RU-2861361-C1 - METHOD FOR QUANTITATIVE DETERMINATION OF DESLORATADINE IN BIOLOGICAL SAMPLES

Abstract

FIELD: analytical chemistry. SUBSTANCE: invention can be used for the quantitative determination of desloratadine content in biological samples, including transdermal therapeutic systems, using HPLC-MS/MS. The method for quantitative determination of desloratadine in biological samples is based on multi-stage sample preparation, including liquid-liquid extraction with a mixture of ethyl acetate and dichloromethane, subsequent evaporation, reconstitution in methanol with ammonium formate and analysis in isocratic mode on a reversed-phase C18 column with MRM detection of characteristic ion transitions of desloratadine: 311.1 m/z - 259.1 m/z. EFFECT: increasing the sensitivity of the method to 0.01-0.05 ng/ml. 1 cl, 2 dwg, 57 tbl

Inventors

• Zaborovskij Andrej Vladimirovich
• Yunina Dina Vladimirovna
• Tararina Larisa Anatolevna
• Tsaregorodtsev Sergej Vadimovich
• Sokolov Filipp Sergeevich
• Ageev Valentin Pavlovich
• Markushova Evgeniya Vitalevna
• Sinichkina Anna Denisovna

Dates

Publication Date

20260505

Application Date

20251226

Claims (1)

1. A method for the quantitative determination of desloratadine in biological samples by high-performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS), comprising: sample preparation by extracting desloratadine with an organic solvent, centrifuging and collecting the supernatant, evaporating the extract, restoring the residue in the mobile phase, chromatographic separation on a reversed-phase C18 column, mass spectrometric detection in the MRM mode with the transition 311.1 m/z → 259.1 m/z, quantitative determination using a calibration curve, characterized in that: sample preparation for samples of skin, plasma and transdermal patches is carried out at room temperature of 20-25°C by homogenizing a sample of the analyzed matrix weighing 0.3 ± 0.04 g in a mixture of ethyl acetate and dichloromethane in a ratio of 80:20 parts by volume, the homogenized mixture is vortexed for 2 minutes at 2500 rpm, centrifuged at 10,000 g for 10 minutes, 900 μl of the supernatant are collected and evaporated to dryness at a temperature of 60 ° C, the dry residue is reconstituted in a mixture of 480 μl of methanol and 120 μl of 10 mM ammonium formate, centrifuged again at 10,000 g for 10 minutes, the resulting supernatant is diluted with a mobile phase, methanol: 10 mM ammonium formate 80:20, 1000 times, while the chromatographic separation is carried out in isocratic mode at a flow rate of 0.5 ml / min, a column temperature of 40 ° C and a sample injection volume of 10 μl, a calibration curve is plotted in the concentration range 0.1-100 ng/ml with a determination coefficient R2 ≥ 0.99, calibration samples are incubated after adding the working solution for 15-20 minutes at room temperature of 20-25°C for uniform distribution of the analyte, standard solutions of desloratadine are stored at -20°C for no more than 30 days, working solutions are stored at +4°C for no more than 24 hours, long-term stability of samples is ensured by storage at -80°C for 30 days.

Description

The invention relates to the field of analytical chemistry, pharmaceutical analysis and can be used to control the quality of dosage forms of desloratadine, as well as to study its pharmacokinetics and biodistribution in blood plasma and tissues at the preclinical and clinical stages of development. The prevalence of allergic diseases worldwide remains extremely high, making the development and improvement of antiallergic medications a pressing issue. Desloratadine, an active metabolite of loratadine, is one of the most effective and widely used latest-generation H1-histamine receptor antagonists. Its use is associated with minimal sedative activity and prolonged action. In addition to standard dosage forms (tablets, syrups), new forms of desloratadine are currently being actively developed, such as transdermal patches, designed to provide controlled release and improve patient compliance. The development of such formulations requires careful study of their pharmacokinetics, particularly their skin penetration and distribution in biological environments. High-performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS) is the gold standard for the quantitative determination of desloratadine in biological samples and dosage forms. This method offers high selectivity and sensitivity, but is associated with high equipment and operating costs and requires complex sample preparation, especially for complex matrices such as skin. The main challenge in determining desloratadine in skin samples is the complexity of sample preparation, due to the need to isolate the analyte from a complex solid-phase matrix rich in proteins, lipids, and other interfering substances. Existing sample preparation methods for blood plasma are not applicable to skin samples, as they do not provide effective analyte extraction and result in significant matrix effects. A method is known for the quantitative determination of desloratadine and 3-hydroxydesloratadine in human blood plasma by HPLC-MS/MS with a range of measured concentrations of 0.05-10 ng/ml, using liquid-liquid extraction with ethyl ether [https://www.sciencedirect.com/science/article/pii/S0731708507003949]. A drawback of this method is its inability to determine desloratadine in skin samples, due to the inability of the described sample preparation to effectively extract the analyte from a complex solid-phase matrix. Furthermore, the available scientific literature lacks any information on methods for determining desloratadine in skin. The objective of the present invention is to develop a highly sensitive and selective method for the quantitative determination of desloratadine in a wide range of matrices, including complex biological samples (skin, plasma) and dosage forms (transdermal patches). Technical result and essence of the invention The technical result of the invention is to achieve a lower limit of quantitative determination of desloratadine at the level of 0.01-0.05 ng/ml, which exceeds the sensitivity of known methods for plasma, due to the use of a new multi-stage sample preparation procedure for skin samples. The essence of the invention is as follows. For skin samples, sample preparation is carried out, including: - homogenization of a sample of skin in a mixture of ethyl acetate and dichloromethane; - subsequent centrifugation and collection of supernatant; - evaporation and reduction of the residue in methanol and ammonium formate; - repeated centrifugation and dilution of the resulting supernatant. Sample preparation: A skin sample (0.3 ± 0.04 g) remaining after desloratadine diffusion studies was manually ground in the presence of 1600 μl of a mixture of ethyl acetate and dichloromethane (80:20), vortexed for 2 min at 2500 rpm, and centrifuged for 10 min at 10,000 g. 900 μl of the supernatant were collected in a clean tube and evaporated to dryness in a centrifuge concentrator at 60°C. 480 μl of methanol and 120 μl of 10 mM ammonium formate were added to the dry residue. The resulting mixture was centrifuged for 10 min at 10,000 g. The supernatant was collected in a clean test tube and diluted 1000-fold with the phase (a mixture of methanol and 10 mM ammonium formate, 80:20). 500 µl of the resulting solution was collected and placed in a chromatography vial. For plasma samples and dosage forms (transdermal patches), similarly optimized sample preparation procedures are used to ensure complete extraction of the analyte. For plasma samples, sample preparation is carried out, including: - homogenization of the plasma sample in a mixture of ethyl acetate and dichloromethane; - subsequent centrifugation and collection of supernatant; - evaporation and reduction of the residue in methanol and ammonium formate; - repeated centrifugation and dilution of the resulting supernatant. Sample preparation: To a plasma sample (300 μl) was added 1600 μl of a mixture of ethyl acetate and dichloromethane (80:20), vortexed for 2 min at 2500 rpm, centrifu