INTRODUCTION: Human breast cancer resistance protein (BCRP, ABCG2) is a transport protein of ABC superfamily of transporters that uses ATP energy of for its work. BCRP plays an important role in the pharmacokinetics of drugs, therefore all new drugs are recommended to be tested for belonging to its substrates, inducers and inhibitors. The influence of substances on the activity of this transport protein in vitro is evaluated by a change of transmembrane transfer of its substrates, one of which being quercetin. This, in turn, requires the development and validation of a method for its quantitative analysis in an appropriate matrix. AIM: To develop a method for the quantitative determination of quercetin using high performance liquid chromatography (HPLC) with tandem mass selective detection (MS/MS) with full validation. MATERIALS AND METHODS: The method was developed on Ultimate 3000 HPLC equipped with TSQ Fortis (Thermo Fisher, USA) MS/MS detector. The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm*100 mm 5μm, 100 A, Selectra C18 Guard Cartridges SLC-18 GDC46-5UM. The analysis time was 11 min at separation temperature 35°С, flow rate 0.5 ml/min, injected sample volume 5 μl. Gradient elution regime was used: on 0th minute, the ratio of 0.1% formic acid solution and acetonitrile was 70% and 30%; 0.3 min — 30% and 70%; 4 min — 1% and 99%; 9 min — 70% and 30%. Quercetin retention time was 3.91 minutes. Detection conditions: negative ionization mode, 301 m/z → 150.9 m/z with collision energy 22 V, 301 m/z →178.9 m/z with collision energy 17 V, source fragmentation 5 V, electrospray voltage 3 000 V, CID gas pressure 1 mTorr, Sheath gas 50 Arb, Aux gas 10 Arb, Sweep gas 10 Arb, ion transfer tube temperature 300°C, vaporizer temperature 350°C. The matrix was transport medium after incubation for 3 hours with Caco-2 cells overexpressing BCRP. In this medium, the transport of quercetin through the cell monolayer was further evaluated in vitro. Precipitation of protein and isolation of quercetin from the transport medium was realized by a mixture of water and acetonitrile in a ratio of 1:1. RESULTS: The developed method was validated by the following parameters: selectivity, linearity, accuracy, precision, limit of quantification, sample transfer, matrix effect, sample stability. The analytical range of the technique was 5–500 nmol/l. In this case, the correlation coefficient was more than 0.99. The limit of detection and the lower limit of quantification of quercetin (LLQQ) were 1 and 5 nmol/l, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for other concentrations. The analyte demonstrated stability in triple freeze-defreeze cycle at -80°C, in storage at -80°C for 60 days, after sample preparation and being kept in the autosampler for 24 hours. There was no sample transfer and no matrix effect. CONCLUSION: A method for the quantitative determination of quercetin using HPLC-MS/MS in a transport medium was developed and fully validated.

