RNA drug stabilizer suitable for mass spectrometry and fluorescence detection and applications thereof

Abstract

The application discloses an RNA drug stabilizer suitable for mass spectrometry and fluorescence detection and application thereof, and the RNA drug stabilizer is an ammonium acetate aqueous solution, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 3-5 mol / L, the pH value of the ammonium acetate aqueous solution is 5.5-6, and the ammonium acetate aqueous solution is used for stabilizing the to-be-detected RNA drug in the storage and processing process of a biological matrix sample. The RNA drug stabilizer suitable for mass spectrometry and fluorescence detection can stabilize ASO drugs and siRNA drugs in various biological matrices, does not interfere with LC-MS / MS and LC-FLD detection, effectively solves the quantitative analysis bottleneck of RNA drugs, promotes the research and development efficiency of the RNA drugs, and improves the data reliability of the RNA drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an RNA drug stabilizer suitable for mass spectrometry and fluorescence detection and its application. BACKGROUND

[0002] In recent years, RNA drugs represented by antisense oligonucleotides (ASO) and small interfering RNAs (siRNA) have shown great potential in the treatment of single-gene genetic diseases and tumors due to their precise gene silencing mechanism. These drugs are usually 15-30 base RNA fragments that enhance their stability through 2' hydroxyl modification, locked nucleic acid (LNA) insertion, and phosphorothioate backbone modification, thereby degrading specific mRNA and inhibiting protein expression in vivo. Although the RNA drugs with structural modifications have better stability than endogenous RNA, they still face severe degradation challenges during storage and processing in biological matrices (such as plasma, tissue homogenate, and excreta) — the ubiquitous RNases (such as RNase A and RNase H) in the biological matrix can initiate drug fragmentation by recognizing phosphodiester bonds or modification sites; meanwhile, endogenous components such as proteins and lipids in the matrix may change the spatial conformation of the RNA drug through non-specific binding, further accelerating its degradation.

[0003] In the development process of RNA drugs, pharmacokinetic (PK) and toxicokinetic (TK) studies rely on the accurate quantification of drug concentrations in biological matrices, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) and liquid chromatography-fluorescence detection (LC-FLD) are currently the mainstream analysis techniques. However, the application of existing RNA stabilizers has significant limitations, making it difficult to meet the needs of these two detection platforms, specifically:

[0004] 1. Interference of traditional stabilizers with detection platforms

[0005] In existing technologies, some stabilizers use functionalized nanomaterials (such as polystyrene microspheres) as core components to stabilize RNA drugs by adsorption, but such materials are prone to residual during liquid-liquid extraction or solid-phase extraction, leading to decreased ionization efficiency of mass spectrometry or quenched fluorescence signals; another type of stabilizer containing urea and metal chelators can inhibit RNase activity, but high concentrations of urea can damage the stationary phase of the chromatographic column, and metal ions may interfere with the formation of multiple charged ions in mass spectrometry, affecting quantitative accuracy.

[0006] 2. Balance between stability and detection compatibility

[0007] To improve stability, some solutions use strong denaturants (such as guanidine salts) or extreme pH conditions (pH < 4 or pH > 9), which can quickly inactivate RNases but can also cause secondary structure disruption of RNA drugs or reduce the binding efficiency of fluorescent probes (such as SYBR Green), making it difficult to adapt to LC-FLD detection. At the same time, strong denaturants can easily produce ion suppression effects in mass spectrometry detection, resulting in a quantitative deviation of more than 20% for low-concentration samples, which does not meet the methodological requirements of PK / TK studies (recovery rate should be within 85%-115%).

[0008] 3. Insufficient specificity of biological matrix adaptation

[0009] Most existing stabilizers are designed for a single matrix (such as blood) or a specific RNA type (such as mRNA), without considering the impact of different biological matrices (such as high levels of proteases in tissue homogenate and high salt environments in urine) on stability. For example, a stabilizer developed for plasma cannot effectively neutralize the high activity of RNase H in tissue, resulting in a recovery rate of less than 50% for RNA drugs stored at 4°C for 12 hours, which cannot meet the needs of multi-organ distribution studies.

