A quantitative analytical method for the blood concentrations of HYQ-169 and prednisolone and its application.

Abstract

This invention discloses a quantitative analysis method for the blood concentrations of HYQ-169 and prednisolone and its application, belonging to the fields of medical testing and pharmaceutical analysis. The quantitative analysis method includes the following steps: preparing standard curve samples and quality control samples; adding internal standard solution; extracting with ethyl acetate; drying the supernatant and reconstituteing; separating using SPE; drying the fraction and reconstituteing again; detecting using UPLC-MS / MS; establishing a standard curve and obtaining a regression equation; adding internal standard solution to the plasma sample to be tested and processing it in the same way; calculating the blood concentrations of HYQ-169 and prednisolone according to the regression equation. This invention can simultaneously perform quantitative detection of two drug components with significantly different chemical properties in a single analysis, with high sensitivity, high accuracy, and fast analysis speed, providing a foundation for preclinical and clinical pharmacokinetic studies of HYQ-169.

Description

Technical Field

[0001] This invention belongs to the field of medical testing and pharmaceutical analysis technology, and in particular relates to a quantitative analysis method for the blood concentration of prednisolone and its metabolite after administration of HYQ-169 formulation. Background Technology

[0002] Ulcerative colitis is a chronic, nonspecific inflammatory bowel disease of unknown etiology. The lesions primarily affect the colonic mucosa and submucosa, clinically manifesting as persistent or recurrent diarrhea, bloody and mucous stools, and abdominal pain. The disease has a long course, is prone to relapse, severely impacts patients' quality of life, and can increase the risk of colon cancer. Glucocorticoids, such as prednisolone, are core drugs for inducing remission in moderate to severe active ulcerative colitis, effectively suppressing intestinal inflammation and controlling the disease. However, long-term or high-dose systemic use of glucocorticoids can lead to numerous serious adverse reactions, including immunosuppression, metabolic disorders (such as hypertension, hyperglycemia, and osteoporosis), increased risk of infection, and suppression of the hypothalamic-pituitary-adrenal axis, limiting their clinical application.

[0003] To improve treatment efficacy and reduce systemic side effects, local drug delivery (such as colon-targeted delivery) has become an important strategy for treating ulcerative colitis. Current local drug delivery methods mainly include rectal administration (enemas, suppositories) and oral colon-targeted formulations. However, rectal administration is only suitable for distal colonic lesions and has limited efficacy for proximal colonic lesions; while traditional oral colon-targeted formulations, although achieving drug release in the colon to some extent, still suffer from insufficient targeting precision, uneven drug distribution within the colon, and unstable local concentrations, affecting the sustainability and predictability of therapeutic effects.

[0004] To overcome the aforementioned limitations, by modifying prednisolone into the prodrug HYQ-169, which is activated only under specific colonic environments (such as specific pH, enzyme, or reducing conditions), more precise targeted release of active prednisolone at the colonic site can be achieved, increasing local drug concentration in the lesion while minimizing systemic exposure and side effects. Compared to existing formulations, this prodrug promises to achieve more stable and longer-lasting local colonic drug delivery, thereby significantly enhancing anti-inflammatory effects and substantially reducing systemic side effects. This provides a safer and more effective treatment option for patients with ulcerative colitis, possessing significant clinical development value and market potential.

[0005] However, the characteristics of such locally released drugs and the drugs themselves present significant challenges to preclinical and clinical drug development evaluation. HYQ-169 itself, as well as its metabolite prednisolone, have very low in vivo concentrations. Furthermore, because HYQ-169 has undergone structural modifications based on prednisolone, it is necessary to investigate the blood concentrations of both HYQ-169 and prednisolone in humans and animals to understand their absorption, distribution, metabolism, and excretion. Accurate, rapid, and convenient analytical methods are fundamental to understanding the in vivo properties of drugs. HYQ-169 is compound 1 in Table 1 of the specification in patent CN110650966A, as detailed below:

[0006] .