Mitoxantrone is a marker substrate of breast cancer resistance protein (BCRP). BCRP is involved in a number of pharmacokinetic drug–drug interactions. The transporter’s possible saturability makes it advisable to use low concentrations of mitoxantrone for in vitro studies. Consequently, mitoxantrone quantification requires a method with high sensitivity.The aim of the study was to develop and validate a procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.Materials and methods. The authors used an Ultimate 3000 HPLC system and a TSQ Fortis triple quadrupole mass spectrometer by Thermo Fisher Scientific and a Selectra C18 column (4.6×100 mm, 5 µm, 100 Å) by United Chemical Technologies. The elution ran in a gradient mode with a mobile phase of 1% formic acid solution and methanol. Experimental parameters were as follows: eluent flow rate, 0.3 mL/min; separation column temperature, 35 °C; injection volume, 5 µL; analysis time, 10 min; approximate mitoxantrone retention time, 5.51 min. The sample preparation involved protein precipi tation from the culture medium with methanol, followed by centrifugation at 13,000 g for 10 min. The detection was performed using electrospray ionisation in the positive ion mode. Detection parameters were as follows: electrospray voltage, 3700 V; sheath gas flow rate, 50 L/min; auxiliary gas flow rate, 10 L/min; sweep gas flow rate, 1 L/min; ion-transfer tube temperature, 300 °C; and evaporator temperature, 350 °C. The detection was set at mass transitions of m/z 455 to 88.2 and m/z 455 to 358.1, with the collision energy for these transitions amounting to 25 V and 18 V, respectively. The source fragmentation was at 0, and the CID gas pressure was at 2 mTorr.Results. The analytical procedure showed selectivity, high sensitivity (limit of detection, 10 nmol/L; lower limit of quantification, 50 nmol/L), accuracy, precision, and linearity in the concentration range of 50–1000 nmol/L. The authors observed no carryover or matrix effects. A simulation of real-life storage conditions demonstrated high stability of mitoxantrone samples. Thus, the analytical procedure enables preclinical evaluation of medicinal product effects on the functional activity of BCRP, based on assessing the transcellular mitoxantrone transport in the presence of a test product.Conclusion. The authors developed and validated the analytical procedure for mitoxantrone quantification in Caco-2 culture media by HPLC-MS/MS.

Cells of the Caco-2 line have the basic properties of enterocytes of the small intestine, and therefore can be used to study the absorption of medicinal substances. Aim. To characterize the properties of the Caco-2 cell line from the Institute of Cytology of the Russian Academy of Sciences and to evaluate with its help the mechanism of absorption of the original domestic drug - ethylmethylhydroxypyridine succinate (EMGPS). Materials and methods. The study was performed on Caco2 cells that were cultured for 21 days, since at this time their spontaneous differentiation into polarized cells similar to enterocytes of the small intestine occurs. The density of the cell monolayer was estimated by the value of transepithelial resistance. The number of major efflux proteins of glycoprotein-P transporters (Pgp) and breast cancer resistance protein (BCRP) in Caco-2 cells was analyzed using enzyme immunoassay. In specialized transwell systems, the transport of the Pgp substrate fexofenadine (40, 150 and 300 microns), the BCRP substrate methotrexate (5, 10, 50 microns) and EMGPS (10, 100 and 250 microns) through the cell monolayer was studied. The results of the study. By day 21 of cultivation, cells of the Caco2 line formed a merging monolayer with pronounced dense contacts. The amount of Pgp and BCRP was 110.8±14.1 ng/mg and 4.39±0.12 ng/mg, respectively, which correlates with the amount of these proteins in the human small intestine. Transport of fexofenadine (40, 150 and 300 microns) and methotrexate (5 microns) from the basolateral chamber to the apical chamber (corresponding to transport from enterocytes to the intestinal lumen) prevailed over transport in the opposite direction, which is associated with the work of Pgp and BCRP. The transport of EMGPS significantly exceeded the transport of fexofenadine and methotrexate and was symmetrical with respect to the cellular monolayer. Conclusion. Thus, the cells of the Caco-2 line, commercially available in the Russian Federation, have the basic properties of enterocytes of the small intestine, and can be used to study the absorption of medicinal substances in vitro. EMGPS quickly passes through the cellular monolayer, and the mechanism of its absorption is passive diffusion, without the participation of specific transporters.

