Method for diagnosing poisoning with antipsychotics alimemazine and / or olanzapine and / or risperidone

Abstract

FIELD: clinical laboratory diagnostics; toxicology; pharmacology.SUBSTANCE: used to diagnose poisoning with antipsychotics alimemazine and / or olanzapine and / or risperidone. Blood sampling and quantitative determination of alimemazine, olanzapine and risperidone in the blood are carried out using the HPLC-MS / MS method. Sample preparation is carried out using butyl acetate and Tris buffer, and the specified substances are determined in one matrix. If olanzapine levels are above 60 ng / ml, or risperidone levels are above 80 ng / ml, or alimemazine levels are above 120 ng / ml in the blood, poisoning with the corresponding antipsychotic is diagnosed; or with a mixture of them, if the level of two or more antipsychotics is above the specified level.EFFECT: method enables the accurate and rapid diagnosis of poisoning with antipsychotics – alimemazine, olanzapine and risperidone – with high analytical reliability and practical applicability due to their quantitative determination in the blood.1 cl, 2 ex

Description

[0001] The invention relates to medicine, in particular to clinical laboratory diagnostics, toxicology, pharmacology, and can be used in the diagnosis of acute poisoning, pharmacological research and therapeutic monitoring for individuals taking antipsychotics for a long period.

[0002] A method is known for the quantitative determination of alimemazine in human plasma using liquid-liquid extraction with 100% ethyl acetate for sample preparation [Singh B. et al. Bioanalytical method development and validation of alimemazine in human plasma by LC-MS / MS and its application in bioequivalence studies / / Journal of Pharmacy and Bioallied Sciences. - 2013. - Vol. 5. - No. 4. - Pp. 257-264.], followed by determination by HPLC-MS / MS with electrospray ionization and the use of a deuterated internal standard.

[0003] This method has several limitations: it is only applicable to plasma and is not suitable for whole blood analysis. It only detects alimemazine, making it unsuitable for screening multiple antipsychotics. It also requires a rare and expensive deuterated standard (alimemazine D6), increasing the cost and complexity of the analysis.

[0004] The closest to the claimed method is the simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in human plasma. [Zhou Z. et al. Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry / / Journal of Chromatography B. - 2004. - V. 802. - No. 2. - P. 257-262.] The method uses HPLC-MS / MS with electrospray ionization. Sample preparation includes liquid-liquid extraction with ether after alkalization of the samples. Chromatographic separation is performed on a C18 column with a mobile phase consisting of water (with added formic acid and ammonium acetate) and acetonitrile. Detection is performed in selected ion recording (SIR) mode.

[0005] The disadvantages of the method chosen as a prototype are as follows: it does not allow the determination of alimemazine, which reduces its value for the comprehensive monitoring of atypical antipsychotics; it is designed exclusively for plasma analysis and is unsuitable for whole blood testing; it requires the use of diazepam as an internal standard, which makes it inapplicable in clinical laboratory diagnostics, since diazepam is often used in therapy and may be present in the patient's biological material.

[0006] The objective of the invention is the accurate and rapid diagnosis of poisoning with antipsychotics – alimemazine, olanzapine and risperidone – by their quantitative determination in the blood.

[0007] The technical result of the stated problem is achieved in that in the method for diagnosing poisoning with antipsychotics alimemazine and / or olanzapine and / or risperidone, which includes blood sampling, quantitative determination of alimemazine, olanzapine and risperidone in the blood by HPLC-MS / MS, sample preparation is carried out using butyl acetate and Tris buffer and the said substances are determined in one matrix, and at olanzapine levels above 60 ng / ml or risperidone above 80 ng / ml or alimemazine above 120 ng / ml in the blood, poisoning with the corresponding antipsychotic is diagnosed; or a mixture thereof, if the level of two or more antipsychotics is above the specified mark.

[0008] The use of butyl acetate and Tris buffer in sample preparation ensures high extraction of target compounds, minimizes matrix effects, and ensures analyte stability. This ensures a linear dependence of the analytical signal on concentration within a given range and increases the accuracy of quantification. When taking the listed antipsychotics at maximum therapeutic doses, their concentrations in the blood do not exceed the specified limits. For olanzapine, risperidone, and alimemazine, these are 60, 80, and 120 ng / mL in the blood, respectively. Blood concentrations of antipsychotics above these limits can indicate poisoning.