[0007] However, due to the inherent chemical structure of HYQ-169, its mass spectrometry response is extremely low, and the compound exhibits extremely high polarity. Its extraction efficiency, stability, and that of its metabolite prednisolone show drastically different characteristics. Furthermore, its polarity leads to strong matrix interference in mass spectrometry, severely impacting analytical accuracy. Achieving a rapid and convenient method for simultaneously detecting and analyzing the concentrations of both compounds using the same technique is challenging. Currently, there are no specific analytical methods for detecting HYQ-169 available domestically or internationally; therefore, there is a need for an accurate, efficient, simple, and rapid detection method. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to solve the problems of poor accuracy, low sensitivity and low efficiency in the detection of blood drug concentrations of HYQ-169 and prednisolone.

[0009] The present invention solves the above-mentioned technical problems through the following technical means: The first aspect of this invention provides a method for quantitative analysis of the blood concentrations of HYQ-169 and prednisolone, comprising the following steps: Standard curve samples and quality control samples were prepared using HYQ-169 standard, prednisolone standard, and blank biological matrix. After adding internal standard solution, the samples were extracted with ethyl acetate, the supernatant was dried and reconstituted, and then separated using SPE. The fraction was dried and reconstituted again, and the samples were detected using UPLC-MS / MS. A standard curve was established and a regression equation was obtained. The plasma samples to be tested were treated in the same way after adding internal standard solution (i.e., extraction with ethyl acetate, drying and reconstituted with supernatant, separation using SPE, drying and reconstituted again, and detection using UPLC-MS / MS). The blood drug concentrations of HYQ-169 and prednisolone were calculated using the regression equation. The high-performance liquid chromatography (HPLC) conditions for UPLC-MS / MS were as follows: the column was a Kinetex 5μm Biphenyl 100Å LC column (50×4.6mm); mobile phase A was 0.05–0.2% formic acid aqueous solution, and mobile phase B was methanol; the flow rate was 0.45–0.55 mL / min; and the column temperature was 40–46 °C. Gradient elution is performed, and the gradient elution procedure is as follows: Within 0-2.3 min, the volume ratio of mobile phase A to B is 30:70; Within 2.3-2.5 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 30:70 to 0:100; Within 2.5-3.5 min, the volume ratio of mobile phase A to mobile phase B is maintained at 0:100; Within 3.5-3.51 min, the volume ratio of mobile phase A to mobile phase B gradually changed from 0:100 to 30:70; Within 3.51-4 min, the volume ratio of mobile phase A to mobile phase B was maintained at 30:70; The mass spectrometry conditions for UPLC-MS / MS were as follows: ESI ion source; negative ion scanning mode; spray voltage of 3-4 kV; interface temperature of 280-320℃; DL temperature of 220-270℃; heating block temperature of 390-410℃; nebulizer gas flow rate of 2.2-2.8 L / min; heating gas flow rate of 8-12 L / min; and drying gas flow rate of 8-12 L / min.

[0010] Prednisolone: ​​Its foreign name is prednisolone, and its chemical name is 11β,17α,21-trihydroxypregn-1,4-diene-3,20-dione.

[0011] The standard curve samples for HYQ-169 and prednisolone blood concentrations were prepared using HYQ-169 standard, prednisolone standard, and a blank biological matrix. The quality control samples were prepared using a blank biological matrix. Preferably, the blank biological matrix is ​​whole blood from healthy humans or animals that has been anticoagulated and separated by EDTA-K2, stored at -80°C, and thawed naturally at room temperature before use.

[0012] Preferably, the plasma sample to be tested is whole blood from humans or animals after oral administration of HYQ-169 preparation, which is then anticoagulated and separated by EDTA-K2, stored at -80°C for testing, and thawed naturally at room temperature before use.

[0013] Preferably, the internal standard in the internal standard solution is HYQ-169-d3 and prednisolone-d3.

[0014]

[0015] Prednisolone-d3 Internal standard HYQ-169-d3 was used for the analysis of HYQ-169; internal standard prednisolone-d3 was used for the analysis of prednisolone.

