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JAC Advance Access originally published online on June 20, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):557-563; doi:10.1093/jac/dkl258
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Determination of gentamicin in different matrices by a new sensitive high-performance liquid chromatography-mass spectrometric method

Concepción Lecároz1, Miguel A. Campanero2, Carlos Gamazo1 and María J. Blanco-Prieto3,*

1 Department of Microbiology, University of Navarra Pamplona, Spain 2 Department of Clinical Pharmacology, Clínica Universitaria Pamplona, Spain 3 Department of Pharmacy and Pharmaceutical Technology, University of Navarra 31080 Pamplona, Spain

Received 3 April 2006; returned 8 May 2006; revised 23 May 2006; accepted 30 May 2006

*Corresponding author. Tel: +34-948-425600; Fax: +34-948-425649; E-mail: mjblanco{at}unav.es

Objectives: The aim of this work was to develop and validate an HPLC method for gentamicin quantification in different types of biological samples such as animal tissues and cellular material and also in pharmaceuticals.

Methods: Poly(lactide-co-glycolide) microparticles (MP) of gentamicin (PLGA 502H MP), THP-1 cells, and plasma and tissue samples of mice treated with the antibiotic either free or loaded into PLGA 502H MP were processed by a simple preparation procedure, subjected to chromatography on a reversed-phase column and measured by mass spectrometry detection. The developed method was compared with bioassay and fluorimetric assay methods previously used for gentamicin determination.

Results: The HPLC method was linear over the ranges 40–800 ng/mL and 0.1–100 µg/mL and showed good accuracy (average accuracy < 5.59%) and reproducibility (CV < 6.13%). Encapsulation of gentamicin in PLGA 502H MP was determined by the three methods. Good correlation was observed between bioassay (reference method) and HPLC. Extra- and intracellular in vitro antibiotic accumulation was determined by bioassay and chromatography. Both methods gave similar extracellular concentrations but the HPLC-MS technique demonstrated an improved accuracy (5.59% versus 14%) and precision (6.13% versus 15%) compared with bioassay. However, only the HPLC-MS method was sensitive enough to detect the drug, intracellularly and in tissues.

Conclusions: All these data favour the use of chromatography-mass spectrometry as a versatile technique not only suitable for gentamicin quantification loaded in drug delivery systems, but also sensitive and specific enough for in vivo and intracellular studies.

Keywords: HPLC-MS , bioassay , fluorimetric assay , cellular quantification , biological matrices , drug delivery systems , PLGA


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M. C. Lecaroz, M. J. Blanco-Prieto, M. A. Campanero, H. Salman, and C. Gamazo
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