Method for detecting blood concentration of aripiprazole

Abstract

The invention relates to the field of blood drug detection, in particular to a method for detecting blood drug concentration of aripiprazole. According to the present invention, the blood concentration detection accuracy of aripiprazole in the hyperlipidemia sample is improved in the manner of primary centrifugation, clear liquid precipitation, lipid low-temperature extraction with tert-butyl methyl ether, and lipid centrifugal separation.

Description

Technical Field

[0001] This application relates to the field of blood drug detection, and in particular to a method for detecting aripiprazole blood concentration. Background Technology

[0002] Aripiprazole is an atypical antipsychotic drug with the chemical name 7-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydroquinolone. It has high lipophilicity and can cross the blood-brain barrier. It exerts its therapeutic effect through partial agonist activity of dopamine D2 receptors and 5-HT1A receptors and antagonist activity of 5-HT2A receptors. It is often used to treat mental illnesses such as schizophrenia and bipolar I disorder.

[0003] The detection of aripiprazole blood concentration is usually performed by liquid chromatography or liquid chromatography-mass spectrometry (LC-MS), and pretreatment is typically performed using two methods. One is precipitation, which uses a precipitating agent to precipitate proteins in the blood. This method is simple and quick, but has low impurity removal rates and is prone to matrix effects. The other method is solvent extraction, which has the advantage of good impurity removal and reduced residual impurities. However, its disadvantages include low recovery rates and a tendency for emulsification to occur in high-lipid samples, making aripiprazole separation difficult. Summary of the Invention

[0004] Based on the above problems, the purpose of this application is to provide a detection method that can achieve both high recovery rate and high accuracy in hyperlipidemia samples.

[0005] First, this application provides a method for detecting aripiprazole blood concentration, comprising the following steps:

[0006] S1. Prepare samples and standards;

[0007] S2. Control the temperature at 0-10℃ and perform preliminary centrifugation to separate the lipid layer and the first supernatant;

[0008] S3. Use a precipitating agent to precipitate the first clear liquid, then centrifuge to remove impurities to obtain the second clear liquid;

[0009] S4. Add tert-butyl methyl ether to the lipid layer, then control the temperature to be less than 0°C, and centrifuge again to obtain the second lipid and the third supernatant.

[0010] S5. Combine the second and third supernatants, perform chromatographic analysis, plot a standard curve using standards, and compare the samples.

[0011] The above scheme adopts a three-step treatment method of preliminary centrifugation to remove lipids, precipitation to remove proteins, and secondary centrifugation to remove lipids. For systems with high blood lipids, lipids can be removed first to reduce the influence of lipids on the precipitation process during subsequent separation, making the precipitation process easier. At the same time, during precipitation in step S3, the influence of lipids on the precipitation process can also be reduced, thereby reducing the loss of aripiprazole caused by precipitation.

[0012] After the protein and other impurities are precipitated by the precipitant, a second centrifugation is performed. The preferred method is that the difference between the first and second centrifugation is that a mixed solution of water and tert-butyl methyl ether is added during the second centrifugation. The organic phase is then used for extraction against the lipid layer. During this extraction step, at a lower temperature, tert-butyl methyl ether has poor solubility for blood lipid components such as cholesterol and triglycerides, which can redissolve the lipid-coated aripiprazole. At the same time, this process is less likely to produce emulsification reaction at a lower temperature, thus providing better recovery rate and accuracy overall.

[0013] Preferably, in step S2, the centrifugation rate is 1000–3000 rpm and the centrifugation time is 1–5 min. Also preferably, in step S4, the centrifugation rate is 3000–5000 rpm and the centrifugation time is 5–10 min. In this scheme, a lower centrifugation rate is used in the first step and a higher centrifugation rate is used in the second step. Because tert-butyl methyl ether is used for extraction during the second centrifugation process, cholesterol and triglycerides, among other lipid components, coagulate more quickly at lower temperatures, making them more prone to forming microcrystals. Using a higher centrifugation rate and a longer, interleaved centrifugation time helps to ensure complete separation of the components and reduces the influence of triglycerides and other components on subsequent chromatographic detection.

[0014] Further preferably, in step S2, the temperature is controlled at 4±1℃, and in step S4, the temperature is controlled at -20 to -4℃. Within this temperature range, cholesterol and triglycerides coagulate more quickly, making separation easier. Additionally, tert-butyl methyl ether is used as the solvent instead of organic solvents such as methanol, as it has better extraction efficiency and reduces the impact of high lipid content on sample accuracy.

[0015] Preferably, in step S3, the precipitant is an aqueous solution, an organic solvent solution, or a mixed solution of zinc sulfate. More preferably, the precipitant is a water-acetonitrile-methanol mixed solution of zinc sulfate, wherein the volume ratio of water, acetonitrile, and methanol is 1:2 to 4:5 to 8. This approach improves the overall precipitation effect of the system and reduces the loss of aripiprazole during precipitation.

[0016] Preferably, in step S1, the standard is prepared by dissolving aripiprazole in animal serum. More preferably, the standard contains additional tea polyphenols and ascorbic acid at a mass concentration of 0.5–2 mg / mL. This system can be used to simulate the blood conditions in hyperlipidemic samples, and the addition of tea polyphenols can further reduce the influence of metal ions on the sample during precipitation.