[0016] Preferably, the concentration of HYQ-169-d3 and prednisolone-d3 is 3500~4500 ng / mL; more preferably 4000 ng / mL.

[0017] Preferably, after adding the internal standard solution, extraction with ethyl acetate is performed, and the supernatant is dried and then reconstituted. Specifically: Mix the sample with the internal standard solution, add ethyl acetate, vortex and centrifuge, take the supernatant and dry it under nitrogen, then add methanol, vortex, add ultrapure water, vortex, and take all the reconstituted solutions for later use.

[0018] A further preferred method is as follows: Add 45-55 μL of each sample to an EP tube, add 15-25 μL of internal standard solution, then add 950-1050 μL of ethyl acetate, and vortex for 4-7 min; centrifuge at 12000-14000 g for 4-6 min at room temperature; then transfer 850-950 μL of supernatant to a new 96-well plate and dry it under nitrogen at 45-55 °C; then add 48-55 μL of methanol, vortex for 4-6 min (1100-1300 rpm), then add 150-250 μL of ultrapure water, vortex for 4-6 min (1100-1300 rpm), and collect all the reconstituted solutions for later use.

[0019] A further preferred method is as follows: Add 50 μL of each sample to an EP tube, add 20 μL of internal standard solution, then add 1000 μL of ethyl acetate, and vortex for 5 min; centrifuge at 13000 g for 5 min at room temperature; then transfer 900 μL of supernatant to a new 96-well plate and dry it under nitrogen at 50 °C; then add 50 μL of methanol, vortex for 5 min (speed: 1200 rpm), then add 200 μL of ultrapure water, vortex for 5 min (speed: 1200 rpm), and collect all the reconstituted solutions for later use.

[0020] Preferably, separation is performed using SPE, and the fraction is dried and then reconstituted. Specifically, the reconstituted solution is loaded onto an SPE plate and dried under negative pressure. Then, it is eluted sequentially with purified water, 4-6% methanol-water, 18-22% methanol-water, and 68-72% methanol-water, each time dried under negative pressure and collected in a new 96-well plate. After collecting the eluent, the 96-well plate is dried under nitrogen at 55-65°C until completely dry. 45-55% methanol-water is added and mixed at 1100-1300 rpm for 8-12 minutes, and then it is ready for analysis.

[0021] Preferably, the elution is performed sequentially with purified water, 4-6% methanol-water, 18-22% methanol-water, and 68-72% methanol-water, specifically as follows: (0.4-0.6) mL of purified water is rinsed 1-2 times, (0.4-0.6) mL of 4-6% methanol-water is rinsed 2-3 times, and (0.4-0.6) mL of 18-22% methanol-water is rinsed 3-4 times.

[0022] A further preferred method is as follows: the entire reconstituted solution is loaded onto an SPE plate, dried under negative pressure, then rinsed once with 0.5 mL of purified water, twice with 0.5 mL of 5% methanol-water, and three times with 0.5 mL of 20% methanol-water, each time dried under negative pressure; eluted twice with 0.4 mL of 70% methanol-water, dried under negative pressure, and collected entirely in a new 96-well plate; after collecting the eluent, the 96-well plate is dried completely under nitrogen at 60°C; 100 µL of 50% methanol-water is added and mixed at 1200 rpm for 10 minutes, then set aside for analysis.

[0023] Preferably, the SPE plate is a Panthera Deluxe Polymer DVB 96-well SPE plate with a specification of 30 mg / 1 mL; before use, the SPE plate is activated with methanol and then equilibrated with purified water.

[0024] Preferably, the injection volume in high performance liquid chromatography is 8~15 μL; more preferably 10 μL.

[0025] A second aspect of the present invention proposes the application of the above-mentioned quantitative analysis method in monitoring the blood concentrations of HYQ-169 and prednisolone.

