Method for determining bedaquiline concentration in blood serum

Abstract

FIELD: pharmacology.SUBSTANCE invention can be used to determine the concentration of an anti-tuberculosis drug in blood serum. The method for determining the concentration of bedaquiline in the blood serum in patients with tuberculosis or mycobacteriosis is that whole blood samples are centrifuged at 3000 rpm for 20 minutes, then the blood plasma is collected in sterile 1.5 mL Eppendorf tubes, then 900 mcL of acetonitrile are added to 300 mcL of plasma and centrifuged at 13500g for 15 minutes, then 1 mL of the supernatant is transferred to a chromatographic vial and make the assay by UHPLC MS / MS using an Athena UHPLC C18, 1.8 mcM, 120A, 2.1×100 mm chromatography column, when using an aqueous solution containing 5 g / L of ammonium acetate, 25 ml / L of concentrated acetic acid, 2 ml / L trifluoroacetic acid as mobile phase A and 100% acetonitrile as mobile phase B, the concentration of bedaquiline is determined using a pre-plotted calibration curve.EFFECT: determination of bedaquiline in human blood plasma in a time not exceeding 6 minutes.1 cl, 9 dwg, 6 tbl

Description

[0001] Technical field

[0002] This invention relates to medicine and pharmacology. It relates to a method for determining the serum concentration of bedaquiline in patients with tuberculosis or mycobacteriosis. The invention can be used to determine the serum concentration of an anti-tuberculosis drug and prevent dose-dependent toxic reactions.

[0003] Technology Level

[0004] Many countries have signed and ratified documents regulating the conduct of clinical trials, including pharmacokinetic and bioequivalence studies of medicinal products. The key international documents are the World Medical Association (WMA) Declaration of Helsinki (1964), the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine (1997) (Russia is not a party to this Convention), and the Universal Declaration on Bioethics and Human Rights adopted by the UNESCO General Conference in 2005.

[0005] The main validation parameters for evaluating a pharmacokinetic study are: selectivity, accuracy, precision, calibration curve, stability. Guide to the examination of medicinal products. Volume 1. - M.: Grif i K, - 2014 .; Pradaxa® European Medicines Agency. [Electronic resource]. - URL: http: / / www.ema.europa.eu / docs / en_GB / document_library / EPAR-Product_ Information / human / 000829 / WC500041059.pdf; "Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine" (ETSN 164). - 1997 (as amended on 11 / 27 / 2008) [Electronic resource]. - URL: https: / / rm.coe.int / 168007d004.]

[0006] Currently, five clinical trial protocols for comparative pharmacokinetics and bioequivalence of bedaquiline have been approved on the State Register of Medicines portal in the Russian Federation. A key step in studying the pharmacokinetics of drugs or their substances is the development and validation of a bioanalytical method. This paper describes the development and application of a method for determining bedaquiline concentrations in human plasma.

[0007] Methods for determining bedaquiline and its enantiomers for quality control of synthesis are known in the art. A method for analyzing the synthesis product is known, which is performed using HPLC with UV detection (WO2006 / 125769 A1).

[0008] Chromatography conditions:

[0009] YMC-Pack ODS-AQ Column (150*4.6, 3 μm) (or similar in characteristics)

[0010] 1. Flow rate - 1 ml / min

[0011] 2. Mobile phase A - 0.1% trifluoroacetic acid in water

[0012] 3. Mobile phase B - 0.08% trifluoroacetic acid in water

[0013] 4. Elution - gradient

[0014] 5. Detection wavelength: 275 nm

[0015] A method for analyzing the final synthesis product, bedaquiline, is known, which is performed using HPLC MS / MS at m / z 555.1671 (CN 105085395 A).

[0016] Both methods are not applicable for the analysis of the drug in human blood plasma, since they were tested only on chemical substances

[0017] The choice of plasma sample preparation method is also important for analysis. Known plasma sample preparation methods for bedaquiline concentration determination are listed in Table 1.

