Detection method of siRNA drug metabolites
By optimizing the erosion and elution buffer formulation of solid-phase extraction, combined with high-performance liquid chromatography-high resolution mass spectrometry, the problem of low recovery in the detection of siRNA drug metabolites is solved, and higher recovery and detection accuracy is achieved, supporting effective evaluation and optimization of drugs.
Patent Information
- Application Number
- CN202411966507.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-11
AI Technical Summary
In the detection of siRNA drug metabolites, solid phase extraction methods have problems with low recovery rate or loss of target substances, which affects the accuracy and sensitivity of the detection.
The elution and elution buffer formulation of solid-phase extraction was optimized, and the elution and elution were elution and elution were used to elute and elute, combined with high-performance liquid chromatography-high resolution mass spectrometry combined use, and the operation conditions were optimized to improve the recovery rate of siRNA drug metabolites.
It significantly improves the recovery rate of siRNA drug metabolites, ensures the accuracy and sensitivity of the test results, can better evaluate the efficacy and potential toxicity of the drug, and provides a basis for the clinical application and dose adjustment of the drug.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for detecting siRNA drug metabolites. Background Art
[0002] As one of the hot spots in drug development in recent years, oligonucleotide drugs have attracted more and more attention. Oligonucleotide drugs are a class of drugs composed of single-stranded or double-stranded oligonucleotides synthesized by artificial chemical synthesis, including antisense nucleic acids (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), nucleic acid aptamers (aptamer), ribozymes (ribozyme), etc. Among them, siRNA-type oligonucleotide drugs are widely used and have gradually been applied to many aspects such as anti-tumor, virus treatment, and immune regulation.
[0003] Small interfering RNA (siRNA) is a double-stranded RNA with a length of 15 to 25 nucleotides. It is an important type of small nucleic acid drug and is also called short interfering RNA or silencing RNA. Based on the RNA interference mechanism, gene silencing or activation therapy is performed at the post-transcriptional level. Compared with traditional drugs that work at the protein level, it has obvious advantages such as high specificity, high efficiency, and long-term effect.
[0004] In order to more accurately evaluate the absorption, distribution, metabolism and excretion (ADME) characteristics of siRNA drugs in vivo, it is necessary to detect their sense and antisense chains and metabolites. The methods involved include nucleic acid-based enzyme-linked bridging analysis (ELBA) method, real-time reverse transcription polymerase chain reaction (RT-qPCR), hybrid immunoassay, liquid chromatography-mass spectrometry (LC-MS), etc.; among them, the LC-MS method uses mass spectrometry for direct quantification, does not require enzymes and special reagents, has good repeatability, high selectivity, high throughput, and wide linearity. It can simultaneously detect the sense and antisense chains of siRNA and distinguish metabolites from analytes. It has wide applicability and higher sensitivity and specificity.
[0005] Since the drug metabolites of siRNA are usually at low concentrations and the sample matrix is complex, direct analysis may cause signal interference and background noise. Pretreatment is required to improve the detection sensitivity, remove interference, and ensure the accuracy and repeatability of the test results. Conventional sample pretreatment methods include protein precipitation, liquid-liquid extraction, and solid phase extraction. Among them, solid phase extraction can effectively separate and enrich target siRNA molecules from complex biological samples. Compared with traditional liquid phase extraction, this method has the advantages of simple operation, solvent saving, high recovery rate, and easy automation.
[0006] However, solid phase extraction pretreatment also has some disadvantages in improving the recovery rate of siRNA drug metabolites. For example, improper operating conditions may lead to reduced recovery or loss of target. Therefore, it is urgent to develop a detection method for siRNA drug metabolites to maximize the recovery rate and analysis accuracy of siRNA drug metabolites. Summary of the invention
[0007] In view of the problems in the prior art, the present invention provides a method for detecting siRNA drug metabolites.
[0008] A method for detecting siRNA drug metabolites comprises the following steps:
[0009] Step 1: Pre-treat the sample to be tested by solid phase extraction: after the OTX solid phase extraction plate is activated, it is balanced with a balancing buffer NaH2PO4-water solution; NaH2PO4-acetonitrile water solution and NH4HCO3-acetonitrile water solution are used as elution buffers in sequence for elution; elution is performed with an elution buffer NH4HCO3-ethanol-acetonitrile-water solution, the eluate is collected, dried and redissolved to obtain a sample solution; the concentration of the NaH2PO4-water solution is 45-55mmol / L, and the solution pH is 5-6; the NaH2PO4 concentration in the NaH2PO4-acetonitrile water solution is 45-55mmol / L, the volume fraction of acetonitrile is 45-55%, and the solution pH is 5-6; the NH4HCO3 concentration in the NH4HCO3-acetonitrile water solution is 45-55mmol / L, the volume fraction of acetonitrile is 75-85%, and the solution pH is 5-6.
