A method for detecting olanzapine blood concentration

Abstract

The application relates to the field of drug detection, in particular to a method for detecting blood drug concentration of olanzapine. In the process of extracting olanzapine, the method adopts the mode of first precipitation and then extraction, which can improve the recovery degree of olanzapine in blood, and further improve the detection accuracy of olanzapine at a lower concentration.

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 realizing individualized medication.

[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:

[0006] S1, configuring a calibration sample;

[0007] S2, performing chromatographic detection on the standard sample, and recording the standard curve according to the gradient concentration thereof;

[0008] S3, configuring a precipitant, and treating the sample by the precipitant;

[0009] S4, determining the actual sample by the same method, and determining the concentration thereof according to the standard curve;

[0010] In the step S3, the precipitation is firstly carried out, and then the supernatant is extracted and concentrated by using the 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 water to alcohol is 0.02-0.1:1. The extractant is methyl tert-butyl ether.

[0011] In the above scheme, generally, the traditional method is to directly use an organic system such as methanol or acetonitrile to precipitate the protein. In the later stage of protein precipitation, due to the multiple nitrogen rings and the meta nitrogen-sulfur coordination structure in olanzapine, it is easy to form a coordination system with the carboxyl in the protein, causing part of the olanzapine to be adsorbed, and thus causing a small amount of olanzapine to have a reduced concentration, and thus causing the standard curve obtained to have a low accuracy in the low concentration region.

[0012] 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 improve the precipitation rate of the protein itself. At the same time, the use of zinc sulfate to bind 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 mixed system of water and alcohol with a volume ratio of 0.02-0.2:1. Further preferably, the solvent of the precipitant is a mixed system of water and methanol with a volume ratio of 0.05-0.1:1.

[0013] On the basis of the above, methyl tert-butyl ether is used as the extractant to extract the lipophilic olanzapine. It should be noted that in this step, the specific steps are as follows: in the step S2, the precipitant is added to the sample, and then the supernatant is blown and concentrated. Subsequently, without separating the precipitate, methyl tert-butyl ether is added for extraction, and the supernatant is separated by centrifugation. In the case of separating the precipitate, the supernatant is first concentrated and extracted, and then the protein is separated 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 the supernatant is blown and concentrated, most of the methanol is volatilized, so methyl tert-butyl ether can better extract the residual methanol and olanzapine dissolved in the above-mentioned methanol. Overall, it has a better extraction degree for olanzapine compared with directly using a mixed system of methyl tert-butyl ether and water for extraction, or directly using an organic solvent precipitant to precipitate the protein.

[0014] Preferably, the precipitant further comprises magnesium sulfate, and the mass concentration of the magnesium sulfate is 1-5 g / L.

[0015] The small amount of magnesium sulfate as a whole can utilize the complexing and sedimentation properties of magnesium ions to further improve the sedimentation properties of the protein. Since the size of magnesium ions is small, they can better occupy the adsorption of olanzapine in the protein quaternary structure. At the same time, magnesium ions themselves can 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 the ion electrolysis in water and the solubilization of methanol.

[0016] Preferably, the precipitant further contains a buffer, which adjusts the pH of the precipitant to 5-7. Within this range, olanzapine has poorer solubility in water and better solubility in the organic phase, while the protein itself is more easily precipitated, improving the overall separation performance. The buffer is preferably well soluble in the above solvent system and has a more stable pH.

[0017] In the step S3, the volume after concentration is 20-40% of the supernatant volume. Within 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 due to coordination and other phenomena is reduced. 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.

[0018] Preferably, during the chromatographic detection process, the mobile phase comprises mobile phase A and 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. In the above scheme, the tailing phenomenon in liquid chromatography can be reduced, thereby improving the accuracy of the standard curve.

[0019] In summary, the present application provides a method for detecting the blood concentration of olanzapine, which uses the method of precipitation first and then extraction in the process of extracting olanzapine, which can improve the recovery of olanzapine in blood, thereby improving the detection accuracy of olanzapine at low concentrations. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the detection peak of olanzapine in chromatography in step S2 of Example 4.

[0021] Figure 2 is the standard curve drawn in step S2 of Example 4. DETAILED DESCRIPTION

[0022] The scheme in the present application is further described in the following specific embodiments.

[0023] In the present application, the recovery rate of olanzapine is mainly determined by the following method: adding olanzapine with a mass of m0 into a sample containing protein, then treating the sample in the manner of step S2, then removing the solvent by distillation under reduced pressure, then washing with water, and weighing after drying to obtain m1.

[0024] The recovery rate P = m1 / m0 x 100%

[0025] In the following examples, the recovery rates of high-concentration olanzapine and low-concentration olanzapine are 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.

[0026] Example 1, the present embodiment is a method for treating an olanzapine sample, and the sample involved in the present embodiment is a large bovine serum containing 2 mg / mL citric acid and 1 mg / mL glutathione. Specifically, in the present embodiment, the sample is treated as follows:

[0027] 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, mixed uniformly to obtain the precipitant, and the pH of the precipitant is 7.