[0017] Preferably, in step S5, a Phenyl-Hexyl column is used, and gradient elution is performed using a water-methanol mixture as the eluent, at a volume ratio of:

[0018] Initial composition: 95% water + 5% methanol

[0019] 0.9 min: 60% water + 40% methanol

[0020] 1.8 min: 50% water + 50% methanol

[0021] 2 min: 40% water + 60% methanol

[0022] 4.3 min: 50% water + 50% methanol

[0023] 4.5 min: 95% water + 5% methanol.

[0024] More preferably, in step S5, the pH of the eluent is controlled to be 8-10 by a buffer solution.

[0025] The above method can adjust the peak shape, resulting in a narrower peak shape and higher detection accuracy.

[0026] Preferably, in step S3, separation is performed using distributed centrifugation, which includes a first centrifugation separation and a second centrifugation separation. The first centrifugation separation rate is 1000–2000 rpm for 3–6 min, and the second centrifugation separation rate is 3000–6000 rpm for 5–20 min. Distributed centrifugation allows for a strategy of flocculation followed by compaction, removing large particles first and then compacting fine precipitates, thereby improving the clarity of the supernatant and facilitating subsequent detection and filtration.

[0027] In summary, this application provides a method for detecting the blood concentration of aripiprazole, which utilizes... Attached Figure Description

[0028] Figure 1 This is the LC-MS detection result of aripiprazole in Example 1.

[0029] Figure 2 The curve is a linear equation curve of aripiprazole blood concentration and LC-MS peak area obtained in Example 1.

[0030] Figure 3 The curve is a linear regression equation obtained from the linear regression experiment of aripiprazole blood concentration in Example 1. Detailed Implementation

[0031] The technical solution of this application will be further described through the following specific embodiments.

[0032] In this application, the recovery of aripiprazole is mainly improved by the following method: adding risperidone of mass m0 to a specific protein-containing sample, then processing the sample according to the method in step S2, then removing the solvent by vacuum distillation, washing with water, drying and weighing to obtain m1.

[0033] Recovery rate P = m1 / m0 × 100%

[0034] Example 1: This example provides a method for detecting aripiprazole blood concentration, specifically comprising the following steps:

[0035] S1. Prepare samples and standards, wherein the samples are actual samples collected from the patient's blood. In the following examples, the samples are quality control samples prepared using the same preparation method as the standards, and 60 μL / mL of 20% fat emulsion is added to the samples to simulate a hyperlipidemia state.

[0036] Specifically, the standards and samples were prepared according to the concentrations shown in Table 1. For the specific preparation, the aripiprazole standard was dissolved in methanol to prepare a solution. This working solution was then added to horse serum containing 1 mg / mL tea polyphenols and 1 mg / mL ascorbic acid. The concentrations of the prepared standards and quality control samples are shown in Table 1.

[0037] Table 1

[0038] name Aripiprazole ng / mL Calibrator 1 19.0 Calibrator 2 45.0 Calibrator 3 120.0 Calibrator 4 350.0 Calibrator 5 800.0 Calibrator 6 1320.0 Quality control product 1 70 Quality control product 2 280 Quality control product 3 950

[0039] S2. Take 60 μL of sample and keep it at 4℃. Then, centrifuge at 2000 rpm for 3 min to separate the lipid layer and the first supernatant.

[0040] S3. Add a precipitant to the first supernatant. The precipitant formula is as follows: Weigh 8g of zinc sulfate, dissolve it in 100mL of purified water, then add 600mL of methanol and 300mL of acetonitrile, and mix well to obtain the precipitant. Add 300μL of the precipitant to 1.6mL of the first supernatant in a 96-well plate, vortex initially, then centrifuge at 1500rpm for 5min; centrifuge at 5000rpm for 10min to obtain the second supernatant.

[0041] S4. Add 300 μL of tert-butyl methyl ether to the lipid layer, centrifuge at 4000 rpm and a temperature of -10 °C for 8 min to obtain the second lipid and the third supernatant.

[0042] 5. Combine the second and third supernatants, take 30 μL, add 150 μL of ultrapure water, vortex at 500 rpm for 5 min, and perform LC-MS detection. The detection parameters are shown in Table 2.

[0043] Table 2

[0044]

[0045] The chromatogram of aripiprazole under the above conditions is as follows: Figure 1 As shown. Figure 2 This is the standard curve obtained in Example 1. Its linear equation is y = 0.0238x + 0.0145, and the r value is 0.9984.

[0046] The accuracy of the samples was tested using quality control materials, and the specific results are shown in Table 3.

[0047] Table 3

[0048] sample Theoretical concentration ng / mL Detection concentration ng / mL Accuracy % Low quality control 70 74.20 106.00％ Central Quality Control 280 274.00 97.86％ High quality control 950 897.00 94.42％

[0049] In this embodiment, several linear sample quality control samples are also configured, and their configuration method is the same as that of the quality control samples. These samples are used to detect the regressivity of the above-mentioned linear equation, as shown in Table 4.