[0018] Table 1. Main parameters of plasma sample preparation during reversed-phase chromatography with detection by a triple-quadrupole mass spectrometer

[0019] Drug (form) biomaterial Sample preparation Mobile phase links Bedaquiline Plasma Treatment with a mixture of acetonitrile and methanol (84:16), centrifugation at 11000 g for 15 minutes A: Solution of 5 g / L ammonium acetate, 25 ml / L conc. acetic acid, 2 ml / L trifluoroacetic acid in HPLC grade water B: 100% Acetonitrile Jan-Willem C Alffenaar, Mathieu Bolhuis, Kai van Hateren, Marieke Sturkenboom, Onno Akkerman, Wiel de Lange, Ben Greijdanus, Tjip van der Werf, Daan Touw Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry PMID: 26149993 PMCID: PMC4538542 DOI: 10.1128 / AAC.00276-15 Bedaquiline dry blood stain Treatment of a portion of the sample with a mixture of water, methanol and 0.3 M ZnSO4 (66:30:4), then transfer to extraction cartridges. Dissolution in the mobile phase. A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Gerard Aime Kenfack Teponnou, Anton Joubert, Saskia Spaltman, Marthinus van der Merwe, Edda Zangenberg, Sharon Sawe, Paolo Denti, Sandra Castel, Francesca Conradie, Richard Court, Gary Maartens, Lubbe Wiesner a Development and validation of an LC-MS / MS multiplex assay for the quantification of bedaquiline, n-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine in dried blood spots https: / / doi.org / 10.1016 / j.jchromb.2025.124470 Bedaquiline Plasma Treatment with a 4% phosphoric acid mixture, centrifugation at 10,000 g for 10 minutes. Solid-phase extraction of the resulting solution. Dissolution in the mobile phase. A: Solution of 10 mmol ammonium acetate buffer, 2.5 ml / l conc. acetic acid, 0.2 ml / l trifluoroacetic acid in HPLC grade water B: 100% Acetonitrile AK HEMANTH KUMAR, V. SUDHA, A. VIJAYAKUMAR, C. PADMAPRIYADARSINI Simultaneous method for the estimation of bedaquiline and delamanid in human plasma using high-performance liquid chromatography Print ISSN: 2656-0097 | Online ISSN: 0975-1491 Bedaquiline Plasma Sample treatment with 5 mM ammonium formate in 0.1% aqueous formic acid. Treatment with methyl tert-butyl ether after vortexing. Centrifugation at 4000 g for 5 minutes. Drying of the supernatant under a stream of nitrogen and dissolution in the mobile phase. A: Methanol 100% B: 5 mM ammonium formate in 0.1% aqueous formic acid Viritha Bezawada, Padma Mogili, Srinivasa Rao Polagani, Sireesha Dodda Bioanalysis of bedaquiline in human plasma by liquid chromatography-tandem mass spectrometry: Application to pharmacokinetic study https: / / doi.org / 10.1016 / j.jmsacl.2024.01.001

[0020] Thus, the technical problem solved by the claimed invention is the need to overcome the disadvantages inherent in analogues by creating a simple and time-efficient method for determining bedaquiline in human blood plasma.

[0021] Disclosure of invention

[0022] The technical result of the proposed invention is the determination of bedaquiline in human blood plasma in a time not exceeding 6 minutes.

[0023] The technical result is achieved by the claimed method of determining the concentration of bedaquiline in human blood plasma using the UHPLC-MS / MS method.

[0024] A method for determining the concentration of bedaquiline in the blood serum of patients with tuberculosis or mycobacteriosis, consisting of the following: whole blood samples are centrifuged at 3000 rpm for 20 min, plasma is collected in sterile 1.5 ml Eppendorf tubes, then 900 μl of acetonitrile are added to 300 μl of plasma, centrifuged at 13,500 g for 15 minutes, then 1 ml of the supernatant is transferred to a chromatographic vial and analyzed by UHPLC MS / MS using an Athena UHPLC C18, 1.8 uM, 120A, 2.1x100 mm column, using as mobile phase A an aqueous solution containing 5 g / L ammonium acetate, 25 ml / L conc. acetic acid, 2 ml / l trifluoroacetic acid and mobile phase B - 100% acetonitrile, standard samples are prepared for constructing a calibration graph, the concentration of bedaquiline is determined using a previously constructed calibration curve.