[0028] In the above sample, the sample and the precipitant are mixed at a volume ratio of 1:10, first mixed uniformly, then concentrated by nitrogen blowing to a concentration of 30% of the initial volume, 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 then weighed after drying.

[0029] The control is set as follows, for example:

[0030] Control Example 1: After the precipitant is configured, the sample and the precipitant are mixed at a volume ratio of 1:10, 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 then weighed.

[0031] 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.

[0032] Table 1

[0033]

[0034] From the above experimental data, it can be seen that compared with Comparative Example 1, the recovery rate of olanzapine can be obviously improved by the method of the present application, and good recovery can be achieved at both high and low concentrations. The volume ratio of water to methanol is preferably controlled in 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. When the water content is too low, it will easily reduce the phase separation performance after extraction, resulting in a decrease in the recovery rate at low concentration.

[0035] In Example 2, the salt component in the precipitant was adjusted, as shown in Table 2.

[0036] Table 2

[0037]

[0038] From the above experiments, it can be seen that under the combined action of zinc sulfate and magnesium sulfate, the adsorption of protein on olanzapine can be reduced on the basis of good precipitation of protein. 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.

[0039] In Example 3, the purpose of this example is to study the effect 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.

[0040] Table 3

[0041]

[0042] From the above experiments, by comparing Comparative Example 3-1 and Example 1, it can be seen that when no concentration is performed, extraction is easy to cause 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-settle, thereby forming a more stable chelation system inside. Especially at a higher concentration, olanzapine may precipitate in the precipitant, thereby resulting in a lower recovery rate at high concentration.

[0043] In Example 4, the raw material liquid is treated according to the extraction method in Example 1, and a detection method for the blood concentration of olanzapine is provided, which specifically comprises the following steps:

[0044] S1, preparing a standard: olanzapine standard is dissolved in methanol to prepare a solution for preparing a standard working solution. Take the working solution to prepare 6 series of calibration samples with concentrations as shown in Table 4 (unit: ng / mL).

[0045] Table 4

[0046]

[0047] S2, chromatographic detection was performed on the standard, and a standard curve was drawn according to the gradient concentration thereof; the specific chromatographic detection parameters and conditions are shown in Table 5.

[0048] Table 5

[0049]

[0050] The detection peak of olanzapine in liquid chromatography is shown in Figure 1 The standard curve drawn is shown in Figure 2

[0051] The linear range verification results of the above standard curve are shown in Table 6.

[0052] Table 6

[0053]

[0054] S3, a precipitant was configured, and the sample was treated by the precipitant;

[0055] In this step, the sample was configured by configuring olanzapine into a solution containing 2 mg / mL citric acid and 1 mg / mL glutathione in large bovine serum. The treatment method refers to Example 1.

[0056] S4, the actual sample was measured according to the same method, and the concentration thereof was determined according to the standard curve;

[0057] 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.

[0058] Table 7

[0059]

[0060] In addition, the following sample was configured to verify the precision of olanzapine on the linear range, and the specific results are shown in Table 8.

[0061] Table 8

[0062]

[0063] From the above experiments, it can be seen that the scheme in the present application still has good detection accuracy and precision at a lower concentration.

[0064] ​The embodiments are only illustrative of the present application, and are not intended to limit the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for detecting blood concentration of olanzapine, characterized by, The method comprises the following steps S1, preparing a standard sample; S2, performing chromatographic detection on the standard sample, and drawing a standard curve according to the gradient concentration; S3, preparing a precipitant, and treating the sample by using the precipitant; S4, determining the actual sample by using the same method, 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 by using an extractant to improve the content of olanzapine, the solvent of the precipitant is a mixed system of water and methanol, the volume ratio of the water and the alcohol is 0.02-0.1:1, and the extractant is methyl tert-butyl ether; The step S3 is specifically as follows: the precipitant is added to the sample, and then the supernatant is blown and concentrated, the volume after the concentration is 20-40% of the volume of the supernatant, then methyl tert-butyl ether is added to extract without separating the precipitate, and the supernatant is separated by centrifugation, the precipitant contains at least zinc sulfate, the mass concentration of the zinc sulfate is 5-20 g / L, and the precipitant further contains magnesium sulfate, the mass concentration of the magnesium sulfate is 1-5 g / L; The precipitant further contains a buffer, and the buffer adjusts the pH of the precipitant to 5-7.

2. The method of claim 1, wherein the method is for detecting an olanzapine blood concentration. The buffer is ammonium acetate.

3. The method for detecting olanzapine blood concentration according to claim 1, characterized in that, In the step S3, the volume of the extractant added is 5-10 times of the volume of the supernatant after the concentration.

4. The method of claim 1, wherein the method is for detecting blood concentration of olanzapine. The volume ratio of the water and the methanol in the solvent of the precipitant is 0.05-0.1:1.

Images