INTRODUCTION: Metoprolol is a selective β1-adrenoblocker without intrinsic sympathomimetic activity. The effectiveness of metoprolol has been proven in numerous clinical studies in the treatment of arterial hypertension, stable angina, myocardial infarction, chronic heart failure. To improve the efficiency and safety of the therapy, it is advisable to carry out therapeutic drug monitoring of metoprolol, which requires a sensitive method for its quantitative determination. AIM: To develop, validate and test a method for the determination of metoprolol in human plasma using high performance liquid chromatography (HPLC) with tandem mass spectrometric detection (MS/MS). MATERIALS AND METHODS: The work was performed on Ultimate 3000 TSQ Fortis HPLC-MS/MS (Thermo Fisher, USA). For sample preparation, acetonitrile was used with fexofenadine hydrochloride at a concentration of 10 ng/ml as an internal standard, which was added to plasma samples in 3:1 ratio. The volume of the injected sample was 5 μl. Separation was performed on UCT Selectra C18 4.6 mm × 100 mm, 3 um, 100 A column with a similar pre-column at 35°C, in gradient elution mode in proportion of 0.1% formic acid solution/acetonitrile: 0 min — 80%/20%, 0.1 min — 45%/55%, 5 min — 10%/90%, 10 min — 80%/20%, at flow rate of 300 µl/min. Detection was performed in positive electrospray ionization mode, electrospray voltage 4000 V, sheath gas 50 arb, auxiliary gas 10 arb, purge gas 1 arb, evaporator temperature 350°C, ion transport tube temperature 300°C, using the multiple reaction monitoring mode (MRM) at argon flow rate 2 mTorr, 268 m/z → 115.5 m/z, Collision Energy 18 V, Tube lens 95 V, 268 m/z → 191 m/z, Collision Energy 17 V, Tube lens 95 V. The blood plasma of healthy volunteers served as matrix. RESULTS: The analytical range of the technique was 2–1000 ng/ml. The developed technique was tested on a patient with arterial hypertension. During the analysis, an equilibrium concentration of metoprolol of 12.0 ng/ml was detected in the patient's blood plasma (previously, the patient took metoprolol tartrate at a dose of 12.5 mg twice a day for a week), and in 2 hours after taking the drug — 31.0 ng/ml. CONCLUSION: A method for the quantitative determination of metoprolol in human blood plasma using HPLC-MS/MS has been developed, validated and tested.

INTRODUCTION: Despite the recently established evidence-based and systemic approach to treatment for arterial hypertension (AH), not in all cases its control can be achieved. AIM: To conduct a comparative analysis of the concentration of antihypertensive drugs (AHTDs) in blood serum of patients with controlled and uncontrolled AH. MATERIALS AND METHODS: Fifty six patients were included. Inclusion criteria: age 18 years, signing of informed consent, established diagnosis of AH, regular intake of any two of study antihypertensive drugs (lisinopril, amlodipine, valsartan) and also of indapamide at stable doses, for women adequate contraception. According to the results of daily monitoring of the arterial pressure (AP), patients were divided into two groups: the first group controlled hypertension (AP 140/90 mmHg; n = 39), the second uncontrolled hypertension (AP 140/90 mmHg; n = 17). The mean age of patients in the first group was 65.03 10.80 years, in the second 63.50 8.31 (p = 0.576). In the first group, women prevailed (64.1% vs. 35.3%, p = 0.047) and the mean body mass index was lower (26.30 1.38 kg/m2 vs. 32.20 4.15 kg/m2, p = 0.02). In patients of both groups, venous blood was taken in fasting condition in the morning and 2 hours after intake of AHTDs to assess their concentration by high-performance liquid chromatography. The analytical range for lisinopril, indapamide, amlodipine was 5500 ng/ml, for valsartan 1010,000 ng/ml. RESULTS: In the first group, equilibrium concentration of lisinopril was 2.67 times higher (p = 0.053), and concentration of indapamide in 2 hours after intake was 1.83 times higher (р = 0,084); when normalized to the dose, the differences were leveled out (p 0.05). Concentrations of amlodipine and valsartan did not differ between the groups both before and 2 hours after intake (p 0.05). In 3 of 39 (7.7%) patients with controlled hypertension and in one of 17 patients (5.9%, p = 1.0) with uncontrolled hypertension, AHTDs were detected in blood serum, which were not administered to them. CONCLUSIONS: Results of the pilot part of the study (n = 56) demonstrated the absence of difference between the mean concentrations of the study AHTDs in the blood serum of patients with controlled and uncontrolled AH, and in some cases the presence of traces of AHTDs not administered by the doctor.