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

Abstract

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.

Keywords

atorvastatin / HPLC-MS/MS / transporter proteins / OATP1B1 / HepG2 cell line

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