[0010] Step 2: The sample solution is detected by high performance liquid chromatography-high resolution mass spectrometry.
[0011] Preferably, in step 1, the volume fraction of ethanol in the elution buffer NH4HCO3-ethanol-acetonitrile-water solution is 10-20%, the volume fraction of acetonitrile is 30-40%, the concentration of NH4HCO3 is 90-110 mmol / L, and the pH of the elution buffer is 8-10.
[0012] Preferably, the volume fraction of the ethanol is 20%.
[0013] Preferably, the volume fraction of the acetonitrile is 30%.
[0014] Preferably, the concentration of NH4HCO3 is 100 mmol / L; and the pH of the elution buffer is 9.5.
[0015] Preferably, in step 1, the method for preparing the sample to be tested is: adding siRNA drug working solution to the biological matrix to obtain a mixed solution 1; adding a lysis buffer to the mixed solution 1 to obtain; the volume ratio of the biological matrix to the siRNA drug working solution is ≥9:1, and the volume ratio of the mixed solution 1 to the lysis buffer is 1:1-1:2.
[0016] Preferably, the biological matrix is at least one of feces, bile and urine.
[0017] Preferably, in step 2, the chromatographic conditions of the high performance liquid chromatography-high resolution mass spectrometry are as follows: the chromatographic column is selected from Thermo Scientific DNAPacTM RP column (4μm, 2.1×50mm) or XTerra MS C18 column (125A, 2.5μm, 2.1×50mm); the mobile phase A is (0.1%-0.5%) TEA-(0.1%-0.5%) HFIP-aqueous solution, and the mobile phase B is (0.1%-0.5%) TEA-(0.1%-0.5%) HFIP-methanol solution, and gradient elution is performed at a flow rate of 0.2-0.3mL / min; the injection volume is 1-5μL, the column temperature is 60-80°C, and the wash solution is 0-50% isopropanol aqueous solution.
[0018] Preferably, the gradient elution conditions are shown in the following table:
[0019]
[0020] Preferably, in step 2, the mass spectrometry conditions of the high performance liquid chromatography-high resolution mass spectrometry are as follows: the ion source is a heated electrospray ion source, the spray voltage is 2000-3000 V, the sheath gas is 10-50 mL / min, the auxiliary gas is 10-50 mL / min, the sweep gas is 0-1 mL / min, the ion transfer tube temperature is 300-400°C, the solvation temperature is 300-400°C, the resolution is 60000-120000, and the scanning range is 570-2000 m / z.
[0021] The present invention optimizes the formula of the elution buffer and the elution buffer in the solid phase extraction (SPE) pretreatment method, thereby improving the recovery rate of siRNA drug metabolites. The detection method of the present invention can better evaluate the efficacy and potential toxicity of the drug, which is of great significance to the clinical application and dosage adjustment of the drug.
[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.
[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION
[0024] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.
[0025] Example 1 Detection Method of siRNA Drug Metabolites
[0026] 1. Use solid phase extraction method to pretreat the sample to be tested
[0027] 1) Preparation of test samples: Take 10 μL of 50 μM Inclisiran working solution, add 990 μL of feces, urine and bile respectively, and then add an equal volume of lysis buffer, mix well, and use as the test sample.
[0028] 2) Activation: Use 1 mL of methanol to activate the OTX solid phase extraction plate.
[0029] 3) Equilibration: Use 1 mL of equilibration buffer (50 mM NaH2PO4-aqueous solution (pH 5.5)) for equilibration.
[0030] 4) Sample loading: 300 μL of feces, urine and bile samples were loaded respectively.
[0031] 5) Elution: First, elute with an elution buffer (50 mM NaH2PO4-50% acetonitrile aqueous solution (pH 5.5)) for 3 times, 1 mL each time; then, elute with an elution buffer (50 mM NH4HCO3-80% acetonitrile aqueous solution (pH 5.5)) for 2 times, 1 mL each time.
[0032] 6) Elution: Use 1 mL of elution buffer (100 mM NH4HCO3-20% ethanol-30% acetonitrile aqueous solution (pH 9.5)) for elution, and collect the eluate.
[0033] 7) Drying: Take 800 μL of the eluent and blow it to near dryness with nitrogen.
