Fast LC–MS Electrospray Ionization for the Quantification of Digoxin in Human Plasma and Its Application to Bioequivalence Studies
- 232 Downloads
A fast, sensitive and specific liquid chromatography-mass spectrometry method has been developed for quantification of digoxin in human plasma. The method was optimized to bioequivalence studies aiming higher sensitivity and selectivity than previously published methods, in addition to shorter run time allowing high-throughput sample analyses from volunteers. Chromatographic separation was achieved by an RP-18e column hyphenated to an API 5000 mass spectrometer system set at negative electrospray ionization and operating in the MRM mode. Calibration curve was linear over a wide range of concentration (50.0–6000.0 pg mL−1), with the lower limit of quantification at 50.0 pg mL−1 and without interfering peaks at the retention time of digoxin (2.09 min). Dexamethasone was used as internal standard and samples were cleaned up by liquid-liquid extraction obtaining a mean recovery of 73.8%. Validation results confirmed inter-batch accuracy (−8.66 to 5.78%), precision (4.1–10.6%) and stability, in accordance with the U.S. Food and Drug Administration and the Brazilian National Health Surveillance Agency guidelines. The developed analytical method could be successfully applied to a single oral dose (0.25 mg), one-way, randomized, two-sequence, crossover bioequivalence study validating, up to date, the fastest analysis and the most sensitive and specific method already published for digoxin quantification.
KeywordsColumn liquid chromatography-mass spectrometry Bioequivalence and pharmacokinetic study Method validation Digoxin
All financial support was provided by FUNAPE and the Institute of Pharmaceutical Sciences, Goiânia, GO, Brazil.
- 1.Silva P (2006) Farmacologia. Guanabara Koogan, Rio de Janeiro, pp 63–67, 642Google Scholar
- 6.Garteiz D, Din S, Morrow R (2003) Quantification of digoxin in human plasma using negative ion electrospray ionization LC/MS. Varian Inc., HoustonGoogle Scholar
- 8.Baselt RC, Cravey RH (1995) Disposition of toxic drugs and chemicals in man. Chemical Toxicology Institute, Foster City, p 802Google Scholar
- 9.Stone JA, Soldin SJ (1989) Clin Chem 35:1326–1331Google Scholar
- 10.Stoll RG, Christensen MS, Sakmar E, Wagner JG (1972) Res Commun Mol Pathol Pharmacol 4:503–510Google Scholar
- 13.ANVISA, Agência Nacional de Vigilância Sanitária (2003) RE no 899, de 29 de Maio de 2003. Guia para validação de métodos analíticos e bioanalíticosGoogle Scholar
- 14.FDA, Center for Drug Evaluation and Research (2001) U S Department of Health and Human Services, Guidance for Industry, Bionalytical Method ValidationGoogle Scholar
- 15.United States Pharmacopeia Convention (2003) United States pharmacopeia, USP 26. National Publishing, PhiladelphiaGoogle Scholar
- 19.Jedlička A, Grafnetterová T, Miller V (2003) J Chromatogr B Anal Technol Biomed Life Sci 33:109–115Google Scholar