[0025] Brief description of drawings

[0026] The invention is explained by the following drawings.

[0027] Fig. 1 shows the sequence of sample preparation steps.

[0028] Fig. 2 shows the preparation of bedaquiline standards for constructing a calibration curve and determining the concentration in the test samples.

[0029] Fig. 3 shows calibration curves.

[0030] Fig. 4-9 shows the concentration spectrograms.

[0031] Implementation of the invention

[0032] The concentration in patients' plasma was determined by LC-MS / MS on an EXPEC 5210 UHPLC-MS / MS system based on a triple quadrupole, manufactured by EXPEC TECHNOLOGY, China. The application of this method is based on a literature review [Jan-Willem C Alffenaar, Mathieu Bolhuis, Kai van Hateren, Marieke Sturkenboom, Onno Akkerman, Wiel de Lange, Ben Greijdanus, Tjip van der Werf, Daan Touw Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry PMID: 26149993 PMCID: PMC4538542 DOI: 10.1128 / AAC.00276-15].

[0033] Step 1. Sample preparation

[0034] Plasma collection. Whole blood collected from patients at the National Medical Research Center for Clinical Pathology, Ministry of Health of the Russian Federation, was centrifuged at 3000 rpm for 20 min. Plasma was then collected into sterile 1.5 ml Eppendorf tubes. 900 μl of acetonitrile (CAS# 75-05-8, gradient grade for liquid chromatography, Merck, Germany) was added to 300 μl of plasma, thoroughly mixed, and centrifuged for 15 minutes at 11,000 g. Next, 1 ml of the supernatant was transferred to a chromatographic vial (borosilicate glass type I, class A, ANPEL, China). The selection of whole blood sample preparation conditions was based on literature sources [Aziz MY, Hoffmann KJ, Ashton M. LC-MS / MS quantitation of antimalarial drug piperaquine and metabolites in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 15;1063:253-258. doi: 10.1016 / j.jchromb.2017.06.035. Epub 2017 Jun 28. PMID: 28863865; Zhao Y, Guo YJ, Chen XL, Yang YL, Ma H, Wang YQ, Sun LN. Determination of Orelabrutinib in Human Plasma Using LC-MS / MS. Ther Drug Monit.2023 Oct 1;45(5):599–605. doi: 10.1097 / FTD.00000000000001106. Epub 2023 May 5. PMID: 37199420; Saha, A., Jangala, H., Vats, P., Thakur, R., Khuroo, A., & Monif, T. (2015). Stability indicating LC-MS / MS method for estimation of lovastatin in human plasma: application to a bioequivalence study. Journal of Analytical Science and Technology, 6(1). doi:10.1186 / s40543-015-0061-6;]. The main conditions for determining the concentration of bedaquiline are:.

[0035] - simplicity of the method

[0036] - sample acquisition speed

[0037] - compliance with the chromatographic separation parameters, chemical and physical properties of bedaquiline.

[0038] The choice of acetonitrile as a solvent for plasma sample preparation is determined by the mobile phase (MP) selection, described further in the development of the protocol for measuring bedaquiline concentrations in plasma. Furthermore, based on an analysis of literature data on plasma sample preparation, the versatility of acetonitrile as a solvent when using reversed-phase chromatography (RPC) was demonstrated. The analytical data are presented in Table 1.

[0039] Preparation of standard solutions of bedaquiline. A weighed sample (10 mg) of pure bedaquiline substance (CAS# 845533-86-0, Nanjing Pars Biochem CO., China) was dissolved in 5 ml of a mixture of MetOH and DMSO (50:50) (CAS# 67-56-1, special purity grade for gradient chromatography, Khimmed, Russian Federation). Then, by serial dilution, a series of 5 standards from 4 μg / ml to 0.05 μg / ml were obtained in individual chromatographic vials.

