax. The internal standard stock solution has a molar concentration of 50 ng / mL. When used, the internal standard stock solution needs to be diluted 10-fold to prepare the internal standard working solution. For example, 100 μL of the internal standard stock solution and 900 μL of the sample dilution treatment solution 50% (v / v) methanol aqueous solution are mixed and diluted 10-fold to prepare an internal standard-sample release agent with a concentration of 5 ng / mL.

[0044] Sample dilution solution: 50% (v / v) methanol in water.

[0045] Mobile phase: The mobile phase of this kit includes mobile phase A and mobile phase B, wherein mobile phase A is 5mM ammonium acetate aqueous solution and mobile phase B is methanol.

[0046] Example 2 Standard curve drawing 10 μL of calibrators and quality control products of each concentration were added to 90 μL of blank cerebrospinal fluid, mixed evenly, and then 200 μL of internal standard-sample release agent was added for HPLC-MS / MS detection to draw a standard curve.

[0047] In this example, the liquid chromatography conditions for HPLC-MS / MS detection are as follows: chromatographic column: Chromcore 120 C18 2.1 mm × 100 mm, 3 μm analytical column; column temperature 45° C.; flow rate 0.55 mL / min; injection volume 2 μL; mobile phase A is 5 mM ammonium acetate aqueous solution; mobile phase B is methanol; gradient elution program is: 0-1min: 95% mobile phase B, the balance is mobile phase A; 1-1.5 min: 95%-100% mobile phase B, the balance is mobile phase A; 1.5-2 min: 100% mobile phase B, the balance is mobile phase A; 2-2.1 min: 100%-70% mobile phase B, the balance mobile phase A; 2.1~3.5min: 70% mobile phase B, the balance mobile phase A.

[0048] HPLC-MS / MS mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) of venetoclax and its internal standard was performed using an electrospray ionization (ESI) source in positive ion mode. MS / MS spectra of venetoclax and its internal standard were first acquired in Q3 full scan mode. Full scan mass spectral data were then acquired in product ion scan mode. A collision energy gradient (-10 V to -40 V) was set to optimize fragment ion yields. Product ion scans confirmed that the characteristic ion pairs of the target and internal standard exhibited good specificity and no crosstalk. The ion transition for venetoclax was 868.2 → 321, and the ion transition for d-8 venetoclax was 876.2 → 329.0. The ion source voltage was 4500 V, the interface temperature was 300°C, the collision gas was argon, the collision pressure was 230 kPa, the heating gas was air, and both the nebulizing and drying gases were nitrogen. The heating gas flow rates were 10 L / min, the nebulizing gas flow rate was 3 L / min, and the drying gas flow rate was 10 L / min. The collision energy was 37 V, and the scan time was 50 ms.

[0049] The concentration (ng / mL) is the horizontal axis (X), the ratio of the measured peak area of ​​the 7 calibrators to the peak area of ​​the internal standard As / Ais is the vertical axis (Y), and 1 / x 2 The weight coefficient was regressed to establish the standard curve of Venetoclax. The standard curve is as follows: Figure 1 As shown, the linear regression equation is Y=0.104606X+0.00514873, r=0.9983003, and the linear relationship is good in the range of 0.1~10.0ng / mL.

[0050] Example 3 Detection of venetoclax concentration in human cerebrospinal fluid S1: Preparation of internal standard-sample release agent: dilute the internal standard d8-venetoclax stock solution (50 ng / mL) with a sample dilution solution of 50% (v / v) methanol in water to prepare an internal standard-sample release agent with a d8-venetoclax concentration of 5 ng / mL; S2: Sample pretreatment: Take 100 μL of cerebrospinal fluid containing venetoclax, add 200 μL of the internal standard-releasing agent prepared in step S1, vortex mix for 3-5 minutes, and centrifuge at 4°C and 14,000 × g for 10 minutes; S3: Pipette the supernatant into a clean centrifuge tube and centrifuge again under the conditions in step S2. The final supernatant is used as the test solution and subjected to HPLC-MS / MS detection. S4: Compare the results of HPLC-MS / MS detection with the standard curve and calculate the concentration of venetoclax in the sample.

[0051] In this example, the liquid chromatography conditions for HPLC-MS / MS detection are as follows: chromatographic column: Chromcore 120 C18 2.1 mm × 100 mm, 3 μm analytical column; column temperature 45° C.; flow rate 0.55 mL / min; injection volume 2 μL; mobile phase A is 5 mM ammonium acetate aqueous solution; mobile phase B is methanol; gradient elution program is: 0-1min: 95% mobile phase B, the balance is mobile phase A; 1-1.5 min: 95%-100% mobile phase B, the balance is mobile phase A; 1.5-2 min: 100% mobile phase B, the balance is mobile phase A; 2-2.1 min: 100%-70% mobile phase B, the balance mobile phase A; 2.1~3.5min: 70% mobile phase B, the balance mobile phase A.