[0010] 4. Complexity of operation limits practical application

[0011] Some stabilizers require low-temperature pretreatment (such as pre-freezing at -80°C) or centrifugation steps (such as separation after magnetic microsphere adsorption), which increases the time cost and operational errors of sample processing. In large-scale PK / TK sample analysis (such as processing hundreds of plasma samples in a single experiment), such operations can cause the time difference of sample degradation to expand, reducing data reproducibility (CV values often exceed 15%).

[0012] Therefore, there is a need in the art to develop an RNA drug stabilizer that can stabilize ASO drugs and siRNA drugs in various biological matrices without interfering with LC-MS / MS and LC-FLD detection, solving the bottleneck of RNA drug quantitative analysis, improving the efficiency of RNA drug research and development, and improving data reliability. SUMMARY

[0013] To solve the above technical problems, the present application provides an RNA drug stabilizer suitable for mass spectrometry and fluorescence detection, which is an aqueous ammonium acetate solution, the concentration of ammonium acetate in the aqueous ammonium acetate solution is 3-5 mol / L, the pH value of the aqueous ammonium acetate solution is 5.5-6.0, and the aqueous ammonium acetate solution is used to stabilize the RNA drug to be tested during the storage and processing of biological matrix samples.

[0014] Specifically, the mass spectrometry and fluorescence detection are quantitative analysis performed by liquid chromatography-tandem mass spectrometry or liquid chromatography-fluorescence detection.

[0015] Specifically, the RNA drug is an antisense oligonucleotide drug, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 3-5 mol / L, and the pH value of the ammonium acetate aqueous solution is 5.5-6.0.

[0016] Specifically, the RNA drug is small interfering RNA, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 3~5 mol / L, and the pH value of the ammonium acetate aqueous solution is 5.5~6.0.

[0017] Specifically, the biological matrix sample is selected from one of plasma, tissue homogenate, urine or feces, and the plasma is selected from one of human, rat or mouse.

[0018] A second aspect of this application provides a method for stabilizing an RNA drug in a biological matrix sample, the method being based on the aforementioned RNA drug stabilizer suitable for mass spectrometry and fluorescence detection, the method comprising the following steps:

[0019] Step 1: Mix the RNA drug stabilizer with the biological matrix sample to be tested and ensure thorough mixing;

[0020] Step 2: Store the mixed sample for 16-20 hours. The stored RNA drug remains stable during storage and is subsequently validated by quantitative analysis.

[0021] Specifically, in step 1, the mixing ratio of the RNA drug stabilizer to the biological matrix sample to be tested is 1:1 by volume.

[0022] Specifically, in step 2, the storage temperature conditions are selected from one of the following: wet ice conditions, room temperature conditions, -60°C freezing conditions, or repeated freeze-thaw conditions.

[0023] Specifically, in step 2, the quantitative analysis is liquid chromatography-tandem mass spectrometry or liquid chromatography-fluorescence detection quantitative analysis.

[0024] A third aspect of this application also provides the application of the aforementioned RNA drug stabilizer suitable for mass spectrometry and fluorescence detection in pharmacokinetic or toxicokinetic studies for stabilizing RNA drugs in biological matrices. The application involves quantitative analysis of RNA drugs in stored biological matrix samples by liquid chromatography-tandem mass spectrometry or liquid chromatography-fluorescence detection, wherein the RNA drug is an oligonucleotide drug or a small interfering RNA drug.

[0025] The RNA drug stabilizer of the present invention, suitable for mass spectrometry and fluorescence detection, has the following beneficial effects:

[0026] 1. The RNA drug stabilizer provided by this invention uses ammonium acetate as the core component. Through the exploration of optimal concentration and pH value, a dual stabilization mechanism is achieved: on the one hand, ammonium acetate maintains the appropriate ionic strength of the biological matrix, effectively reducing the non-specific binding of ASO and siRNA to endogenous proteins, and avoiding the destruction of the spatial conformation of RNA drugs; on the other hand, ammonium acetate has a relatively suitable pH buffer range, which makes it easy to control the acidity and alkalinity within the range of low RNase activity by adjusting the pH, directly inhibiting the degradation of RNase A and RNase H, etc., and effectively solving the core problem of easy degradation of RNA drugs in biological matrices.