[0026] The beneficial effects of this invention are as follows: 1. The quantitative analysis method for the blood concentration of HYQ-169 original drug and metabolites proposed in this invention adopts liquid-liquid extraction and SPE mixed extraction method, and uses UPLC-MS / MS technology and develops suitable detection conditions. Through the optimization of extraction method conditions, chromatographic column, mobile phase, gradient elution conditions and other detection conditions, the method ingeniously achieves the extraction and separation of two components in a single injection, and successfully retains the two components in the chromatographic system under specific conditions, greatly reducing matrix effects.

[0027] 2. This analytical method can be used for quantitative detection. The linear range of HYQ-169 is 0.1-200 ng / mL, and the linear range of prednisolone is also 0.1-200 ng / mL. It has high detection sensitivity, high accuracy, low matrix effect, and fast analysis speed, making it convenient and quick (HYQ-169 and prednisolone can be analyzed simultaneously within 4 minutes after sample processing). This provides a solid and reliable analytical basis for preclinical and clinical pharmacokinetic studies of HYQ-169.

[0028] 3. This invention can simultaneously perform quantitative detection of two drug components with significantly different chemical properties in a single analysis. It features high detection sensitivity, high accuracy, and fast analysis speed, providing a foundation for preclinical and clinical pharmacokinetic studies of HYQ-169.

[0029] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0030] Figure 1 This is a typical liquid chromatography-mass spectrum of HYQ-169 in the human plasma standard curve sample of Example 1 of the present invention; Figure 2 This is a typical liquid chromatography-mass spectrum of the internal standard HYQ-169-d3 in human plasma in Example 1 of this invention; Figure 3 This is a typical liquid chromatography-mass spectrum of prednisolone in the human plasma standard curve sample of Example 1 of the present invention; Figure 4 This is a typical liquid chromatography-mass spectrum of the internal standard prednisolone-d3 in human plasma in Example 1 of the present invention; Figure 5 This is a standard curve of HYQ-169 in human plasma in Example 1 of the present invention; Figure 6 This is a standard curve of prednisolone in human plasma in Example 1 of the present invention; Figure 7 This is a typical liquid chromatography-mass spectrum of HYQ-169 in the rat plasma standard curve sample of Example 2 of the present invention; Figure 8 This is a typical liquid chromatography-mass spectrum of the internal standard HYQ-169-d3 in rat plasma in Example 2 of this invention; Figure 9 This is a typical liquid chromatography-mass spectrum of prednisolone in the rat plasma standard curve sample of Example 2 of the present invention; Figure 10 This is a typical liquid chromatography-mass spectrum of prednisolone-d3 in rat plasma in Example 2 of the present invention; Figure 11 This is a standard curve of HYQ-169 in rat plasma in Example 2 of the present invention; Figure 12 This is a standard curve of prednisolone in rat plasma in Example 2 of the present invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0032] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0033] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0034] Example 1: A method for quantitative analysis of the blood concentrations of HYQ-169 (original form) and its metabolite prednisolone, comprising the following steps: I. Solution preparation: 1. Preparation of standard curve solutions 1.1 Preparation of Standard Stock Solution Weigh approximately 10 mg of HYQ-169 standard and prednisolone standard into the same glass sample vial. Add a certain volume of methanol solution to prepare a mixed stock solution of HYQ-169 standard and prednisolone standard with a concentration of 1 mg / mL. Vortex the mixed stock solution thoroughly until completely dissolved and store it at -20℃ as a linear standard stock solution (SS-TA-A-1). Similarly, prepare a mixed stock solution as a quality control sample stock solution (SS-TA-A-2).

[0035] 1.2 Preparation of working solutions for plasma standard curve and quality control working solutions Plasma standard curve working solutions: Dilute linear standard stock solution (SS-TA-A-1) with methanol to prepare a series of plasma standard curve working solutions (WS-TA, for mixed quantitative standard working solutions of HYQ-169 and prednisolone) with concentrations of 2ng / mL, 4ng / mL, 10ng / mL, 40ng / mL, 100ng / mL, 200ng / mL, 400ng / mL, 1000ng / mL and 4000ng / mL.