[0052] HPLC-MS / MS mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) of venetoclax and its internal standard was performed using an electrospray ionization (ESI) source in positive ion mode. MS / MS spectra of venetoclax and its internal standard were first acquired in Q3 full scan mode. Full scan mass spectral data were then acquired in product ion scan mode. A collision energy gradient (-10 V to -40 V) was set to optimize fragment ion yields. Product ion scans confirmed that the characteristic ion pairs of the target and internal standard exhibited good specificity and no crosstalk. The ion transition for venetoclax was 868.2 → 321, and the ion transition for d-8 venetoclax was 876.2 → 329.0. The ion source voltage was 4500 V, the interface temperature was 300°C, the collision gas was argon, the collision pressure was 230 kPa, the heating gas was air, and both the nebulizing and drying gases were nitrogen. The heating gas flow rates were 10 L / min, the nebulizing gas flow rate was 3 L / min, and the drying gas flow rate was 10 L / min. The collision energy was 37 V, and the scan time was 50 ms. The HPLC-MS / MS test results were incorporated into the standard curve to obtain the concentration of venetoclax in the sample.

[0053] Example 4 Anti-interference performance verification Under the conditions of Example 2 and Example 3, the retention time of venetoclax was 1.656 min, and the retention time of d8-venetoclax was 1.652 min.

[0054] Six cerebrospinal fluid clinical samples from patients receiving venetoclax were collected for actual sample verification. After the same pretreatment, the samples were injected and analyzed to obtain the chromatograms as shown in the following figure. Figure 2 As shown. Figure 2It can be seen that the chromatographic peak shape of the target compound is symmetrical, and no metabolite or drug interference peaks were found in the patient samples, which confirms the applicability of the method to real complex biological samples and its strong anti-interference ability.

[0055] Example 5 Sensitivity testing The standard working solution of 1 ng / mL of venetoclax and the quality control product were added to the blank cerebrospinal fluid, and the whole process was processed and the sample was analyzed. The chromatogram was obtained as follows: Figure 3 As shown. Figure 3 It can be seen that the elution time of venetoclax and the internal standard is around 1.65 min, and the chromatographic peak shape is good, which proves that this method can meet the needs of trace detection and can achieve venetoclax detection as low as 0.1 ng / mL.

[0056] Example 6 Accuracy and precision verification 10 μL of each calibrator of varying concentrations from Example 1 was added to 90 μL of blank cerebrospinal fluid, mixed thoroughly, and then 200 μL of the internal standard-sample release agent was added. The mixture was shaken and centrifuged to prepare low, medium, and high quality control samples containing venetoclax at concentrations of 0.2 ng / mL, 2 ng / mL, and 8 ng / mL. The samples were then injected and analyzed using the HPLC-MS / MS detection conditions described in Example 2. Five replicates were run to assess intra-day precision. Measurements were repeated for 2-3 consecutive days, and the measured concentrations of the samples were calculated based on the standard curve for each day to assess the inter-day precision of the assay. The measured concentrations of the quality control samples were compared with the standard concentrations to assess the accuracy of the assay. The results are shown in Table 1. As can be seen from Table 1, the relative standard deviations (RSDs) for intra-day and inter-day precision were less than 15%, and the standard errors (REs) were less than 15%, meeting the basic requirements of the relevant guidelines.

[0057] Table 1 Example 7 Extraction recovery and matrix effect detection The quality control product in Example 1 was measured, 90 μL of blank cerebrospinal fluid was taken, 10 μL of working solution was added to prepare low, medium and high quality control solutions (0.1, 2, 8 ng / mL), vortexed thoroughly, 200 μL of internal standard-sample release agent was added, vortexed and centrifuged again, the supernatant was collected, and then sampled and analyzed according to the detection conditions of high performance liquid chromatography tandem mass spectrometry in Example 2, and the peak area A was determined; another 100 μL of blank cerebrospinal fluid was taken, 200 μL of methanol was added, vortexed and centrifuged for 15 minutes, and then 90 μL of the entire supernatant solution was taken into a new EP tube, 10 μL of working solution was added to prepare low, medium and high quality control solutions (0.1, 2, 8 ng / mL), and the sample was analyzed in the same way to measure the chromatographic peak area B; low, medium and high standard solutions (0.1, 2, 8 ng / mL) were prepared in the same way using methanol as the solvent, and the sample was analyzed to measure the chromatographic peak area C. The peak areas A\B obtained by the above measurements were compared with the chromatographic peak area C to evaluate the extraction recovery and matrix effect. The results are shown in Table 2.

[0058] Table 2 In Table 2, the extraction recovery rate and matrix effect are the means of five groups of data. It can be seen from Table 2 that the matrix effect has little influence on the kit of the present invention. At the same time, the recovery rate of the present invention is high.

