Risperidone blood concentration detection method
By combining low-temperature extraction and a specific solvent system with chromatographic technology, the problems of risperidone loss and decomposition during the extraction process were solved, achieving high accuracy and stability in risperidone blood concentration detection.
Patent Information
- Application Number
- CN202511091764.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-05
- Publication Date
- 2025-09-26
AI Technical Summary
In existing methods for detecting risperidone blood concentration, risperidone is easily lost during the extraction process and decomposes in acidic and alkaline environments, resulting in a decrease in detection accuracy.
A low-temperature extraction method is adopted, using an ester solvent such as butyl acetate and a water-ethylene glycol mixture system as a precipitant, combined with a Phenyl-Hexyl chromatographic column and gradient elution, controlling the pH value at 3-5, and removing impurities through nitrogen drying and elution to reduce the decomposition and loss of risperidone.
The extraction rate and detection accuracy of risperidone were improved, the interference of impurities was reduced, and the detection accuracy of high and low concentration samples was ensured.
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Figure CN120703270A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of blood drug detection, and in particular to a method for detecting risperidone blood drug concentration. Background Art
[0002] Risperidone is a benzisoxazole derivative with the molecular formula , containing benzene ring, fluorine atom and heterocyclic group. Risperidone is a second generation (atypical) antipsychotic drug with dopamine Receptors and Serotonin It has a receptor antagonist effect and is widely used in the treatment of mental illnesses such as schizophrenia and bipolar disorder. It is usually available in dosage forms such as oral tablets and long-acting injections.
[0003] Testing risperidone blood concentrations can be used to assess drug release and absorption in the body, study the drug's mechanism of action, and adjust drug usage. Currently, risperidone blood concentration testing is primarily achieved through chromatography, which uses a precipitant to precipitate proteins in the blood and then detects the residue in the supernatant. Because risperidone itself has poor stability, weak light resistance, and a high risk of decomposition in both acidic and alkaline environments, the above-mentioned extraction process can easily result in risperidone loss. Summary of the Invention
[0004] In order to reduce the loss of risperidone during the extraction process, the present application provides a method for detecting the blood concentration of risperidone.
[0005] In this scheme, a method for detecting the blood concentration of risperidone is provided, which comprises the following steps: S1. Prepare calibration products; S2. performing low-temperature extraction on the sample to extract risperidone therein to obtain an extract; S3. Perform chromatographic detection on the standard product and obtain a standard curve according to its gradient concentration; S4. Determine the extract using the same method and determine its concentration according to the standard curve; In step S2, the specific steps are as follows: S2-1, controlling the temperature not to exceed 5°C, adding an ester solvent to the system, and mixing thoroughly to obtain a first mixture; S2-2, adding a neutral precipitant to the first mixture, then centrifuging in a low-temperature centrifuge, retaining the supernatant, and removing the precipitate to obtain an extract; S2-3, separating the extract and retaining the organic phase as the extract; All the above steps were performed in a dark place.
[0006] In the above scheme, a low-temperature extraction method is used to treat risperidone. What needs to be solved is the problem that risperidone has poor solubility at low temperatures, resulting in precipitation loss, and the water system is prone to crystallization in the above process, resulting in a decrease in the extraction rate in the sample. Therefore, in this application, risperidone is volumetrically expanded and extracted by an ester solvent, which can not only better extract risperidone in the system, but also better remove the impurity protein system in the system.
[0007] In the above scheme, in step S2-1, the ester solvent is preferably butyl acetate, which has better solubility and a greater solubility difference with the aqueous phase. Compared with materials such as ethyl acetate or ethyl formate, the extraction rate is relatively higher, and a stratified system can be better formed, thereby improving the accuracy of the extraction step of the system.
[0008] Preferably, in step S2-2, the residual solid precipitate is back-extracted with a water-organic solvent mixed system, and then centrifuged again and precipitated to be combined with the extract. Further preferably, the water-organic solvent system is a water-butyl acetate mixed system, wherein the volume ratio of water to butyl acetate is 1: 5-10, and in this step, the overall pH is adjusted to 3-5. The risperidone system remaining in the precipitate can be further separated by the water-butyl acetate system, and a certain alkalinity is used to improve the solubility of risperidone while reducing the dissolution of protein. In this step, a small amount of alkaline solution will not significantly cause the decomposition of risperidone, so the recovery efficiency is better. In the other steps of steps S2-1 and 3-2, a neutral system is used, which also reduces the loss of risperidone under large samples. The volume of the ester solvent is 10 to 50 times the sample volume, which can achieve a good extraction effect.
[0009] Preferably, in step S3, a Phenyl-Hexyl chromatographic column is used, and a water-methanol mixed solution is used as an eluent for gradient elution, according to the volume ratio: Initial: 95% water + 5% methanol; 0.5 min: 90% water + 10% methanol; 1 min: 50% water + 50% methanol; 1.2 min: 20% water + 80% methanol; 2.6 min: 90% water + 10% methanol; 3min: Elution is completed.