[0036] Quality control working solutions: Dilute the quality control sample stock solution (SS-TA-A-2) with methanol to prepare a series of plasma quality control curve working solutions (WS-QC, for mixed quality control working solutions of HYQ-169 and prednisolone) with concentrations of 6 ng / mL, 100 ng / mL, 2000 ng / mL and 3000 ng / mL.

[0037] 1.3 Preparation of standard curve samples and quality control plasma samples Standard curve samples: 10 μL of each of the above standard curve working solutions (WS-TA) were added to 190 μL of blank human plasma matrix to obtain a series of standard curve samples with HYQ-169 and prednisolone concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 200 ng / mL, respectively. Quality control plasma samples: Add 10 μL of each of the above quality control curve working solutions (WS-QC) to 190 μL of blank human plasma matrix to obtain low-concentration quality control samples (QCL: HYQ-169 0.3 ng / mL, prednisolone 0.3 ng / mL), geometrically medium-concentration quality control samples (QCGM: HYQ-169 5 ng / mL, prednisolone 5 ng / mL), medium-concentration quality control samples (QCM: HYQ-169 100 ng / mL, prednisolone 100 ng / mL), and high-concentration quality control samples (QCH: HYQ-169 150 ng / mL, prednisolone 150 ng / mL). The blank biological matrix is ​​plasma from whole blood of healthy individuals that has been anticoagulated and separated by EDTA-K2, stored at -80℃, and thawed naturally at room temperature before use.

[0038] 2. Preparation of internal standard solution Accurately weigh 10.000 mg of internal standard 1 HYQ-169-d3 and 10.000 mg of internal standard 2 prednisolone-d3 into a glass sample vial. After dissolving completely in an appropriate amount of methanol, a mixed internal standard stock solution (MIX-IS-SS, HYQ-169-d3 and prednisolone-d3 concentrations are all 1 mg / mL) is obtained. Take an appropriate amount of internal standard stock solution (MIS-IS-SS) and dilute it with an appropriate amount of methanol to prepare a mixed internal standard working solution with a concentration of 4000 ng / mL (MIX-IS-WS, HYQ-169-d3 and prednisolone-d3 concentrations are all 4000 ng / mL).

[0039] 3. Processing of standard curve samples and quality control samples Add 50 μL of the mixed sample to each EP tube, add 20 μL of internal standard working solution, then add 1000 μL of ethyl acetate, and vortex for 5 min. Centrifuge at 13000 g for 5 min at room temperature. Transfer 900 μL of the supernatant to a new 96-well plate and dry under nitrogen at 50 °C. Add 50 μL of methanol, vortex for 5 min (1200 rpm), then add 200 μL of ultrapure water, vortex for 5 min (1200 rpm), and collect all reconstituted solutions for later use. Use Panthera Deluxe, Polymer DVB 96-well plate, and 30 mg / 1 mL SPE plate. Activate the SPE with 0.6 mL of methanol, then equilibrate with 0.6 mL of purified water. The reconstituted solution was loaded onto an SPE plate and dried under negative pressure. The plate was then washed once with 0.5 mL of purified water, twice with 0.5 mL of 5% methanol-water solution, and three times with 0.5 mL of 20% methanol-water solution, each time dried under negative pressure. The plate was then eluted twice with 0.4 mL of 70% methanol-water solution, dried under negative pressure, and the eluent was collected in a new 96-well plate. After collecting the eluent, the 96-well plate was incubated at 60°C under nitrogen until completely dry. 100 µL of 50% methanol-water solution was added and mixed at 1200 rpm for 10 minutes. The mixture was then injected for LC-MS / MS analysis.