[0009] Simultaneous analysis of the specified substances in one matrix in one chromatographic run reduces the research time, reduces the consumption of sample and reagents, and also provides a rapid assessment of the concentrations of atypical antipsychotics in the patient's blood.

[0010] The method is as follows. Blood is collected from the patient. For sample preparation, 4 ml of demineralized water, 600 µl of Tris buffer, and 1 ml of butyl acetate are added to 1 ml of blood. The sample is vortexed for 10 minutes, then the tube is centrifuged at 3000 rpm for 5 minutes. 500 µl of the organic layer from the top is removed and transferred to a vial, evaporated at room temperature using a vacuum evaporator, and the sample is diluted with 500 µl of the working solution. The vial is shaken well for 1 minute, after which the sample is drawn with a syringe through a 13 mm diameter filter with a 0.45 µm pore size under pressure into a clean vial. The resulting filtered sample is placed in the chromatograph autosampler.

[0011] Reagents used:

[0012] Phase A - Purified deionized water was placed in a 1 L flat-bottomed flask in an amount of 500 ml, ammonium formate (0.63 g) and formic acid (1 ml) were added, and the solution was brought to 1 L with water. The resulting solution was passed through filter paper using a VP-30 vacuum pump (Labtech).

[0013] Phase B - acetonitrile for chromatography.

[0014] Working solution - place 10 ml of acetonitrile in a 100 ml measuring flask and add phase A to the mark.

[0015] Butyl acetate

[0016] Tris-HCl buffer with pH=9.0 consisting of tris-(hydroxymethyl)-aminomethane and hydrochloric acid

[0017] Stock 1 mg / ml solutions of olanzapine, risperidone and alimemazine: 10 mg of the substance (precisely weighed) is placed in a 10 ml flask, brought to the mark with 50% methanol, and mixed.

[0018] A stock solution with a concentration of 300 μg / mL is prepared by mixing 900 μL of each 1 mg / mL stock solution and adding 300 μL of the working solution.

[0019] From the stock solution, by successive dilutions with the working solution, a solution with a concentration of each component of 2 μg / ml is obtained.

[0020] If olanzapine levels are above 60 ng / ml, risperidone levels are above 80 ng / ml, and alimemazine levels are above 120 ng / ml in the blood, poisoning is diagnosed.

[0021] Example 1. Patient G., 22 years old. Admitted to the I.I. Dzhanelidze Research Institute of Emergency Medicine via ambulance on April 14, 2024. Blood was tested using the stated method for diagnosis. Ethanol, alimemazine, and olanzapine were detected in the blood. The stated method was used for the quantitative determination of antipsychotics. The obtained olanzapine blood concentrations were 282 ng / ml, and alimemazine was 144 ng / ml. The diagnosis was antipsychotic poisoning. The patient later admitted to taking approximately 10 olanzapine tablets and a blister pack of alimemazine tablets. The patient received the necessary care for combined poisoning and was discharged from the toxicology department.

[0022] Example 2. Patient L., 57, was admitted to the I.I. Dzhanelidze Research Institute of Emergency Medicine by ambulance on March 28, 2025, in the toxicology department with suspected neuroleptic malignant syndrome. For diagnosis, blood was tested using the stated method. Olanzapine, alimemazine, risperidone, and other antipsychotics were not detected in the blood. The patient was transferred to the neurology department of the I.I. Dzhanelidze Research Institute of Emergency Medicine.

[0023] The proposed method demonstrates high analytical reliability and practical applicability, making it an effective solution for clinical laboratory diagnostics. It is characterized by high accuracy and sensitivity (detection limit of 0.05 ng / ml), ensuring the reliable determination of therapeutic and toxic concentrations of antipsychotics in the range of 0.05-0.3 μg / ml.

[0024] The method is applicable to blood analysis, providing broad diagnostic capabilities for assessing pharmacokinetics and identifying intoxications. Its clinical significance is confirmed by a high correlation with patient dynamics and the ability to monitor detoxification effectiveness.

[0025] The cost-effectiveness of this method is achieved through reduced operating costs, reduced reagent consumption, and the use of standard laboratory equipment, making it accessible to medical institutions of various levels. Implementation of this method optimizes therapy, reduces the incidence of hospitalizations associated with intoxications, and improves the early diagnosis of complications.