[0034] 8) The mixture was reconstituted with 150 μL of 1 mM EDTA-0.5% TEA solution, and centrifuged at 4°C and 18,000 g for 10 minutes. 120 μL of the supernatant was taken for sample analysis.
[0035] 2. Detection by high performance liquid chromatography-high resolution mass spectrometry
[0036] 1) Chromatographic conditions (see Table 1, Table 2)
[0037] Table 1
[0038]
[0039] Table 2. Gradient elution program
[0040]
[0041] 2) Mass spectrometry conditions (see Table 3)
[0042] Table 3
[0043]
[0044] The samples were tested using a high performance liquid chromatography-high resolution mass spectrometer (OE 120) and analyzed using BiopharmFinder software to calculate the recovery rate. Recovery rate (%) = chromatographic peak area of the analyte 含生物基质的样本 ÷Chromatographic peak area of the analyte 同浓度标准工作溶液 ×100, where the standard working solution was diluted with enzyme-free water.
[0045] The technical solution of the present invention is further illustrated by experiments below.
[0046] Experimental Example 1 Optimization of operating conditions in solid phase extraction of siRNA drug metabolites
[0047] In this experimental example, the elution buffer is 50 mM NaH2PO4-50% acetonitrile aqueous solution (pH 5.5), and the elution buffer is one of the elution buffers 1-3. The rest of the operation method is the same as Example 1.
[0048] Elution buffer 1: 100 mM NH4HCO3-10% THF / 40% acetonitrile-water solution (pH 9);
[0049] Elution buffer 2: 100 mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9);
[0050] Elution buffer 3: 10% THF / 40% acetonitrile-water solution (pH 9).
[0051] 1. Experimental Methods
[0052] Calculation method of AS chain and SS chain recovery rate:
[0053] Recovery rate (%) = AS or SS chain chromatographic peak area 含生物基质的样本 ÷AS or SS chain chromatographic peak area 同浓度标准工作溶液 ×100.
[0054] 2. Experimental Results
[0055] The results are shown in Table 4. Compared with elution buffer 3, elution buffer 1 has a higher recovery rate due to the addition of 100mM NH4HCO3 to the eluent composition; compared with elution buffer 1, elution buffer 2 has a higher recovery rate due to the use of ethanol in the eluent composition; the above results show that elution buffer 2 (100mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9)) has the highest recovery rates of AS chain and SS chain in feces and urine samples. Therefore, the composition of the eluent is 100mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9).
[0056] Table 4
[0057]
[0058] Experimental Example 2 Optimization of operating conditions in solid phase extraction of siRNA drug metabolites
[0059] In this experimental example, the elution buffer is selected as elution buffer 1 or elution buffer 1 is first used and then elution buffer 2 is used; the elution buffer is selected as one of elution buffers 4 and 5, and the rest of the operation method is the same as Example 1.
[0060] Elution buffer 1: 50 mM NaH2PO4-50% acetonitrile aqueous solution (pH 5.5);
[0061] Elution buffer 2: 50 mM NH4HCO3-80% acetonitrile aqueous solution (pH 5.5);
[0062] Elution buffer 4: 100 mM NH4HCO3-10% ethanol / 40% acetonitrile-water solution (pH 9.5);
[0063] Elution buffer 5: 100 mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9.5).
[0064] 1. Experimental Methods
[0065] Calculation method of AS chain and SS chain recovery rate:
[0066] Recovery rate (%) = AS or SS chain chromatographic peak area 含生物基质的样本 ÷AS or SS chain chromatographic peak area 同浓度标准工作溶液 ×100.
[0067] 2. Experimental Results
[0068] The results are shown in Table 5. Compared with elution buffer 4, elution buffer 5 has a higher recovery rate of AS chain and SS chain when 30% acetonitrile and 20% ethanol are used in the eluent composition; SPE2 has a higher recovery rate when elution buffer 1 is used followed by elution buffer 5 and drying and re-dissolving, compared with SPE3 which uses elution buffer 1 and then elution buffer 5 but not drying, and drying and re-dissolving; SPE4 has a higher recovery rate when elution buffers 1 and 2 are used in sequence and then elution buffer 5 is used, compared with SPE2 which uses only elution buffer 1 and then elution buffer 5, and elution with two elution buffers. The above results show that, taking bile samples as an example, elution buffer 5 (100mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9.5)) is the optimal elution buffer composition, and the recovery rate of AS chain and SS chain is higher when the elution buffer is first eluted with elution buffer 1 and then eluted with elution buffer 2.