[0040] The choice of solvent for standard preparation is also based on the physicochemical properties of bedaquiline and literature data. According to the scientific article [SJ Rajput & PJ Vanavi, Evaluation of physicochemical stability and degradation kinetics of bedaquiline in hydrolytic solutions of different pH, Future Journal of Pharmaceutical Sciences, volume 7, Article number: 88 (2021)], bedaquiline is poorly soluble in water but readily soluble in organic solvents, such as methanol.

[0041] The use of a reversed phase has a number of advantages over unmodified silica gel when applying the RPCh method: better reproducibility of the retention times of the components being separated, rapid establishment of equilibrium in the system. In RPCh, as a rule, the adsorbent (i.e. the stationary phase) is non-polar - silica gel with alkyl chains grafted onto its surface (C1-C22), the eluent is polar (alcohols, acetonitrile, water), and the substances being separated can be of any nature. This type of chromatography is currently used for about 2 / 3 of separations in HPLC, due to the simplicity of optimizing conditions, conducting analysis and versatility [Krasnov, E. A. Modern chromatographic methods (GLC, HPLC) in pharmaceutical analysis: a tutorial for students studying in the specialty 040500 "Pharmacy" / E. A. Krasnov, A. A. Blinnikova; Ministry of Education and Science of the Russian Federation, Federal Agency for Healthcare and Social Development, State Educational Institution of Higher Prof.[education Siberian State Medical University. - Tomsk: Siberian State Medical University, 2006. - 154 p. - ISBN 5-98591-019-9. - EDN QLMXIJ].

[0042] Step 2. Development of a protocol for measuring bedaquiline concentrations in plasma.

[0043] Optimization of the protocol for conducting analysis using standards.

[0044] Based on scientific literature [Jan-Willem C Alffenaar, Mathieu Bolhuis, Kai van Hateren, Marieke Sturkenboom, Onno Akkerman, Wiel de Lange, Ben Greijdanus, Tjip van der Werf, Daan Touw Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry PMID: 26149993 PMCID: PMC4538542 DOI: 10.1128 / AAC.00276-15] optimization of chromatographic separation was carried out according to the following parameters:

[0045] - selection of stationary phase;

[0046] - selection of the mobile phase composition;

[0047] - elution mode.

[0048] - chromatographic conditions

[0049] The nature of the stationary phase affects the selectivity of the column and is the main factor that determines the sequence of components leaving the column. The choice of the stationary phase is determined by the nature of the substances being separated, their polarity and boiling point [Guskova, V. P. Chromatographic methods of separation and analysis: a tutorial / Guskova V. P., Sizova L. S. - 2nd ed., corrected. and add. - Kemerovo: KemSU, 2015. - 148 p. - ISBN 978-5-89289-888-1].

[0050] An analytical chromatographic column Athena UHPLC C18, 1.8uM, 120A, 2.1x100 mm (ANPEL Laboratory technologies Inc, China) was selected for the analysis as the most similar in characteristics to the column Hypurity C18, 3 uM, 2.1x100 mm from the article [Jan-Willem C Alffenaar, Mathieu Bolhuis, Kai van Hateren, Marieke Sturkenboom, Onno Akkerman, Wiel de Lange, Ben Greijdanus, Tjip van der Werf, Daan Touw Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry PMID: 26149993 PMCID: PMC4538542 DOI: 10.1128 / AAC.00276-15] and The OFC method was used. The flow rate was 0.5 ml / min, and the analysis time was 5 minutes.