[0010] More preferably, in the water-methanol mixed system, ammonium acetate is added to water and methanol at a concentration of 1 to 5 mM respectively.
[0011] In the above scheme, the chromatographic elution is performed with a water-methanol system, which has better elution properties as a whole and has a large difference in elution time between impurity peaks, thereby improving the purity of the peak type and reducing the influence of impurity peaks on the experimental results.
[0012] Preferably, the precipitant is a mixture of an organic solvent and a neutral inorganic salt, wherein the organic solvent is a mixture of water and ethylene glycol, wherein the volume ratio of water to ethylene glycol is 1:5-10, and the mass concentration of the neutral inorganic salt in the precipitant is 5-10 g / L. Further preferably, the amount of the precipitant added is 10-20 times the sample volume.
[0013] In the above scheme, a water-ethylene glycol composite system is used as the precipitant. Compared to conventional methanol or acetonitrile systems, ethylene glycol provides a certain degree of crosslinking. Due to its greater solubility difference with ester solvents such as butyl acetate, it facilitates separation during the extraction process and achieves a higher extraction efficiency. Ethylene glycol also has greater hydrophobicity, making it more effective in stripping inorganic phases.
[0014] Preferably, in step S2-3, the sample is blown dry by nitrogen at a temperature not exceeding 10°C, then rinsed with a water-inorganic salt solution system, and finally redissolved in an organic solvent.
[0015] By drying with nitrogen and then eluting, a small amount of residual protein in the system can be removed, and residual alkaline substances can be washed away at the same time, reducing the impact of the system in subsequent chromatographic analysis.
[0016] In summary, the present application provides a method for detecting risperidone blood concentration based on a low-temperature extraction scheme, which reduces the decomposition and loss of risperidone during the removal of impurities and has better detection accuracy in high and low concentration samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the standard curve drawn in Example 1.
[0018] Figure 2 This is the peak shape of risperidone determined by LC-MS in Example 1.
[0019] Figure 3 The linear regression equation obtained in the linear regression experiment in Example 1 is y=1.0192x-1.5857, and the correlation coefficient R² is 0.9996. DETAILED DESCRIPTION
[0020] The solution in this application is further described through the following specific implementation methods.
[0021] In the present application, the recovery of risperidone is mainly improved by the following method: adding risperidone with a mass of m0 to a specific protein-containing sample, then treating the sample according to the method in step S2, then distilling under reduced pressure to remove the solvent, washing with water, drying and weighing to obtain m1.
[0022] Recovery rate P = m1 / m0 × 100% In the following examples, the recoveries of high-concentration risperidone and low-concentration risperidone were tested respectively, wherein the concentration of the low-concentration risperidone was 1.0 ng / mL and the concentration of the high-concentration risperidone was 200 ng / mL.
[0023] Example 1: This example is a detection scheme for detecting the concentration of risperidone in blood, which specifically includes the following steps: S1. Prepare standards. In this step, risperidone was first dissolved in a small amount of methanol and then added to bovine serum containing 1 mg / mL glutathione and 0.5 mg dithiothreitol to prepare six concentration series of standards. In addition, three concentrations of quality control products were prepared, as shown in Table 1.
[0024] Table 1 S2. performing low-temperature extraction on the sample to extract risperidone therein to obtain an extract; Specifically, this step includes the following sub-steps: S2-1. Take 20 μL of sample, add 2 mL of butyl acetate to the system in an ice-water bath, and mix thoroughly to obtain a first mixture; S2-2. A neutral precipitant is added to the first mixture, followed by centrifugation in a low-temperature centrifuge. The supernatant is retained and the precipitate is removed to obtain an extract. In this step, the precipitant is a mixture of an organic solvent and a neutral inorganic salt, wherein the organic solvent is a water-ethylene glycol mixture at a volume ratio of 1:10. The neutral inorganic salt is sodium sulfate, and its mass concentration in the precipitant is 10 g / L. The remaining solid precipitate is back-extracted with a water-butyl acetate mixture, then centrifuged again and combined with the extract, wherein the volume ratio of butyl acetate to water is 8:1. Hydrochloric acid is added to adjust the pH to a range of 3 to 5. The centrifugation process is as follows: vortexing at 1400 rpm for 5 minutes; centrifuging at 5000 rpm for 10 minutes, and then collecting the supernatant.
[0025] S2-3. Separate the combined extracts and retain the organic phase as the extract.
[0026] All the above steps were performed in a dark place.
[0027] S3. Perform chromatographic detection on the calibrator. Specifically, the mass spectrometry parameters are shown in Table 2.
[0028] Table 2 The elution parameters are shown in Table 3.
[0029] Table 3 The standard curve was drawn for the standard product, and the results are shown in Table 4. Figure 1 The chromatogram of risperidone is shown in Figure 2 shown.
[0030] S4. Prepare quality control products using the same method to simulate actual samples to verify accuracy, and perform separation and determination according to steps S2 and S3. The determination results are shown in Table 4.
