Analytical and Bioanalytical Chemistry

, Volume 400, Issue 2, pp 423–433 | Cite as

Development and validation of a sensitive, simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Joyce S. Macwan
  • Ileana A. Ionita
  • Miroslav Dostalek
  • Fatemeh AkhlaghiEmail author
Original Paper


The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R 2 ≥ 0.9975, n = 3) over the concentration range of 0.05–100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6–111%. Intra- and inter-run mean percent accuracy were between 85–115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at −80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.


Chromatogram of atorvastatin and metabolites obtained with postcolumn infusion shows no matrix effect at the retention times of analytes and IS. Arrow indicates region where the signal of compounds infused post-column is suppressed during the elution of endogenous matrix components


Assay Atorvastatin Concentration Lactones LC-MS/MS Metabolites Pharmacokinetics 



Atorvastatin acid


Peak plasma concentration


Cytochrome P450 3A4


3-Hydroxy-3-methylglutaryl-coenzyme A


High-performance liquid chromatography


High-level quality control


Internal standard


Liquid chromatography-tandem mass spectrometry


Lower limit of quantitation


Low-level quality control


Multiple-reaction monitoring




Quality control samples


Signal-to-noise ratio





The authors gratefully acknowledge the financial support of American Heart Association award #0855761D. Use of an API 4000 mass spectrometer provided as a collaboration agreement with AB Sciex is gratefully acknowledged.


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Joyce S. Macwan
    • 1
  • Ileana A. Ionita
    • 1
  • Miroslav Dostalek
    • 1
  • Fatemeh Akhlaghi
    • 1
    Email author
  1. 1.Clinical Pharmacokinetics Research Laboratory, Department of Biomedical and Pharmaceutical SciencesUniversity of Rhode IslandKingstonUSA

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