[0051] The choice of PF is often more important than the choice of NF [Guskova, V. P. Chromatographic methods of separation and analysis: a tutorial / Guskova V. P., Sizova L. S. - 2nd ed., corrected. and add. - Kemerovo: KemSU, 2015. - 148 p. - ISBN 978-5-89289-888-1]. Mixtures of water and an organic modifier - acetonitrile, methanol, isopropanol, tetrahydrofuran and others - are used as PF in OFC [Krasnov, E. A. Modern chromatographic methods (GLC, HPLC) in pharmaceutical analysis: a tutorial for students studying in the specialty 040500 "Pharmacy" / E. A. Krasnov, A. A. Blinnikova; Ministry of Education and Science of the Russian Federation, Federal Agency for Healthcare and Social Development, State Educational Institution of Higher Professional Education Siberian State Medical University. - Tomsk: Siberian State Medical University, 2006. - 154 p. - ISBN 5-98591-019-9. - EDN QLMXIJ].Based on the analysis of literature data, it was revealed that, taking into account the physicochemical properties of bedaquiline and to achieve acceptable resolution and symmetry, an eluent based on acetonitrile and aqueous solutions with an acidic medium is selected, in various ratios of components and flow rates of the mobile phase.

[0052] Table 2. Main parameters of plasma chromatographic separation for determination of bedaquiline concentration with detection by triple quadrupole tandem mass spectrometer

[0053] Parameter Characteristic Chromatography method Reversed-phase chromatography Column Athena UHPLC C18, 1.8uM,120A, 2.1x100 mm (ANPEL Laboratory technologies Inc, China) Scanned masses 555,1 → 58,4 Electric potential for ion fragmentation (cone voltage) 3.5 Retention time (the time it takes for the analyte to pass through the chromatographic column) 1,9 Ionization mode ESI+ The release time of bedaquiline on the chromatogram 2,1 Column temperature 20°C Temperature in the sample box 4°C Mobile phase A Solution of 5 g / L ammonium acetate, 25 ml / L conc. acetic acid, 2 ml / L trifluoroacetic acid in HPLC grade water Mobile phase B 100% Acetonitrile Detection method MRM

[0054] Table 3. Chromatographic separation program (protocol)

[0055] Time, min Flow rate, ml / min PF A content, % PF B content, % 0 0,5 5 95 5 0,5 5 95

[0056] Step 3. Measurement of bedaquiline concentration in patients' plasma.

[0057] Construction of a calibration (standard) curve.

[0058] To construct a calibration (standard) curve, a series of bedaquiline standards with known concentrations were measured using the method described above. The standard solutions had the following concentrations: 4 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, and 50 ng / mL. After measuring and integrating the peaks using the built-in software, the following results were obtained and are described in Table 4.

[0059] Table 4. Measurements of the standard series

[0060] standard Peak area Detector signal K1 (4 mcg / ml) 4215457 5.53e5 K2 (2 mcg / ml) 2436299 3.3e5 K3 (1 mcg / ml) 1331515 1.38e5 K4 (0.5 mcg / ml) 706363 9.85e4 K5 (0.05 μg / ml) 159116 2.14e4

[0061] Based on the peak area and the known concentration of dilutions of the pharmacopoeial standard of bedaquiline, a calibration (standard curve) was constructed (Fig. 3):

[0062] The obtained calibration curve has a linear shape and passes through the origin. R2 (the approximation reliability value) corresponds to 0.9967. The obtained parameters of the calibration curve are an indicator of the reliability of the calibration curve. The obtained linear equation allows calculating the concentration values ​​of the studied samples. Figs. 4 - 9 show the obtained spectrograms of standard (control) samples, where Fig. 4 shows the spectrogram of the control sample of bedaquiline with a concentration of 4 μg / ml; Fig. 5 - the spectrogram of the control sample of bedaquiline with a concentration of 2 μg / ml; Fig. 6 - the spectrogram of the control sample of bedaquiline with a concentration of 1 μg / ml; Fig. 7 - the spectrogram of the control sample of bedaquiline with a concentration of 0.5 μg / ml; Fig. 8 - spectrogram of the control sample of bedaquiline with a concentration of 0.05 μg / ml; in Fig. Fig. 9 shows superimposed spectrograms of the control samples of bedaquiline.

[0063] The present invention is explained by specific examples of implementation, which are not the only possible ones, but clearly demonstrate the possibility of achieving the required technical result.

[0064] Clinical example 1.