[0040] 4. Processing of the plasma to be tested The plasma to be tested is whole blood from a person who has been orally administered HYQ-169, which has been anticoagulated and separated by EDTA-K2. It is stored at -80℃ and thawed naturally at room temperature before use. Take 50 μL of plasma sample, add 20 μL of internal standard working solution, then add 1000 μL of ethyl acetate, and vortex for 5 min. Centrifuge at 13000 g for 5 min at room temperature. Transfer 900 μL of supernatant to a new 96-well plate and dry under nitrogen at 50 °C. Add 50 μL of methanol, vortex for 5 min (1200 rpm), then add 200 μL of ultrapure water, vortex for 5 min (1200 rpm), and collect all reconstituted solutions for later use. Select Panthera Deluxe, Polymer DVB 96-well plate, 30 mg / 1 mL SPE plate, activate SPE with 0.6 mL of methanol, and then equilibrate with 0.6 mL of purified water. The reconstituted solution was loaded onto an SPE plate and dried under negative pressure. The plate was then rinsed once with 0.5 mL of purified water, twice with 0.5 mL of 5% (v / v) methanol-water, and three times with 0.5 mL of 20% methanol-water, each time dried under negative pressure. The plate was then eluted twice with 0.4 mL of 70% methanol-water, dried under negative pressure, and the eluent was collected in a new 96-well plate. After collecting the eluent, the 96-well plate was incubated at 60°C under nitrogen until completely dry. 100 µL of 50% methanol-water was added and mixed at 1200 rpm for 10 minutes. The mixture was then injected for LC-MS / MS analysis.

[0041] II. Testing: Detection was performed using a Shimadzu LCMS-8045 liquid chromatography-mass spectrometry (LC-MS) system. The high-performance liquid chromatography (HPLC) conditions were as follows: chromatographic column: Kinetex 5μm Biphenyl 100Å LC column. The column consisted of 50×4.6 mm particles with a particle size of 5 μm; mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was methanol; the flow rate was 0.5 mL / min; the column temperature was 45 °C; gradient elution was performed, and the gradient elution program was as follows: from 0 to 2.3 min, the volume ratio of mobile phase A to mobile phase B was 30:70; from 2.3 to 2.5 min, the volume ratio of mobile phase A to mobile phase B gradually changed from 30:70 to 0:100; from 2.5 to 3.5 min, the volume ratio of mobile phase A to mobile phase B was maintained at 0:100; from 3.5 to 3.51 min, the volume ratio of mobile phase A to mobile phase B gradually changed from 0:100 to 30:70; from 3.51 to 4 min, the volume ratio of mobile phase A to mobile phase B was maintained at 30:70; the washing solution was a mixed solution of methanol, isopropanol, and water in a volume ratio of 1:1:1. The mass spectrometry conditions were as follows: ESI ion source; negative ion scanning mode; spray voltage 3.5 kV; interface temperature 300℃; DL temperature 250℃; heating block temperature 400℃; nebulizer gas flow rate 2.5 L / min; heating gas flow rate 10 L / min; drying gas flow rate 10 L / min.

[0042] The ion pairs, residence times, CE, and other parameters of HYQ-169, prednisolone, internal standard HYQ-169-d3, and prednisolone-d3 are as follows:

[0043] Typical atlases are shown below Figure 1-6 .

[0044] The HYQ-169 standard curve regression equation is Y = (0.0378811)X + (0.00500738), r2=0.9980582, and the limit of quantitation is 0.1 ng / mL; The prednisolone monoclonal antibody curve regression equation is Y = (0.0603633)X + (0.000444055), r2=0.9993723, and the limit of quantitation is 0.1 ng / mL.

[0045] Results of precision and accuracy data for three consecutive batches of HYQ-169 quality control:

[0046] Precision and accuracy results for three consecutive batches of prednisolone:

[0047] In plasma, the concentrations of HYQ-169 and prednisolone in the range of 0.1-200 ng / mL showed good linearity with the analyte-to-internal-standard ratio, with correlation coefficients (r) greater than 0.999. Precision and accuracy were well-matched, and the method met the analytical requirements.