[0027] 2. Existing commercial stabilizers often interfere with detection systems due to their components. For example, nanomaterials can cause ion suppression in mass spectrometry, while metal ions can affect fluorescence signals. However, the RNA drug stabilizer of this invention has unique chemical compatibility, does not affect the ionization efficiency of mass spectrometry detection, and does not interfere with the binding of fluorescent probes. Therefore, it can be applied to quantitative analysis on different technology platforms: For LC-MS / MS detection, ammonium acetate, as a volatile salt, can be completely vaporized in the ion source without leaving solid particles, avoiding contamination of the chromatographic column and ion source, and will not interfere with the ionization efficiency of the RNA drug to be tested, ensuring accurate quantification of low-concentration samples (such as 30 ng / mL LQC); For LC-FLD detection, ammonium acetate does not competitively bind to fluorescent probes (such as specific RNA probes) and does not quench the fluorescence signal, ensuring a stable linear relationship between fluorescence intensity and RNA drug concentration.

[0028] 3. The RNA drug stabilizer provided by this invention is not limited to a single matrix and can be widely used in various biological matrices, such as plasma from different species like rats, mice, and humans. It solves the problem of high protein content in plasma adsorbing RNA drugs. In addition, it is also suitable for tissue homogenates, such as liver and kidney tissue homogenates. The RNA drug stabilizer of this invention can antagonize the decomposition effect of highly active RNases in tissues. For excretory samples such as feces and urine, the RNA drug stabilizer of this invention can still maintain the stability of RNA drugs in high-salt and high-metabolite environments. In summary, the RNA drug stabilizer of this invention can cover the entire process of drug development from animal experiments to clinical sample analysis, reducing the operation and economic costs of changing RNA stabilizers for different biological matrices.

[0029] 4. The method and application of stabilizing RNA drugs in biological matrix samples provided by the present invention involves mixing RNA drug stabilizers and biological matrix in an appropriate ratio. The operation is simple and cost-controllable, without the need for complex preparation processes and pre- and post-treatment processes. It can be directly introduced into the liquid-liquid extraction or solid-phase extraction process, which greatly shortens the sample processing time. It is not only suitable for analysis on both liquid chromatography-tandem mass spectrometry and liquid chromatography-fluorescence detection platforms, but also helps to improve detection throughput and detection efficiency, which is conducive to large-scale application. Attached Figure Description

[0030] Figure 1 This is a typical standard curve diagram from Example 1.

[0031] Figure 2 This is a typical blank matrix sample chromatogram from Example 1.

[0032] Figure 3 This is a typical lower limit of quantitation (LLOQ) sample chromatogram from Example 1.

[0033] Figure 4 This is a typical standard curve diagram from Example 2.

[0034] Figure 5 The chromatograms are typical blank matrix and lower limit of quantitation (LLOQ) sample chromatograms from Example 2. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0036] Unless otherwise specified, all reagents used in the following examples are commercially available reagents. Table 1 shows the reagents and consumables used in all the following examples, and Table 2 shows the instruments and models used in all the following examples.

[0037] Table 1. Reagent and Consumable Information

[0038]

[0039] Table 2 Instruments

[0040]

[0041] Example 1

[0042] This embodiment illustrates, using rat plasma as an example, the use of the RNA drug stabilizer and administration method provided in this application to stabilize the siRNA drug to be tested in rat plasma, and to perform LC-MS / MS quantitative analysis of the siRNA antisense strand (AS). The specific operation is as follows:

