Olanzapine blood concentration detection method

Abstract

The invention relates to the field of medicine detection, in particular to an olanzapine blood concentration detection method. In the process of extracting olanzapine, a mode of firstly carrying out precipitation and then carrying out extraction is adopted, so that the recovery degree of olanzapine in blood can be improved, and the detection accuracy of olanzapine at a relatively low concentration is further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug detection, in particular to a method for detecting blood concentration of olanzapine. BACKGROUND

[0002] Olanzapine is an antipsychotic drug widely used in the treatment of schizophrenia, bipolar disorder and other diseases, and its blood concentration is closely related to efficacy and adverse reactions. Clinical studies have shown that the effective therapeutic concentration range of olanzapine is 20-80 ng / mL, and a concentration lower than 20 ng / mL may lead to insufficient treatment, and a concentration higher than 80 ng / mL may cause metabolic abnormalities, drowsiness and other side effects213. Therefore, accurate detection of blood concentration is the key to individualized drug use.

[0003] At present, the detection methods of olanzapine blood concentration mainly include high performance liquid chromatography (HPLC), ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and the like. Among them, sample pretreatment is the core link affecting the sensitivity and accuracy of detection. The conventional method usually uses organic solvents combined with precipitants for protein precipitation, and the protein interference in the blood plasma is removed by centrifugal separation. However, the existing technology has the following limitations: olanzapine has high liposolubility (logP=4.1), and it is easy to form a complex with denatured protein or precipitant (such as metal ions) during protein precipitation, resulting in part of the drug being wrapped or adsorbed in the precipitate, and the recovery rate is significantly reduced, which in turn reduces the detection limit of olanzapine in blood. SUMMARY

[0004] In order to improve the detection limit of olanzapine, reduce its adsorption in protein or loss in the protein separation process, and reduce background noise, the present application provides a method for detecting blood concentration of olanzapine.

[0005] Firstly, the method for detecting blood concentration of olanzapine in the present application comprises the following steps: S1, configuring a calibration sample; S2, performing chromatographic detection on the standard sample, and recording the standard curve according to the gradient concentration; S3, configuring a precipitant, and treating the sample by the precipitant; S4, determining the actual sample by the same method, and determining its concentration according to the standard curve; In the step S3, the precipitation is performed first, and then the supernatant is extracted and concentrated by using an extractant to improve the content of olanzapine, the solvent of the precipitant is a mixed system of water and alcohol, and the volume ratio of the water to the alcohol is 0.02-0.1:1, and the extractant is methyl tert-butyl ether.

[0006] In the above scheme, the conventional method is usually to directly use methanol, acetonitrile and other organic systems to precipitate the protein. In the later stage of protein precipitation, due to the multiple nitrogen rings in olanzapine and the meta nitrogen-sulfur coordination structure, it is easy to form a coordination system with the carboxyl group in the protein, causing part of the olanzapine to be adsorbed, thereby causing a small amount of olanzapine to have a reduced concentration, and thus resulting in a lower accuracy of the standard curve obtained at a low concentration region.

[0007] In the present scheme, a water-alcohol mixed system is first used. In this system, the overall precipitation process is slower. In the precipitant, a certain amount of zinc sulfate is added to precipitate, which can improve the precipitation rate of the protein itself. At the same time, the combination of zinc sulfate with the protein can better achieve precipitation while reducing the impact on olanzapine. In this system, the mass concentration of zinc sulfate is preferably 5-20 g / L. The solvent of the precipitant is preferably a mixture of water and alcohol with a volume ratio of 0.02-0.2:1. Further preferably, the solvent of the precipitant is a mixture of water and methanol, wherein the volume ratio of water to methanol is 0.05-0.1:1.

[0008] On the basis of the above, methyl tert-butyl ether is used as an extractant to extract lipophilic olanzapine. It should be noted that in this step, the specific steps are as follows: the step S2 is specifically as follows: adding a precipitant to the sample, then blowing and concentrating the supernatant, then adding methyl tert-butyl ether for extraction without separating the precipitate, and then centrifuging and separating the supernatant. In the case of separating the precipitate, first concentrate and extract, then separate the protein by centrifugation. The olanzapine adsorbed in the protein can be back-extracted, and then separated by centrifugation. In this process, low-concentration olanzapine can be better dissolved in the organic phase system formed by methyl tert-butyl ether, and since methyl tert-butyl ether itself has strong liposolubility, it has good solubility for olanzapine. After blowing and concentrating the supernatant, most of the methanol is volatilized, so methyl tert-butyl ether can better extract the residual methanol and olanzapine dissolved in the above methanol. Overall, it has better extraction degree for olanzapine compared with directly using a mixture of methyl tert-butyl ether and water or directly using an organic solvent precipitant to precipitate the protein.

[0009] Preferably, the precipitant further comprises magnesium sulfate, and the mass concentration of the magnesium sulfate is 1-5 g / L.