[0065] Patient Zh., born in 1998 (27 years old). Weight - 61 kg. Height - 164 cm. Admitted to the department of the NMIC FPI with a diagnosis of: Infiltrative tuberculosis of the upper lobe of the right lung in the decay and dissemination phase, MBT (+), preXDR MBT (SHRE Am Cm Eto Lfx Mfx). 1 GDU (+). It is known from the anamnesis that he did not previously have tuberculosis. Tuberculosis contact with his sister. Consulted a therapist in 10.2023 with respiratory complaints (pain in the right half of the chest when breathing). Additional examination with MSCT of the chest on 10.23.2023 - a picture of focal infiltrative changes in the upper lobe of the right lung. A FBS was performed, and AFB-1 (+) was detected in the BAL fluid using fluorescence microscopy. Real-time PCR detected MBT DNA with resistance to HR Fq. He was hospitalized in the 3rd therapeutic department of the National Medical Research Center for Pedagogical Infections of the Russian Ministry of Health, where he remained from December 15, 2023, to June 26, 2024.Treatment was initiated according to the chemotherapy regimen for pre-XDR-TB: Bedaquiline 0.6 / day, Bedaquiline (as per the regimen), Cycloserine 0.75 / day, Delamanid 0.2 / day, Pyrazinamide 1.5 / day. After 4 months of taking anti-TB drugs, an increase in liver enzymes was noted in laboratory blood tests (April 24, 2024): ALT - 185 U / L, AST - 129 U / L, total bilirubin - 20.8 μmol / L, direct bilirubin - 9.2 μmol / L. Considering the absence of complaints, a 3-5-fold excess of the level of liver enzymes, anti-tuberculosis therapy was not canceled. Detoxification and hepatoprotective therapy (Remaxol intravenously, Ademetionine 400 mg / day intravenously) were added to the treatment. By 06.05.2024 (after 12 days), a further increase in the level of liver enzymes was noted): ALT - 265 U / L (above 6.6 ULN), AST - 157 U / L (above 4.6 ULN), total bilirubin - 24.2 μmol / L, direct bilirubin - 10.2 μmol / L. The levels of GGT and ALP were within the reference values. Anti-tuberculosis therapy was discontinued. The dose of ademetionine was increased to 800 mg / day.The patient underwent 3 plasmapheresis sessions (05 / 15 / 2024; 05 / 21 / 2024; 05 / 24 / 2024).

[0066] Blood samples for pharmacokinetic (PK) studies were collected at 2, 5, and 24 hours after bedaquiline administration. Following plasmapheresis, the patient was given bedaquiline one hour after a meal for subsequent PK studies.

[0067] Whole blood samples were centrifuged at 3000 rpm for 20 min, and plasma was collected in sterile 1.5 ml Eppendorf tubes. 900 μl of acetonitrile (CAS# 75-05-8, gradient grade for liquid chromatography, Merck, Germany) was added to 300 μl of plasma, mixed thoroughly, and centrifuged for 15 min at 13,500 g. Next, 1 ml of the supernatant was transferred to a chromatographic vial (borosilicate glass type I, class A, ANPEL, China). The resulting supernatants were analyzed using UHPLC MS / MS.

[0068] The patient's plasma bedaquiline concentration was determined using a developed method and a calibration (standard) curve. The data obtained after chromatography with triple-quadrupole tandem mass spectrometry detection, as well as peak integration using the built-in software, are presented in Table 5.

[0069] Table 5. Obtained data for patient 1.

[0070] Sample Detector signal Peak area Calculated concentration based on the calibration (standard) graph, converted to µg / ml Solution of the equation y = 1102.3209x with known y (peak area) Dilution correction (initially plasma was diluted 4-fold), μg / ml 1 2 3 4 5 1_0 4.94e3 43580 0,039535 0,158139 1_1 2.9e3 20928 0,018985 0,075942 1_2 3.06e3 25363 0,023009 0,092035 1_3 2.64e3 21847 0,019819 0,079276 1_4 3.1e3 25834 0,023436 0,093744 1_5 3.82e3 31889 0,028929 0,115716 2_0 4.78e3 41714 0,037842 0,151368 2_1 3.02e3 24232 0,021983 0,087931 2_2 2.55e3 21711 0,019696 0,078783 2_3 2.54e3 21350 0,019368 0,077473 2_4 2.48e3 20787 0,018857 0,07543 2_5 3.4e3 28710 0,026045 0,10418 3_0 4.23e3 36335 0,032962 0,131849