[0048] Example 2: A method for quantitative analysis of the blood concentrations of HYQ-169 original drug and its metabolites, comprising the following steps: I. Solution preparation: 1. Preparation of standard curve solutions 1.1 Preparation of Standard Stock Solution Same as Example 1 1.2 Preparation of working solutions for plasma standard curve and quality control working solutions Same as Example 1 1.3 Preparation of standard curve samples and quality control plasma samples Standard curve samples: 10 μL of each of the above standard curve working solutions (WS-TA) were added to 190 μL of blank rat plasma matrix to obtain a series of standard curve samples with HYQ-169 and prednisolone concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 200 ng / mL, respectively. Quality control plasma samples: 10 μL of each of the above quality control curve working solutions (WS-QC) were added to 190 μL of blank rat plasma matrix to obtain low-concentration quality control samples (QCL: HYQ-169 0.3 ng / mL, prednisolone 0.3 ng / mL), geometric medium-concentration quality control samples (QCGM: HYQ-169 5 ng / mL, prednisolone 5 ng / mL), medium-concentration quality control samples (QCM: HYQ-169 100 ng / mL, prednisolone 100 ng / mL), and high-concentration quality control samples (QCH: HYQ-169 150 ng / mL, prednisolone 150 ng / mL). The blank biological matrix was obtained by centrifuging whole blood from healthy SD rats after anticoagulation with EDTA-K2, storing it at -80℃, and thawing it naturally at room temperature before use.

[0049] 2. Preparation of internal standard solution Same as Example 1 3. Processing of standard curve samples and quality control samples Same as Example 1 4. Processing of the plasma to be tested The plasma to be tested was whole blood from rats that had been orally administered HYQ-169, which was then anticoagulated with EDTA-K2 and separated. The plasma was stored at -80°C and thawed naturally at room temperature before use. Take 50 μL of plasma sample, add 20 μL of internal standard working solution, then add 1000 μL of ethyl acetate, and vortex for 5 min. Centrifuge at 13000 g for 5 min at room temperature. Transfer 900 μL of supernatant to a new 96-well plate and dry under nitrogen at 50 °C. Add 50 μL of methanol, vortex for 5 min (1200 rpm), then add 200 μL of ultrapure water, vortex for 5 min (1200 rpm), and collect all reconstituted solutions for later use. Select Panthera Deluxe, Polymer DVB 96-well plate, 30 mg / 1 mL SPE plate, activate SPE with 0.6 mL of methanol, and then equilibrate with 0.6 mL of purified water. The reconstituted solution was loaded onto an SPE plate and dried under negative pressure. The plate was then rinsed once with 0.5 mL of purified water, twice with 0.5 mL of 5% methanol-water solution, and three times with 0.5 mL of 20% methanol-water solution, each time dried under negative pressure. The plate was then eluted twice with 0.4 mL of 70% methanol-water solution, dried under negative pressure, and the eluent was collected in a new 96-well plate. After collecting the eluent, the 96-well plate was incubated at 60°C under nitrogen until completely dry. 100 µL of 50% methanol-water solution was added and mixed at 1200 rpm for 10 minutes. The mixture was then injected for LC-MS / MS analysis.

[0050] II. Testing: Detection was performed using a Shimadzu LCMS-8045 liquid chromatography-mass spectrometry (LC-MS) system. Same as Example 1 Typical atlases are shown below Figure 7-12 .

[0051] The HYQ-169 standard curve linear regression equation is Y = (0.00148786)X + (0.00193353), r2=0.9892005; The prednisolone cyclophosphamide curve regression equation is Y = (0.0171834)X + (0.000368094), r2=0.9967825.