[0010] Overall, a small amount of magnesium sulfate can use the complexing and settling properties of magnesium ions to further improve the settling properties of the protein. Since the size of magnesium ions is small, it can better occupy the adsorption of olanzapine in the protein quaternary structure. At the same time, magnesium ions can also form a coordination system with olanzapine, reducing the dissolution and retention of a small amount of olanzapine in the water system and protein adsorption system due to ion electrolysis in water and the solubilizing effect of methanol.

[0011] Preferably, the precipitant further contains a buffer, which adjusts the pH of the precipitant to 5-7. In this range, olanzapine has a poorer solubility in water and a better solubility in the organic phase, and the protein itself is more easily precipitated, improving the overall separation performance. The buffer is preferably one that has good solubility in the above-mentioned solvent system and is more stable in pH.

[0012] In the step S3, the volume after concentration is 20-40% of the supernatant volume. In this range, the miscibility caused by the residual methanol in the system and the influence on the extraction performance of olanzapine can be reduced as much as possible. The residual olanzapine in the water system caused by coordination and other phenomena is reduced. Meanwhile, preferably, in the step S3, the volume of the added extractant is 5-10 times the volume of the supernatant after concentration. Therefore, it can better purify the olanzapine therein.

[0013] Preferably, in the process of chromatographic detection, the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is a 5nM aqueous solution of ammonium acetate, and the mobile phase B is a mixed system with a volume ratio of methanol: acetonitrile: formic acid = 1: 1: 0.04. In the above-mentioned scheme, the tailing phenomenon in liquid chromatography can be reduced, and the accuracy of the standard curve is improved.

[0014] In summary, the present application provides a method for detecting the blood concentration of olanzapine, which uses the method of first precipitation and then extraction in the process of extracting olanzapine, which can improve the recovery of olanzapine in blood, and thus improve the detection accuracy of olanzapine at a lower concentration. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the detection peak of olanzapine in chromatography in step S2 of Example 4.

[0016] Figure 2 is the standard curve drawn in step S2 of Example 4. DETAILED DESCRIPTION

[0017] The scheme in the present application is further described in the following specific embodiments.

[0018] In the present application, the recovery of olanzapine is mainly determined by the following method: adding olanzapine with a mass of m0 to a sample containing a specific protein, then treating the sample according to the method in step S2, then removing the solvent by distillation under reduced pressure, then washing with water, and then weighing after drying to obtain m1.

[0019] Recovery rate P = m1 / m0 x 100% In the following examples, the recovery rate of high concentration olanzapine and low concentration olanzapine is tested respectively, wherein the concentration of low concentration olanzapine is 5.5 ng / mL, and the concentration of high concentration olanzapine is 1000 ng / mL.

[0020] Example 1, this example is a method for processing olanzapine sample, the sample involved in this example is large bovine serum containing 2 mg / mL citric acid and 1 mg / mL glutathione, specifically, in this example, the sample is processed as follows: The precipitant is configured, specifically, 10 g of zinc sulfate, 2.5 g of magnesium sulfate, and 0.15 g of ammonium acetate (as a buffer) are dissolved in 100 mL of water, and then 900 mL of methanol is added, and the mixture is uniformly mixed to obtain the precipitant, and the pH of the precipitant is 7.

[0021] In the above sample, the sample and the precipitant are mixed at a volume ratio of 1:10, uniformly mixed first, and then concentrated by nitrogen blowing to a concentration of 30% of the initial volume, and then methyl tert-butyl ether is added as an extractant, the amount of the extractant is 10 times the volume of the supernatant after concentration, and after sufficient shaking, it is vortexed at 1400 rpm for 5 min; 5000 rpm centrifugation for 10 min, then the supernatant is washed with water and dried, the residue is washed with water and dried, and then weighed.

[0022] The control is set as follows: Control Example 1: After the precipitant is configured, the sample and the precipitant are mixed at a volume ratio of 1:10, and then vortexed at 1400 rpm for 5 min; 5000 rpm centrifugation for 10 min, then the supernatant is washed with water and dried, the residue is washed with water and dried, and then weighed.

[0023] On the basis of Example 1, the volume ratio of water and methanol in the precipitant is verified, and the experimental results are shown in Table 1.

[0024] Table 1 From the above experimental data, it can be seen that compared with Comparative Example 1, the recovery rate of olanzapine can be significantly improved by using the method of the present application, and good recovery can be achieved at high and low concentrations. The volume ratio of water and methanol is preferably controlled within the range of 1:10 to 1:50, too much water will cause the solubility of olanzapine in the system to decrease, which will affect the subsequent extraction, mainly affecting the recovery rate at high concentration, and too little water will easily reduce the phase separation performance after extraction, resulting in a decrease in the recovery rate at low concentration.

[0025] Example 2, in this example, the salt component in the precipitant is adjusted, as shown in Table 2.

[0026] Table 2 From the above experiments, it can be seen that under the combined action of zinc sulfate and magnesium sulfate, the adsorption of olanzapine by protein can be reduced on the basis of good protein precipitation. Specifically, when the mass concentration of zinc sulfate is controlled in the range of 5-20 g / L and the mass concentration of magnesium sulfate is controlled in the range of 1-5 g / L, the overall recovery rate is better.