[0059] Example 8 Stability test The high, medium, and low concentrations of the quality control products in Example 1 were used to perform room temperature stability tests (20°C for 4 hours), post-treatment stability tests (4°C for 24 hours), frozen stability tests (-80°C for 50 days), and freeze-thaw stability tests (freeze-thaw cycles 3 times). The peak area ratios of the measured results were substituted into the standard curve to calculate the measured values, and the room temperature, post-treatment, frozen, and repeated freeze-thaw stability tests were evaluated. The experimental results are shown in Table 3. As can be seen from Table 3, the measured values ​​are basically consistent with the added amounts, indicating that venetoclax is stable under all conditions.

[0060] Table 3 As can be seen from the above examples, the kit and detection method of the present invention are simple to operate, have accurate results, good linearity and stability, good extraction recovery, and the RSDs of intra-batch and inter-batch precision and accuracy are both less than 10%, which meets the requirements of clinical testing and can realize the monitoring of clinical batch samples.

[0061] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A kit for detecting the concentration of venetoclax, characterized in that: include: Calibrators: including several venetoclax methanol solutions with concentrations ranging from 1 to 100.0 ng / mL; Internal standard stock solution: including d8-Venetradecanoic acid methanol solution; Quality control products: including low-concentration venetoclax methanol solution, medium-concentration venetoclax methanol solution, and high-concentration venetoclax methanol solution. The venetoclax concentration range of the low-concentration venetoclax methanol solution is 1-5 ng / mL; the venetoclax concentration range of the medium-concentration venetoclax methanol solution is 10-50 ng / mL; and the venetoclax concentration range of the high-concentration venetoclax methanol solution is 60-100 ng / mL. Internal standard-sample release agent: including 5 ng / mL d8-venetoclax methanol solution; Sample dilution solution: 50% (v / v) methanol in water; Mobile phase: includes mobile phase A and mobile phase B, wherein mobile phase A is 5 mM ammonium acetate aqueous solution and mobile phase B is methanol.

2. The kit according to claim 1, wherein The calibrator includes seven concentration gradients, specifically 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

3. The kit according to claim 1, wherein The internal standard mother solution is a 10× internal standard solution, and the concentration of d8-venetoclax in the 10× internal standard solution is 50 ng / mL.

4. A method for detecting the concentration of venetoclax, characterized in that: The concentration of venetoclax is detected using the kit described in any one of claims 1 to 3.

5. The method for detecting the concentration of venetoclax as claimed in claim 4, wherein The following steps are involved: S1: Preparation of internal standard-sample release agent: dilute the internal standard d8-venetoclax stock solution with the sample dilution solution to prepare an internal standard-sample release agent with a d8-venetoclax concentration of 5 ng / mL; S2: Sample pretreatment: Take the sample to be tested, add the internal standard-sample release agent prepared in step S1 at a volume twice that of the sample to be tested, vortex mix, and centrifuge at 4°C for 10 minutes; S3: Transfer the supernatant to a clean centrifuge tube and centrifuge again at 4°C. Take the final supernatant as the test solution and perform HPLC-MS / MS detection. S4: Compare the results of HPLC-MS / MS detection with the standard curve and calculate the concentration of venetoclax in the sample.

6. The method for detecting the concentration of venetoclax as claimed in claim 5, wherein: The liquid chromatography conditions for HPLC-MS / MS detection are: column temperature 45°C; flow rate 0.55 mL / min; injection volume 2 μL, mobile phase A is the mobile phase A described in claim 1, mobile phase B is the mobile phase B described in claim 1, and the gradient elution program is: 0-1min: 95% mobile phase B, the balance is mobile phase A; 1-1.5 min: 95%-100% mobile phase B, the balance is mobile phase A; 5-2 min: 100% mobile phase B, the balance is mobile phase A; 2-2.1 min: 100%-70% mobile phase B, the balance mobile phase A; 1~3.5min: 70% mobile phase B, the balance mobile phase A.

7. The method for detecting the concentration of venetoclax as claimed in claim 5, wherein: The mass spectrometry conditions for HPLC-MS / MS detection are as follows: electrospray ion source-positive ion ionization-multiple reaction ion monitoring for scanning detection, Venetoclax ion pair is 868.2→321, d-8 Venetoclax ion pair is 876.2→329.0, ion source voltage is 4500V; interface temperature is 300℃; collision gas is argon; collision gas pressure is 230kPa; heating gas is air; nebulizing gas and drying hot gas are both nitrogen; heating gas flow rate is 10L / min; nebulizing gas flow rate is 3L / min; drying gas flow rate is 10L / min; collision energy is 37V, and scanning time is 50ms.

8. The method for detecting the concentration of venetoclax as claimed in claim 5, wherein: The sample is selected from any one or more of blood, plasma, cerebrospinal fluid, urine, venetoclax solution, and lymph fluid.

9. The method for detecting the concentration of venetoclax as claimed in claim 5, wherein: The concentration of venetoclax in the sample is 0.1-10 ng / mL.

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