[0043] A. Preparation of siRNA drug standard curve and quality control samples in rat plasma: Accurately weigh 2 mg of siRNA standard and dissolve it in 1 mL of deionized water to obtain a 2 mg / mL standard stock solution. Dilute the standard stock solution with 30% methanol solution to prepare a series of standard curve working solutions, add them to rat blank plasma at a volume ratio of 1:19 and mix well to obtain standard curve samples with concentrations of 10, 20, 100, 1000, 2000, 4000, 8000, and 10000 ng / mL, and quality control samples with concentrations of 10, 30, 200, 5000, and 7500 ng / mL, respectively. Taking low-concentration quality control (LQC, 30 ng / mL) and high-concentration quality control (HQC, 7500 ng / mL) as examples, the RNA drug stabilizer (5 mol / L, pH 5.5) provided in this application was added at a 1:1 volume ratio and mixed well. Three samples were immediately processed to obtain the fresh LQC and HQC sample groups. Three samples from each group were placed on wet ice and at -60°C for 16 hours respectively before processing to obtain the matrix stability test sample group. In addition, a control group without RNA drug stabilizer was set up, with the following conditions: fresh LQC and HQC sample groups, placed at 4°C for 20 hours and placed at -60°C for 20 hours, with three samples under each condition.

[0044] B. Processing and LC-MS / MS Quantitative Analysis of Rat Plasma Samples: 30 μL each of blank, standard curve, quality control, and matrix stability test samples were taken. siRNA was extracted using liquid-liquid extraction, and analyte (AS) was quantitatively analyzed by LC-MS / MS. The HPLC and mass spectrometry parameters are shown in Table 3.

[0045] Table 3

[0046]

[0047] C. Standard Curve Regression: Quantification was performed using the internal standard method. The theoretical concentration of AS in the standard curve sample was plotted on the x-axis, and the ratio of the AS peak area to the internal standard peak area was plotted on the y-axis. A weighted average was calculated using w=1 / x. 2 The least squares method is used to perform regression calculations, and the linear regression equation obtained is the standard curve.

[0048] D. Quantitative analysis of AS and matrix stability study: Quantitative analysis of AS in quality control samples and matrix stability samples was performed using the standard curve from step C. Provided that the method's sensitivity, specificity, precision, and accuracy met the acceptance criteria, the stability of the samples after 16 hours of storage on wet ice and in a -60°C freezer was calculated as the percentage of the mean peak area of ​​AS in the matrix stability study sample group (including LQC and HQC, n=3) to the mean peak area of ​​AS in the fresh sample group (including LQC and HQC, n=3).

[0049] Figure 1 To add the standard curves for the RNA drug stabilizer groups provided in this application. Figure 2 This is the chromatogram of a blank matrix sample. Figure 3 The chromatogram is for the sample at the lower limit of quantitation (LLOQ). This is then compared with the data from the control and experimental groups shown in Tables 4 and 5:

[0050] Table 4

[0051]

[0052] Table 5

[0053]

[0054] Table 4 lists the stability results of LQC and HQC levels in the control group without RNA drug stabilizer. It can be seen that AS was significantly degraded after being placed at 4°C and -60°C for 20 hours. Table 5 lists the stability results of LQC and HQC levels in rat plasma with the RNA drug stabilizer provided in this application (5 mol / L, pH 5.5, added at a 1:1 volume ratio). It can be seen that AS remained stable after being placed on wet ice (WI) and in a -60°C freezer for 16 hours.

[0055] Example 2

[0056] This embodiment illustrates, using mouse plasma as an example, the use of the RNA drug stabilizer and administration method provided in this application to stabilize the siRNA drug to be tested in mouse plasma, and to perform LC-FLD quantitative analysis of the siRNA antisense strand (AS). The specific operation is as follows:

[0057] A. Preparation of siRNA drug standard curve and quality control samples in mouse plasma: Accurately weigh 2 mg of siRNA standard and dissolve it in 1 mL of deionized water to obtain a 2 mg / mL standard stock solution. Dilute the standard stock solution with a 10% methanol solution containing 0.1% BSA to prepare a series of standard curve working solutions. Add these solutions to mouse blank plasma at a volume ratio of 1:19 and mix well to obtain standard curve samples with concentrations of 5, 10, 50, 500, 1000, 2000, 4000, and 5000 ng / mL, and quality control samples with concentrations of 5, 15, 200, 2500, and 3750 ng / mL, respectively. Taking low-concentration quality control (LQC, 15 ng / mL) and high-concentration quality control (HQC, 3750 ng / mL) as examples, the RNA drug stabilizer (3 mol / L, pH 6.0) provided in this application was added at a 1:1 volume ratio and mixed well. Three samples were taken and processed immediately to obtain the fresh LQC and HQC sample groups. Three samples from each sample were placed in a refrigerator at room temperature and -60°C for 20 hours respectively before processing to obtain the matrix stability test sample group.