[0027] Example 3, the purpose of this embodiment is mainly to study the influence of the concentration step on the recovery rate. Specifically, the experimental results of the proportion of the volume of the supernatant after concentration to the initial volume and the recovery rate are shown in Table 3.

[0028] Table 3 From the above experiments, comparing Example 3-1 and Example 1, it can be seen that when no concentration is performed, the extraction is easy to cause more obvious material loss, and with the increase of the concentration volume ratio, the overall recovery rate shows a trend of first increasing and then decreasing. The principle may be that after the transition concentration, olanzapine and protein will co-deposit, thereby forming a more stable chelate system inside, especially at a higher concentration, olanzapine may precipitate in the precipitant, thereby resulting in a lower recovery rate at a higher concentration.

[0029] Example 4, in this embodiment, the raw material liquid is treated according to the extraction method in Example 1, and a detection method for olanzapine blood concentration is provided, which specifically comprises the following steps: S1, preparing a standard sample: preparing a standard sample by dissolving olanzapine standard sample with methanol to prepare a solution for preparing a standard working solution. Take the above working solution and prepare 6 series of calibration samples with different concentrations, and the concentrations are shown in Table 4 (unit: ng / mL).

[0030] Table 4 S2, detecting the standard sample by chromatography, and recording the standard curve according to the gradient concentration; the specific chromatography detection parameters and conditions are shown in Table 5.

[0031] Table 5 The detection peak of olanzapine in liquid chromatography is shown in Figure 1 The standard curve obtained is shown in Figure 2 .

[0032] The linear range verification results of the above standard curve are shown in Table 6.

[0033] Table 6 S3, configure a precipitant, and treat the sample by the precipitant; In this step, the sample was configured by configuring olanzapine into a solution of large bovine serum containing 2 mg / mL citric acid and 1 mg / mL glutathione. The treatment method refers to Example 1.

[0034] S4, determine the actual sample according to the same method, and determine its concentration according to the standard curve; In this step, the accuracy of the above method in the lower limit of quantification was first verified, and the specific measurement results are shown in Table 7.

[0035] Table 7 In addition, the following samples were configured to verify the precision of olanzapine in the linear range, as shown in Table 8.

[0036] Table 8 From the above experiments, it can be seen that the scheme in the present application still has good detection accuracy and precision at lower concentrations.

[0037] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A method for detecting blood concentration of olanzapine, characterized by, The method comprises the following steps S1, preparing a calibration sample; S2, performing chromatographic detection on the standard sample, and recording a standard curve according to the gradient concentration; S3, preparing a precipitant, and treating the sample with the precipitant; S4, determining the actual sample in the same way, and determining the concentration according to the standard curve; In the step S3, the precipitant is first precipitated, and then the supernatant is extracted and concentrated with an extractant to improve the content of olanzapine, the solvent of the precipitant is a mixed system of water and alcohol, the volume ratio of water to alcohol is 0.02-0.1:1, and the extractant is methyl tert-butyl ether.

2. The method of claim 1, wherein the method is for detecting an olanzapine blood concentration. The step S3 is specifically as follows: the precipitant is added to the sample, and then the supernatant is concentrated by blowing, and then methyl tert-butyl ether is added for extraction without separating the precipitate, and the supernatant is centrifuged and separated.

3. The method for detecting olanzapine blood concentration according to claim 2, characterized in that, In the step S3, the precipitant contains at least zinc sulfate, and the mass concentration of zinc sulfate is 5-20 g / L.

4. The method of claim 3, wherein the blood concentration of olanzapine is determined by the following equation: ###0001### wherein AUC is the area under the plasma concentration-time curve, and t is the time after administration of the olanzapine. The precipitant further contains magnesium sulfate, and the mass concentration of magnesium sulfate is 1-5 g / L.

5. The method of claim 3, wherein the blood concentration of olanzapine is determined by the following equation: ###0001### wherein AUC is the area under the plasma concentration-time curve, and t is the time after administration of the olanzapine. The precipitant further contains a buffer, and the buffer adjusts the pH of the precipitant to 5-7.

6. The method of claim 5, wherein the blood concentration of olanzapine is determined by the method comprising the steps of: The buffer is ammonium acetate.

7. The method for detecting olanzapine blood concentration according to claim 2, characterized in that, In the step S3, the volume after concentration is 20-40% of the volume of the supernatant.

8. The method for detecting olanzapine blood concentration according to claim 7, characterized in that, In the step S3, the volume of the extractant added is 5-10 times the volume of the supernatant after concentration.

9. The method for detecting olanzapine blood concentration according to claim 2, characterized in that, The solvent of the precipitant is a mixed system of water and methanol, and the volume ratio of water to methanol is 0.05-0.1:

1.

10. The method of claim 1, wherein the method is for detecting olanzapine blood concentration. In the chromatographic detection process, the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a 5nM ammonium acetate aqueous solution, and the mobile phase B is a mixed system with a volume ratio of methanol:acetonitrile:formic acid=1:1:0.04.

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