[0050] Table 4

[0051]

[0052] The experimental results above show that the standard curve obtained in Example 1 exhibits good regression performance, with a correlation coefficient greater than 0.99 and absolute deviations less than ±45 ng / mL, meeting the requirements of actual detection. The specific linear regression equation is y = 0.9922x + 6.5832, and the linear regression curve is shown below. Figure 3 As shown.

[0053] The meanings of each data point in the table above are as follows:

[0054] CV% = Standard deviation of data / Mean

[0055] Accuracy = (estimated value - mean) / mean

[0056] Estimated value = Target value × Linear parameter A + Linear parameter B

[0057] Comparative Example 1 differs from Example 1 in that step S4 is omitted, and the second clear liquid in step S3 is directly subjected to chromatographic detection.

[0058] Example 2: The difference between this example and Example 1 is that in step S4, the temperature is controlled at 0°C.

[0059] Comparative Example 2: The difference between this embodiment and Example 1 is that in step S4, the temperature is controlled at 4°C.

[0060] Example 3 differs from Example 1 in that the temperature is controlled at -4℃ in step S4.

[0061] Example 4: The difference between this example and Example 1 is that in step S4, the temperature is controlled at -20℃.

[0062] Comparative Example 3 differs from Example 1 in that step S2 is replaced as follows: 300 μL of tert-butyl methyl ether is added to the system, followed by centrifugation at 4000 rpm for 8 min. After separating the lipid layer, the liquid is allowed to stand to separate into layers, retaining the organic phase. Step S4 is also omitted.

[0063] Regression tests were performed on three quality control samples (low, medium, and high) for Examples 2-4 and Comparative Examples 1-3 to verify their accuracy and stability. The results are shown in Table 5.

[0064] Table 5

[0065]

[0066]

[0067] The experimental results above show that, compared with the method in Comparative Example 1 that skips step S4, or compared with the method in Comparative Example 3 that directly uses extraction instead of first centrifuging at low temperature and then extracting, the method in this scheme provides a better overall recovery rate, has a higher accuracy of the average measurement, and has better stability.

[0068] In addition, by comparing Example 1 and Comparative Example 2, it can be seen that in step S4, it is necessary to use an ambient temperature not higher than 0°C for extraction. High temperatures may exacerbate the dissolution of lipids in the organic phase, while at low temperatures, lipids are more likely to precipitate and settle, thereby improving the separation of aripiprazole.

[0069] In summary, the method described in this application can improve the separation and extraction performance of aripiprazole at higher blood lipid levels, thereby achieving higher accuracy in blood drug concentration detection.

[0070] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for detecting aripiprazole blood concentration, characterized in that, The steps include: S1. Prepare samples and standards; S2. Control the temperature at 0-10℃ and perform preliminary centrifugation to separate the lipid layer and the first supernatant; S3. Use a precipitating agent to precipitate the first clear liquid, then centrifuge to remove impurities to obtain the second clear liquid; S4. Add tert-butyl methyl ether to the lipid layer, then control the temperature to be less than 0°C, and centrifuge again to obtain the second lipid and the third supernatant. S5. Combine the second and third supernatants, perform chromatographic analysis, plot a standard curve using standards, and compare the samples.

2. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S2, the centrifugation rate is 1000-3000 rpm and the centrifugation time is 1-5 min.

3. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S4, the centrifugation rate is 3000-5000 rpm and the centrifugation time is 5-10 min.

4. A method for detecting aripiprazole blood concentration according to claim 2 or 3, characterized in that, In step S2, the temperature is controlled at 4±1℃, and in step S4, the temperature is controlled at -20 to -4℃.

5. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S3, the precipitant is an aqueous solution of zinc sulfate, an organic solvent solution, or a mixed solution.

6. The method for detecting aripiprazole blood concentration according to claim 5, characterized in that, The precipitant is a water-acetonitrile-methanol mixed solution of zinc sulfate, wherein the volume ratio of water, acetonitrile, and methanol is 1:2 to 4:5 to 8.

7. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S1, the standard is prepared by dissolving aripiprazole in animal serum. The standard is additionally supplemented with tea polyphenols and ascorbic acid at a mass concentration of 0.5-2 mg / mL.

8. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S5, a Phenyl-Hexyl column is used with a water-methanol mixture as the eluent for gradient elution, according to the volume ratio: Initial composition: 95% water + 5% methanol 0.9 min: 60% water + 40% methanol 1.8 min: 50% water + 50% methanol 2 min: 40% water + 60% methanol 4.3 min: 50% water + 50% methanol 4.5 min: 95% water + 5% methanol.

9. The method for detecting aripiprazole blood concentration according to claim 8, characterized in that, In step S5, the pH of the eluent is controlled to be 8-10 using a buffer solution.

10. The method for detecting aripiprazole blood concentration according to claim 1, characterized in that, In step S3, separation is performed using a distributed centrifugation method, which includes a first centrifugation separation and a second centrifugation separation. The first centrifugation separation rate is 1000-2000 rpm and the time is 3-6 min. The second centrifugation separation rate is 3000-6000 rpm and the centrifugation time is 5-20 min.

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