[0058] B. Processing and LC-FLD Quantitative Analysis of Mouse Plasma Samples: 20 μL each of blank, standard curve, quality control, and matrix stability test samples were taken. siRNA was extracted using liquid-liquid extraction, followed by incubation with a fluorescent probe. α-ascorbic acid (AS) was then quantified using LC-FLD. The liquid chromatography and fluorescence detection parameters are shown in Table 6.

[0059] Table 6

[0060]

[0061] C. Standard Curve Regression: The external standard method is used for quantification. The theoretical concentration of AS in the standard curve sample is used as the abscissa, and the peak area of ​​AS is used as the ordinate. The least squares method with weighted w=1 / x is used for regression calculation, and the linear regression equation is obtained as the standard curve.

[0062] Test results as follows Figure 4 and Figure 5 As shown, Figure 4 For standard curve plot, Figure 5 The images show chromatograms of the blank matrix and the sample at the lower limit of quantitation (LLOQ). Based on the results, the method's sensitivity, specificity, precision, and accuracy met the acceptance criteria. AS in mouse plasma remained stable after 20 hours at both room temperature and -60°C.

[0063] Example 3

[0064] This embodiment illustrates the use of rat liver homogenate as an example, employing the RNA drug stabilizer and administration method provided in this application to stabilize the siRNA drug in rat plasma, and to perform LC-MS / MS quantitative analysis of the siRNA antisense strand (AS). The specific operation is as follows:

[0065] A. Preparation of rat blank liver homogenate: The rat blank liver sample to be homogenized was weighed, and the RNA drug stabilizer (5 mol / L, pH 5.5) provided in this application was added at a weight / volume ratio of 1:9 (e.g., 9 mL per 1 g of tissue). The sample was then placed in a polypropylene tube and ground and homogenized with steel balls to obtain a uniform homogenate.

[0066] B. Standard curve of siRNA drug in rat liver homogenate, preparation of quality control samples, sample processing, and LC-MS / MS quantitative analysis: The steps are the same as in Example 1, but the RNA drug stabilizer provided in this application (already added in the blank liver homogenate preparation step) does not need to be added to the samples. The volume of each sample processing is adjusted to 50 μL to obtain better detection sensitivity. The liquid chromatography and mass spectrometry parameters are shown in Table 7:

[0067] Table 7

[0068]

[0069] C. AS Extraction Recovery and Matrix Stability Study: The extraction recovery was calculated as the percentage of the mean peak area of ​​AS in the extracted quality control samples (including LQC and HQC, n=6) to the mean peak area of ​​AS in the sample group after extraction of blank matrix and addition of corresponding standard solutions. The stability of the samples after 20 hours of storage on wet ice and in a -60°C freezer was calculated as the percentage of the mean peak area of ​​AS in the matrix stability study sample group (including LQC and HQC, n=3) to the mean peak area of ​​AS in the fresh sample group (including LQC and HQC, n=3).

[0070] The extraction recovery results are shown in Table 8, and the matrix stability test results are shown in Table 9.

[0071] Table 8

[0072]

[0073] Table 9

[0074]

[0075] As can be seen, the extraction recoveries of AS in rat liver homogenate treated with the RNA drug stabilizer provided in this application were 91.0% and 85.2% at the LQC (60 ng / mL) and HQC (7500 ng / mL) levels, respectively, with inter-sample CV% values ​​of 5.8% and 1.3%, respectively. AS in liver homogenate remained stable after being placed in a refrigerator at room temperature (RT) and -60°C for 20 hours.