[0069] Table 5
[0070]
[0071] In summary, in the solid phase extraction method of siRNA drug metabolites, the elution buffer first uses 50mM NaH2PO4-50% acetonitrile aqueous solution (pH 5.5), and then uses 50mM NH4HCO3-80% acetonitrile aqueous solution (pH 5.5) for elution, and the elution buffer selects 100mM NH4HCO3-20% ethanol / 30% acetonitrile-water solution (pH 9.5) for elution, and when drying and redissolving, the recovery rate of AS chain and SS chain is significantly improved, and the metabolites of siRNA drugs can be analyzed and identified more comprehensively. Therefore, the detection method of the present invention has the advantage of high recovery rate, can better evaluate the efficacy and potential toxicity of drugs, and is crucial for drug development and optimization.
Claims
1. A method for detecting siRNA drug metabolites, characterized in that: The steps include: Step 1: Pre-treat the sample to be tested by solid phase extraction: after the OTX solid phase extraction plate is activated, it is balanced with a balancing buffer NaH2PO4-water solution; NaH2PO4-acetonitrile water solution and NH4HCO3-acetonitrile water solution are used as elution buffers in sequence for elution; elution is performed with an elution buffer NH4HCO3-ethanol-acetonitrile-water solution, the eluate is collected, dried and redissolved to obtain a sample solution; the concentration of the NaH2PO4-water solution is 45-55mmol / L, and the solution pH is 5-6; the NaH2PO4 concentration in the NaH2PO4-acetonitrile water solution is 45-55mmol / L, the volume fraction of acetonitrile is 45-55%, and the solution pH is 5-6; the NH4HCO3 concentration in the NH4HCO3-acetonitrile water solution is 45-55mmol / L, the volume fraction of acetonitrile is 75-85%, and the solution pH is 5-6. Step 2: The sample solution is detected by high performance liquid chromatography-high resolution mass spectrometry.
2. The detection method according to claim 1, characterized in that: In step 1, the volume fraction of ethanol in the elution buffer NH4HCO3-ethanol-acetonitrile-water solution is 10-20%, the volume fraction of acetonitrile is 30-40%, the concentration of NH4HCO3 is 90-110mmol / L, and the pH of the elution buffer is 8-10.
3. The detection method according to claim 2, characterized in that: The volume fraction of the ethanol is 20%.
4. The detection method according to claim 2, characterized in that: The volume fraction of the acetonitrile is 30%.
5. The detection method according to claim 2, characterized in that: The concentration of NH4HCO3 is 100mmol / L; the pH of the elution buffer is 9.
5.
6. The detection method according to claim 1, characterized in that: In step 1, the preparation method of the sample to be tested is: adding siRNA drug working solution to the biological matrix to obtain a mixed solution 1; adding a lysis buffer to the mixed solution 1 to obtain; the volume ratio of the biological matrix to the siRNA drug working solution is ≥ 9:1, the volume ratio of the mixed solution 1 to the lysis buffer is 1:1-1:
2.
7. The detection method according to claim 6, characterized in that: The biological matrix is at least one of feces, bile and urine.
8. The detection method according to claim 1, characterized in that: In step 2, the chromatographic conditions of the high performance liquid chromatography-high resolution mass spectrometry method are as follows: the chromatographic column is selected from Thermo Scientific DNAPacTM RP column (4μm, 2.1×50mm) or XTerra MS C18 column (125A, 2.5μm, 2.1×50mm); the mobile phase A is (0.1%-0.5%) TEA-(0.1%-0.5%) HFIP-water solution, and the mobile phase B is (0.1%-0.5%) TEA-(0.1%-0.5%) HFIP-methanol solution, and gradient elution is performed at a flow rate of 0.2-0.3mL / min; the injection volume is 1-5μL, the column temperature is 60-80°C, and the wash solution is 0-50% isopropanol aqueous solution.
9. The detection method according to claim 8, characterized in that: The gradient elution conditions are shown in the following table:
10. The detection method according to claim 1, characterized in that: In step 2, the mass spectrometry conditions of the high performance liquid chromatography-high resolution mass spectrometry method are as follows: the ion source is a heated electrospray ion source, the spray voltage is 2000-3000 V, the sheath gas is 10-50 mL / min, the auxiliary gas is 10-50 mL / min, the sweep gas is 0-1 mL / min, the ion transfer tube temperature is 300-400°C, the solvation temperature is 300-400°C, the resolution is 60000-120000, and the scanning range is 570-2000 m / z.