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

Cover Page


Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

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.

Full Text

Restricted Access

About the authors

Pelageya D. Erokhina

Ryazan State Medical University

Email: erokhina.pelageya96@yandex.ru
ORCID iD: 0000-0003-4802-5656
SPIN-code: 1480-6854
Russian Federation, Ryazan

Pavel Yu. Myl’nikov

Ryazan State Medical University

Email: dukeviperlr@gmail.com
ORCID iD: 0000-0001-7829-2494
SPIN-code: 8503-3082
Russian Federation, Ryazan

Svetlana O. Ganina

Ryazan State Medical University

Email: svetlanaganina23@gmail.com
ORCID iD: 0000-0002-4408-6454
Russian Federation, Ryazan

Egor A. Konyakhin

Ryazan State Medical University

Email: egor_konyahin@mail.ru
ORCID iD: 0000-0002-9025-130X
Russian Federation, Ryazan

Aleksey V. Shchul’kin

Ryazan State Medical University

Author for correspondence.
Email: alekseyshulkin@rambler.ru
ORCID iD: 0000-0003-1688-0017
SPIN-code: 2754-1702

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

Russian Federation, Ryazan

Alexandr A. Slepnev

Ryazan State Medical University

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

Cand. Sci. (Biol.), Associate Professor

Russian Federation, Ryazan

Elena N. Yakusheva

Ryazan State Medical University

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

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

Russian Federation, Ryazan

References

  1. Alam K, Crowe A, Wang X, et al. Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions. International Journal of Molecular Sciences. 2018;19(3):855. doi: 10.3390/ijms19030855
  2. König J, Cui Y, Nies AT, et al. Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. The Journal of Biological Chemistry. 2000;275(30):23161–8. doi: 10.1074/jbc.M001448200
  3. Roth M, Obaidat A, Hagenbuch B. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology. 2012;165(5):1260–87. doi: 10.1111/j.1476-5381.2011. 01724.x
  4. Shulkin AV, Popova NM, Chernykh IV. The original and generic drugs: current state of the problem. Nauka Molodykh (Eruditio Juvenium). 2016;(1):30–5. (In Russ).
  5. Chernykh IV, Shchulkin AV, Gatsanoga MV, et al. Development of HPLC method of ethylmethylhydroxypyridine succinate quantification in rat and rabbit plasma. I.P. Pavlov Russian Medical Biological Herald. 2015;(1):62–6. (In Russ).
  6. Izucheniye bioekvivalentnosti vosproizvedennykh lekarstvennykh sredstv. In: Rukovodstvo po ekspertize lekarstvennykh sredstv. Vol. I. Moscow: Grif i K; 2014. P. 174–215. (In Russ).
  7. In Vitro Drug Interaction Studies ― Cytochrome P450 Enzyme- and Transporter Mediated Drug Interactions Guidance for Industry. 2020. Available at: https://www.fda.gov/media/134582/download. Accessed: 2022 February 17.
  8. Guidance for Industry. Drug Interaction Studies ― Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations. 2012. Available at: https://www.xenotech.com/wp-content/uploads/ 2020/07/2012-FDA-DDI-Guidance.pdf. Accessed: 2022 February 17.
  9. Bioanalytical method validation. Guidance for Industry. 2018. Available at: https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. Accessed: 2022 February 17.
  10. Guideline on bioanalytical method validation. 2015. Available at: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf. Accessed: 2022 February 17.
  11. Decision of the Council of the Eurasian Economic Commission of dated 2016, 3 November No. 85 "Ob utverzhdenii Pravil provedeniya issledovaniy bioekvivalentnosti lekarstvennykh preparatov v ramkakh Evraziyskogo ekonomicheskogo soyuza". Available at: https://www.alta.ru/tamdoc/16sr0085/. Accessed: 2022 February 17. (In Russ).
  12. Stieger B., Hagenbuch B. Organic anion-transporting polypeptides. Current Topics in Memranes. 2014;73(205–32). doi: 10.1096/B978-0-12-800223-0.00005-0
  13. Summary of Product Characteristics. Available at: https://www.ema.europa.eu/en/documents/product-information/prezista-epar-product-information_en.pdf. Accessed: 2022 February 17.
  14. Neuvonen PJ, Niemi M, Backman JT. Drug interactions with lipid-lowering drugs: Mechanisms and clinical relevance. Clinical Pharmacology & Therapeutics. 2006;80(6):565–81. doi: 10.1016/j.clpt.2006.09.003
  15. Wadhwa K, Rana AC. A review on liquid chromatographic methods for the bioanalysis of atorvastatin. Journal of Pharmaceutical Sciences. 2021;7:4. doi: 10.1186/s43094-020-00146-7
  16. Crevar–Sakač M, Vujić Z, Brborić J, et al. An Improved HPLC Method with the aid of a chemometric protocol: simultaneous determination of atorvastatin and its metabolites in plasma. Molecules. 2013;18(3):2469–82. doi: 10.3390/molecules18032469
  17. Carter SJ, Ferecskó AS, King L, et al. A mechanistic modelling approach for the determination of the mechanisms of inhibition by cyclosporine on the uptake and metabolism of atorvastatin in rat hepatocytes using a high throughput uptake method. Xenobiotica. 2020;50(4):415–26. doi: 10.1080/00498254.2019.1652781
  18. Shu N, Hu M, Liu C, et al. Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2. Xenobiotica. 2016;46(10):875–81. doi: 10.3109/00498254.2016.1141437

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Structural formula of atorvastatin and main products of its fragmentation.

Download (39KB)
Indexing metadata
3. Fig. 2. Chromatogram of blank sample without addition of atorvastatin standard.

Download (46KB)
Indexing metadata
4. Fig. 3. Chromatogram of blank sample with addition of atorvastatin standard to the final concentration 0.5 nmol/l.

Download (65KB)
Indexing metadata

Copyright (c) 2022 ООО "Эко-Вектор"


Media Registry Entry of the Federal Service for Supervision of Communications, Information Technology and Mass Communications (Roskomnadzor) PI No. FS77-76803 dated September 24, 2019.