Development and Validation of the Quantitative Determination of Atorvastatin in HepG2 Cell Line Using High-Performance Liquid Chromatography with Mass-Spectrometric Detection

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INTRODUCTION: Organic anion transporting polypeptide 1B1 (OATP1B1) is a transporter protein that plays an important role in the pharmacokinetics of substances (its substrates), regulating their penetration into hepatocytes. The functional activity of this protein is evaluated by the transport of marker substrates, for example, atorvastatin, on cell lines overexpressing OATP1B1, such as HepG2 (human hepatocellular carcinoma).

AIM: To develop and validate the technique of the quantitative determination of atorvastatin in HepG2 cell line using high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS).

MATERIALS AND METHODS: The study was performed on HPLC chromatograph with an MS/MS detector. The conditions of the chromatographic analysis were as follows: pre-column Selectra C18 Guard Cartridges SLC-18GDC46-5UM, chromatographic column UCT Selectra C18 4.6 mm × 100 mm 5 µm, 100 А column, flow velocity of 0.3 mL/min, and column thermostatic control of 35°C. The volume of introduced samples was 2 µL, and the analysis time was 10 min. Gradient elution mode was used: the ratios of 0.1% formic acid solution and acetonitrile were 35% and 65% for 0 and 0.3 min, 5% and 95% for 0.6 and 5 min, and 35% and 65% for 5.05 and 8 min, respectively. The retention time of atorvastatin was 4.53 min. Atorvastatin detection conditions were as follows: positive ionization mode; spray voltage,3500 V; detection mode, multiple reaction monitoring 559.30 m/z → 466.20 m/z, 559.30 m/z → 440.20 m/z; collision energy, 17 V; source fragmentation, 0; and gas pressure-inducing dissociation, 2 mTorr.

RESULTS: The developed bioanalytical method was validated by the following parameters: linearity, selectivity, accuracy, precision, lower limit of quantity determination, sample transfer, sample stability, and matrix effect. The confirmed range of the technique in cell lysate was 0.5–200 nmol/L.

CONCLUSION: The results validated the technique for the quantitative determination of atorvastatin in the lysate of HepG2 cell line by HPLC-MS/MS.

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作者简介

Pelageya Erokhina

Ryazan State Medical University

Email: erokhina.pelageya96@yandex.ru
ORCID iD: 0000-0003-4802-5656
SPIN 代码: 1480-6854
俄罗斯联邦, Ryazan

Pavel Myl’nikov

Ryazan State Medical University

Email: dukeviperlr@gmail.com
ORCID iD: 0000-0001-7829-2494
SPIN 代码: 8503-3082
俄罗斯联邦, Ryazan

Svetlana Ganina

Ryazan State Medical University

Email: svetlanaganina23@gmail.com
ORCID iD: 0000-0002-4408-6454
俄罗斯联邦, Ryazan

Egor Konyakhin

Ryazan State Medical University

Email: egor_konyahin@mail.ru
ORCID iD: 0000-0002-9025-130X
俄罗斯联邦, Ryazan

Aleksey Shchul’kin

Ryazan State Medical University

编辑信件的主要联系方式.
Email: alekseyshulkin@rambler.ru
ORCID iD: 0000-0003-1688-0017
SPIN 代码: 2754-1702

MD, Dr. Sci. (Med.), Associate Professor

俄罗斯联邦, Ryazan

Alexandr Slepnev

Ryazan State Medical University

Email: a.slepnev@rzgmu.ru
ORCID iD: 0000-0003-0696-6554

Cand. Sci. (Biol.), Associate Professor

俄罗斯联邦, Ryazan

Elena Yakusheva

Ryazan State Medical University

Email: e.yakusheva@rzgmu.ru
ORCID iD: 0000-0001-6887-4888
SPIN 代码: 2865-3080

MD, Dr. Sci. (Med.), Professor

俄罗斯联邦, Ryazan

参考

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2. Fig. 1. Structural formula of atorvastatin and main products of its fragmentation.

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3. Fig. 2. Chromatogram of blank sample without addition of atorvastatin standard.

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4. Fig. 3. Chromatogram of blank sample with addition of atorvastatin standard to the final concentration 0.5 nmol/l.

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