[0071] 1 2 3 4 5 3_1 2.46e3 20129 0,018261 0,073042 3_2 2.16e3 18186 0,016498 0,065992 3_3 2.14e3 17411 0,015795 0,063179 3_4 2e3 16060 0,014569 0,058277 3_5 3.23e3 27287 0,024754 0,099017

[0072] Samples are listed in the order they were received. The first digit indicates the plasmapheresis procedure, and the digit after the underscore indicates the time point (hours).

[0073] According to ultrasound data, signs of diffuse changes in the liver and reactive changes in the pancreas were noted.

[0074] According to liver elastometry data, the degree of liver rigidity was 5.7 kPa (range 3.4-7.2 kPa), which corresponds to fibrosis stage F 0-1 on the METAVIR scale. The severity of steatosis was 188 dB / m, which may correlate with stage S-0.

[0075] Normalization of liver enzymes and bilirubin levels was noted. Anti-tuberculosis therapy was resumed on May 26, 2024, with the exclusion of the trigger drug, pyrazinamide, from the regimen. A satisfactory therapeutic effect was observed during treatment: sputum was cleared, radiographic examination revealed closure of the lesion cavity, and partial resolution of foci in the upper lobe of the right lung.

[0076] The patient was discharged to the local PTD in satisfactory condition to continue treatment. After discharge, it was recommended to continue therapy according to the following regimen: Bedaquiline 0.6 mg / day, Bedaquiline (as prescribed), Cycloserine 0.75 mg / day, Delamanid 0.2 mg / day.

[0077] Thus, the determination of the concentration of bedaquiline in the blood serum by the claimed method made it possible to adjust the dose of the drug for the treatment of the patient and, moreover, in a short period of time, since the sample analysis time on the equipment was 5 minutes.

[0078] Clinical example 2.

[0079] Patient B. born in 1980 (45 years old). Weight - 73 kg. Height - 187 cm, BMI 20.8. Admitted to the department of the Federal State Budgetary Institution "NMITs FPI" of the Ministry of Health of the Russian Federation with the diagnosis: Infiltrative tuberculosis of the lower lobe of the right lung in the decay and dissemination phase, MBT (+), MDR MBT (SHREZK Eto). 1 GDU (+). Concomitant diseases: Chronic viral hepatitis C since 2014. Did not receive antiviral therapy. Denies a history of episodes of jaundice. It is known from the anamnesis that he has not previously suffered from tuberculosis. Denies contact with tuberculosis. Changes in the lungs were first detected in 03.2024 during a preventive examination. Additional examination by a phthisiatrician. On June 25, 2024, treatment with multidrug RCT was initiated: levofloxacin 0.75, capreomycin 1.0 (IM), cycloserine 0.75 / day, bedaquiline 0.6 / day, bedaquiline (as prescribed). Forty doses of anti-TB drugs were administered in the intensive phase. During treatment, the development of hepatotoxic reactions was noted (an increase in liver enzymes was noted - more than 5 ULN). Anti-TB therapy was discontinued.Detoxification and hepatoprotective therapy was carried out.

[0080] Documents were sent to the Federal State Budgetary Institution "National Medical Research Center for Fundamental Pediatrics and Pathology" of the Ministry of Health of the Russian Federation. The patient was hospitalized in the internal medicine department for therapy selection, where he stayed from August 7, 2024, to November 14, 2024. Upon admission, laboratory tests revealed elevated liver enzyme levels: ALT - 59 U / L, AST - 71 U / L. Total bilirubin, GGT, and ALP levels were within reference values.

[0081] According to the ultrasound examination of the abdominal cavity, signs of enlargement and moderate diffuse changes in the liver parenchyma, diffuse changes in the pancreas, and an increase in the size of the spleen were noted.