[0076] Example 4

[0077] This embodiment illustrates, using rat plasma as an example, the use of the RNA drug stabilizer and administration method provided in this application to stabilize the ASO drug in rat plasma and perform LC-MS / MS quantitative analysis. The specific operation is as follows:

[0078] A. Standard curve of ASO drug in rat plasma, preparation of quality control samples, sample processing and LC-MS / MS quantitative analysis: The steps are as described in Example 1, and the liquid chromatography and mass spectrometry parameters are shown in Table 10.

[0079] Table 10

[0080]

[0081] B. ASO matrix stability study: The stability of the samples after 4 freeze-thaw cycles, 18 hours at room temperature and 5 days at -60°C was calculated by the percentage of the mean ratio of the peak area of ​​ASO to the peak area of ​​the internal standard in the matrix stability study sample group (including LQC and HQC, n=3) to the mean in the fresh sample group (including LQC and HQC, n=3). The results are shown in Table 11.

[0082] Table 11

[0083]

[0084] As can be seen from the data in Table 11, the ASO levels in rat plasma treated with the RNA drug stabilizer provided in this application remained stable at both LQC (30 ng / mL) and HQC (7500 ng / mL) levels after four freeze-thaw cycles (FT), 18 hours at room temperature (RT), and 5 days at -60°C.

[0085] Example 5

[0086] This embodiment illustrates the optimization process of RNA drug stabilizers, using LC-MS / MS analysis of siRNA in rat plasma as an example.

[0087] First, RNA later (mainly composed of ammonium sulfate solution), a typical RNA stabilizer in existing technologies, was used. Results showed that the CV% (cumulative value) of samples treated with RNA later and measured by LC-MS / MS was too high, and the chromatographic column could not tolerate it. Then, four single-component stabilizers relatively friendly to chromatography and mass spectrometry were screened. The results showed that the RNA drug stabilizer provided in this application had the best effect. Finally, the concentration of ammonium acetate solution was optimized, resulting in the RNA drug stabilizer and application method provided in this application. The specific steps are as follows:

[0088] A. First, RNA later was added to freshly prepared rat plasma siRNA low-concentration quality control samples (LQC, 30 ng / mL) at a 1:1 volume ratio. The samples were then incubated on wet ice (WI) and at -60°C for 16 hours, respectively, before being processed simultaneously with the freshly prepared samples and analyzed by LC-MS / MS. The results for LQC samples without stabilizer are shown in Table 12, and the results for LQC samples with RNA later stabilizer are shown in Table 13.

[0089] Table 12

[0090]

[0091] Table 13

[0092]

[0093] The results in the table above show that the mean values ​​of the stability tests met the acceptance criteria, but the measured CV% was too high and did not meet the acceptance criteria. A transient increase in column pressure was also observed during sample analysis. These results indicate that RNA later plays a role in stabilizing the siRNA drug, but its components may have a matrix effect on mass spectrometry detection, leading to a higher measured CV%. The increase in column pressure may also be due to components in RNA later. Therefore, RNA later is not suitable for LC-MS / MS analysis.

[0094] B. Four stabilizers relatively friendly to chromatography and mass spectrometry were selected: Lysis loading buffer (mainly composed of guanidine hydrochloride and Triton X-100), 10% RNA later dilution, 0.1% Triton X-100 in 50 mM Tris-HCl, and 10 mol / L ammonium acetate. The above four stabilizers were added at a 1:1 volume ratio to freshly prepared low-concentration siRNA quality control sample (LQC, 30 ng / mL) in rat plasma. The results are shown in Tables 14-17. Specifically, the results for Lysis loading buffer are shown in Table 14, for 10% RNA later dilution in Table 15, for 0.1% Triton X-100 in 50 mM Tris-HCl in Table 16, and for 10 mol / L ammonium acetate in Table 17. It can be seen that 10 mol / L ammonium acetate exhibited the best stabilizing effect and the smallest measurement bias (CV%).