[0031] Table 4 In addition, the linearity of the above experimental method was also verified in this embodiment, and the results are shown in Table 5. At the same time, the linear regression equation curve is obtained as shown in Table 5. Figure 3 The linear regression equation is y=1.0192x-1.5857, and the correlation coefficient R² is 0.9996.
[0032] Table 5 The above scheme was tested on the lower limit samples, and the results are shown in Table 6.
[0033] Table 6 The upper limit sample test of the above scheme is performed, and the results are shown in Table 7.
[0034] Table 7 It can be seen from each group of samples that the solution in this embodiment has good accuracy and linear regression, a small relative deviation, a low detection limit, and better accuracy at high concentrations.
[0035] Comparative Example 1: This comparative example differs from Example 1 in that, in step S2, an ice-water bath is not used, but the process is carried out at room temperature.
[0036] Example 2: This comparative example differs from Example 1 in that, in step S2-1, ethyl acetate is used as the ester solvent.
[0037] Example 3: The difference between this comparative example and Example 1 is that in step S2-1, butyl formate is used as the ester solvent.
[0038] Example 4: This comparative example differs from Example 1 in that, in step S2-1, methyl formate is used as the ester solvent.
[0039] Example 5: This comparative example differs from Example 1 in that the organic solvent in the precipitant is replaced by a water-ethanol system in equal proportions.
[0040] Example 6: This comparative example differs from Example 1 in that the organic solvent in the precipitant is replaced by a water-methanol system in equal proportions.
[0041] Example 7: This comparative example differs from Example 1 in that, in step S2-2, the residual solid is not back-extracted.
[0042] Example 8: The difference between the comparative example and Example 1 is that in step S2-2, when the residual solid is back-extracted, the pH is controlled to be neutral.
[0043] The upper limit sample accuracy and lower limit sample accuracy of the above embodiments and comparative examples were verified, and the results are shown in Table 8.
[0044] Table 8 It can be seen from the data in the above table that the solution in Example 1 has better stability at both high and low concentrations, and provides a relatively low CV value, indicating that in actual blood drug samples, it can more accurately and stably measure the concentration of the sample.
[0045] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.
Claims
1. A method for detecting risperidone blood concentration, characterized in that: Contains the following steps S1. Prepare calibration products; S2. performing low-temperature extraction on the sample to extract risperidone therein to obtain an extract; S3. Perform chromatographic detection on the standard product and obtain a standard curve according to its gradient concentration; S4. Determine the extract using the same method and determine its concentration according to the standard curve; In step S2, the specific steps are as follows: S2-1, controlling the temperature not to exceed 5°C, adding an ester solvent to the system, and mixing thoroughly to obtain a first mixture; S2-2, adding a neutral precipitant to the first mixture, then centrifuging in a low-temperature centrifuge, retaining the supernatant, and removing the precipitate to obtain an extract; S2-3, separating the extract and retaining the organic phase as the extract; All the above steps were performed in a dark place.
2. The method for detecting risperidone blood concentration according to claim 1, wherein: In step S2-2, the residual solid precipitate is back-extracted with a water-organic solvent mixture system, and then centrifuged again and precipitated and then combined with the extract.
3. The method for detecting risperidone blood concentration according to claim 2, wherein: In step S2-2, the water-organic solvent system is a mixed system of water and butyl acetate, wherein the volume ratio of water to butyl acetate is 1:5-10, and in this step, the overall pH is adjusted to 3-5.
4. The method for detecting risperidone blood concentration according to claim 1, wherein: In step S2-1, the ester solvent is butyl acetate.
5. The method for detecting risperidone blood concentration according to claim 1, wherein: In step S3, a Phenyl-Hexyl column was used and a water-methanol mixed solution was used as the eluent for gradient elution, according to the volume ratio: Initial: 95% water + 5% methanol; 0.5 min: 90% water + 10% methanol; 1 min: 50% water + 50% methanol; 1.2 min: 20% water + 80% methanol; 2.6 min: 90% water + 10% methanol; 3min: Elution is completed.
6. The method for detecting risperidone blood concentration according to claim 5, wherein: In a water-methanol mixture system, ammonium acetate was added to water and methanol at a concentration of 1 to 5 mM.
7. The method for detecting risperidone blood concentration according to claim 1, wherein: The precipitant is a mixed system of an organic solvent and a neutral inorganic salt. The organic solvent is a mixed system of water and ethylene glycol, wherein the volume ratio of water to ethylene glycol is 1:5-10, and the mass concentration of the neutral inorganic salt in the precipitant is 5-10 g / L.
8. The method for detecting risperidone blood concentration according to claim 7, wherein: The amount of the precipitant added is 10 to 20 times the volume of the sample.
9. The method for detecting risperidone blood concentration according to claim 1, wherein: The volume of the ester solvent is 10 to 50 times the volume of the sample.
10. The method for detecting risperidone blood concentration according to claim 1, wherein: In step S2-3, the sample is blown dry by nitrogen at a temperature not exceeding 10°C, then rinsed with a water-inorganic salt solution system, and finally redissolved in an organic solvent.