[0052] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A quantitative analytical method for the blood concentrations of HYQ-169 and prednisolone, characterized in that, Includes the following steps: Standard curve samples and quality control samples were prepared using HYQ-169 standard, prednisolone standard, and blank biological matrix. After adding internal standard solution, the samples were extracted with ethyl acetate, the supernatant was dried, reconstituted, and separated using SPE. The fraction was dried, reconstituted again, and detected by UPLC-MS / MS to establish a standard curve and obtain a regression equation. The plasma samples to be tested were treated with the same method after adding internal standard solution, and the blood drug concentrations of HYQ-169 and prednisolone were calculated according to the regression equation. The high-performance liquid chromatography (HPLC) conditions for UPLC-MS / MS were as follows: the column was a Kinetex 5μm Biphenyl 100Å LCColumn 50×4.6mm column; mobile phase A was 0.05~0.2% formic acid aqueous solution, and mobile phase B was methanol; the flow rate was 0.45~0.55mL / min; and the column temperature was 40~46℃. Gradient elution is performed, and the gradient elution procedure is as follows: Within 0-2.3 min, the volume ratio of mobile phase A to B is 30:70; Within 2.3-2.5 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 30:70 to 0:100; Within 2.5-3.5 min, the volume ratio of mobile phase A to mobile phase B is maintained at 0:100; Within 3.5-3.51 min, the volume ratio of mobile phase A to mobile phase B gradually changed from 0:100 to 30:70; Within 3.51-4 min, the volume ratio of mobile phase A to mobile phase B was maintained at 30:70; The mass spectrometry conditions for UPLC-MS / MS were as follows: ESI ion source; negative ion scanning mode; spray voltage of 3-4 kV; interface temperature of 280-320℃; DL temperature of 220-270℃; heating block temperature of 390-410℃; nebulizer gas flow rate of 2.2-2.8 L / min; heating gas flow rate of 8-12 L / min; and drying gas flow rate of 8-12 L / min.

2. The quantitative analysis method according to claim 1, characterized in that, The blank biological matrix is ​​whole blood from healthy humans or animals that has been anticoagulated and separated by EDTA-K2, stored at -80°C, and thawed naturally at room temperature before use.

3. The quantitative analysis method according to claim 1, characterized in that, The plasma sample to be tested is whole blood from humans or animals after oral administration of HYQ-169 preparation, which is then anticoagulated and separated by EDTA-K2, stored at -80℃ for testing, and thawed naturally at room temperature before use.

4. The quantitative analysis method according to claim 1, characterized in that, The internal standard in the internal standard solution is HYQ-169-d3 and prednisolone-d3; the concentration of HYQ-169-d3 and prednisolone-d3 is 3500~4500 ng / mL.

5. The quantitative analysis method according to claim 1, characterized in that, After adding the internal standard solution, extraction with ethyl acetate was performed, and the supernatant was dried and reconstituted. Specifically: Mix the sample with the internal standard solution, add ethyl acetate, vortex and centrifuge, take the supernatant and dry it under nitrogen, then add methanol, vortex, add ultrapure water, vortex, and take all the reconstituted solutions for later use.

6. The quantitative analysis method according to claim 1, characterized in that, Separation was performed using SPE, and the fraction was dried and then reconstituted. Specifically, the reconstituted solution was loaded onto an SPE plate and dried under negative pressure. Then, it was eluted sequentially with purified water, 4-6% methanol-water, 18-22% methanol-water, and 60-80% methanol-water, each time under negative pressure. Only the 60-80% methanol-water eluent was collected and placed in a new 96-well plate. After collecting the eluent, the 96-well plate was dried under nitrogen at 25-65°C until completely dry. 20-70% methanol-water was added and mixed for 8-12 minutes, and then it was ready for analysis.

7. The quantitative analysis method according to claim 6, characterized in that, The elution was carried out sequentially with purified water, 4-6% methanol-water, 18-22% methanol-water, and 68-72% methanol-water, specifically as follows: (0.4-0.6) mL of purified water was rinsed 1-2 times, (0.4-0.6) mL of 4-6% methanol-water was rinsed 2-3 times, and (0.4-0.6) mL of 18-22% methanol-water was rinsed 3-4 times.

8. The quantitative analysis method according to claim 6, characterized in that, The SPE plate is a Panthera DeluxePolymer DVB 96-well SPE plate with a specification of 30 mg / 1 mL.

9. The quantitative analysis method according to claim 1, characterized in that, Under high performance liquid chromatography conditions, the injection volume is 8~15μL.

10. The use of the quantitative analysis method according to any one of claims 1-9 in monitoring the blood concentrations of HYQ-169 and prednisolone.

Images