[0095] Table 14

[0096]

[0097] Table 15

[0098]

[0099] Table 16

[0100]

[0101] Table 17

[0102]

[0103] Furthermore, the results in Table 17 show that the mean and CV% of the stability test of LQC samples with 10 mol / L ammonium acetate as a stabilizer after 16 hours at room temperature (RT) both meet the acceptance criteria. However, the mean values ​​of the stability test after 16 hours on wet ice (WI) and the CV% values ​​after 16 hours in a -60°C freezer both slightly exceed the acceptance criteria, which may be due to matrix effects. The concentration of the ammonium acetate solution stabilizer needs to be further optimized.

[0104] C. Further optimization of the ammonium acetate solution stabilizer concentration was conducted. Three sets of data are listed in Tables 18-20. Table 18: When the ammonium acetate solution concentration was reduced to 5 mol / L, the CV% of the newly prepared LQC samples was acceptable, while the stable LQC samples all showed significant measurement deviations. Table 19: When the ammonium acetate solution concentration was reduced to 5 mol / L and the pH was adjusted to 5.5, the CV% of the newly prepared LQC samples was acceptable, and the accuracy and CV% of the stable LQC samples were also acceptable (one outlier was removed from the room temperature storage group). Table 20: When the ammonium acetate solution concentration was reduced to 2 mol / L and the pH was adjusted to 5.5, the CV% of both the newly prepared LQC samples and all stable LQC samples were too high. After testing with multiple groups of different RNA analytes, the optimal stabilization effect was found to be achieved with an ammonium acetate concentration of 3–5 mol / L and a pH of 5.5–6.

[0105] Table 18

[0106]

[0107] Table 19

[0108]

[0109] Table 20

[0110]

[0111] In summary, while existing RNA stabilizers such as RNA later can stabilize siRNA drugs in rat plasma, they exhibit problems such as an inflated CV% in LC-MS / MS measurements, possibly due to matrix effects, failing to meet acceptance criteria and causing increased column pressure. However, as demonstrated by the examples above, the RNA drug stabilizer and its application provided in this application can stabilize the siRNA drug in rat plasma without causing the aforementioned problems. It is also more compatible with chromatographic and mass spectrometric analyses, demonstrating significant advantages and promising application prospects.

[0112] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for stabilizing RNA drugs in a biological matrix sample, characterized in that, The method is based on an RNA drug stabilizer suitable for mass spectrometry and fluorescence detection. The RNA drug stabilizer is an aqueous solution of ammonium acetate, the concentration of ammonium acetate in the aqueous solution is 3-5 mol / L, and the pH value of the aqueous solution is 5.5-6.

0. The aqueous solution of ammonium acetate is used to stabilize the RNA drug to be tested during the storage and processing of biological matrix samples. The method includes the following steps: Step 1: Mix the RNA drug stabilizer with the biological matrix sample to be tested and mix thoroughly. The mixing ratio of the RNA drug stabilizer to the biological matrix sample to be tested is 1:1 by volume. Step 2: Store the mixed sample for 16-20 hours. The stored RNA drug remains stable during storage and is subsequently verified by quantitative analysis. The storage temperature conditions are selected from one of the following: wet ice conditions, room temperature, -60℃ freezing, or repeated freeze-thaw conditions.

2. The method for stabilizing RNA drugs in biological matrix samples according to claim 1, characterized in that, In step 2, the quantitative analysis is liquid chromatography-tandem mass spectrometry or liquid chromatography-fluorescence detection quantitative analysis.

3. The method for stabilizing RNA drugs in biological matrix samples according to claim 1, characterized in that, The RNA drug is an antisense oligonucleotide drug, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 3~5 mol / L, and the pH value of the ammonium acetate aqueous solution is 5.5~6.

0.

4. The method for stabilizing RNA drugs in biological matrix samples according to claim 1, characterized in that, The RNA drug is a small interfering RNA, the concentration of ammonium acetate in the ammonium acetate aqueous solution is 3~5 mol / L, and the pH value of the ammonium acetate aqueous solution is 5.5~6.

0.

5. The method for stabilizing RNA drugs in biological matrix samples according to claim 1, characterized in that, The biological matrix sample is selected from one of plasma, tissue homogenate, urine or feces, and the plasma is selected from one of human, rat or mouse.

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