[0082] According to liver elastometry data, the degree of liver rigidity was 5.8 kPa (range 5.3-6.7 kPa), which corresponds to fibrosis stage F 0-1 on the METAVIR scale. The severity of steatosis was 142 dB / m, which may correlate with stage S-0.

[0083] To prevent dose-dependent toxic reactions and determine bedaquiline serum concentrations, the patient underwent three plasmapheresis sessions (September 16, 2024; September 19, 2024; September 23, 2024). Blood samples for pharmacokinetic studies were collected 2, 5, and 24 hours after bedaquiline administration. Following the plasmapheresis procedure, bedaquiline was administered to the patient one hour after a meal for subsequent pharmacokinetic studies.

[0084] Whole blood samples were centrifuged at 3000 rpm for 20 min, and plasma was collected in sterile 1.5 ml Eppendorf tubes. 900 μl of acetonitrile (CAS# 75-05-8, gradient grade for liquid chromatography, Merck, Germany) was added to 300 μl of plasma, mixed thoroughly, and centrifuged for 15 minutes at 13,500 g. Next, 1 ml of the supernatant was transferred to a chromatographic vial (borosilicate glass type I, class A, ANPEL, China). The resulting supernatants were analyzed using UHPLC MS / MS.

[0085] The patient's plasma bedaquiline concentration was determined using a developed method and a calibration (standard) curve. The data obtained after chromatography with triple-quadrupole tandem mass spectrometry detection, as well as peak integration using the built-in software, are presented in Table 6.

[0086] Table 6. Obtained data for patient 2.

[0087] Sample* Detector signal Peak area Calculated concentration based on the calibration (standard) graph, converted to µg / ml Solution of the equation y = 1102.3209x with known y (peak area) Dilution correction (initially plasma was diluted 4-fold), μg / ml 1_0 4.94e3 43580 0,039535 0,158139 1_1 2.21e3 17452 0,015832 0,063328 1_2 2.25e3 18284 0,016587 0,066347 1_3 1.84e3 14135 0,012823 0,051292 1_4 2.04e3 15948 0,014468 0,057871 1_5 3.58e3 29404 0,026675 0,106699 2_0 4.53e3 39638 0,035959 0,143835 2_1 1.94e3 15027 0,013632 0,054529 2_2 1.78e3 13226 0,011998 0,047993 2_3 1.78e3 13785 0,012505 0,050022 2_4 1.78e3 14056 0,012751 0,051005 2_5 3.19e3 27321 0,024785 0,09914 3_0 5.13e3 44523 0,04039 0,161561 3_1 1.78e3 13051,75 0,01184 0,047361 3_2 1.61e3 12497,23 0,011337 0,045349 3_3 1.91e3 15277,79 0,01386 0,055439 3_4 1.85e3 13817,97 0,012535 0,050141 3_5 3.41e3 28458 0,025816 0,103266

[0088] *Samples are listed in the order they were received. The first number indicates the plasmapheresis procedure; the number after the underscore indicates the time point (hours).

[0089] As a result, a treatment regimen was developed according to the MDR-TB regimen: Levofloxacin 0.75 / day, Bedaquiline 0.6 / day, Bedaquiline (according to the regimen), Cycloserine 0.75 / day, Delamanid 0.2 / day.

[0090] During treatment, positive dynamics were noted: sputum negativity was achieved, and radiographically, a tuberculoma-like focus formed in the S-6 section of the right lung. The patient was consulted by a thoracic surgeon: given the nature of the process, surgical treatment was indicated. On November 28, 2024, a VATS resection of the S-6 section of the right lung was performed.

[0091] The postoperative period was uneventful. The patient was discharged to the PTD at his place of residence with recommendations to continue treatment according to the established regimen for at least 180 doses of anti-TB drugs.

[0092] Thus, the determination of the concentration of bedaquiline in the blood serum by the claimed method made it possible to adjust the dose of the drug for the treatment of the patient and, moreover, in a short period of time, since the sample analysis time on the equipment was 5 minutes.

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