ORIGINAL ARTICLE A Rapid High-Performance Liquid Chromatography Method to Measure Linezolid and Daptomycin Concentrations in Human Plasma Marialuisa Polillo, PharmD,* Carlo Tascini, MD,† Marianna Lastella, PhD,* Paolo Malacarne, MD,‡ Laura Ciofi, BSc,* Bruno Viaggi, MD,‡ Guido Bocci, MD, PhD,* Francesco Menichetti, MD,† Romano Danesi, MD, PhD,* Mario Del Tacca, MD, PhD,* and Antonello Di Paolo, MD, PhD* Abstract: Daptomycin and linezolid, recently introduced to treat severe Gram-positive infections, are effective against multidrug- resistant Gram-positive microorganisms such as methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epi- dermidis, and vancomycin-resistant Enterococci bacteria that are less sensitive or frankly resistant, including methicillin-resistant S. aureus. However, alteration of their plasma profile has been described in some patients and this may be associated with toxicities or selection of resistant strains. The measurement of plasma concentrations of both drugs may allow the identification of those subjects at major risk of adverse events. Therefore, a rapid and sensitive high-performance liquid chromatography method for the analysis of daptomycin and linezolid was developed and applied in clinical settings. Drugs were extracted from plasma by adding methanol and, after centrifugation, clear supernatants were injected into the high-performance liquid chromatography system. Isocratic elution (1.5 mL/min) was per- formed using a mobile phase consisting of ammonium phosphate buffer 40 mM, pH 4.0, acetonitrile (70:30, vol/vol) through a BDS C 8 Hypersil stationary phase (250 3 4.6 mm, 5 mm); ultraviolet de- tection was used at 214 nm. Linezolid and daptomycin eluted within 20 minutes from the injection, and mean recoveries ranged between 95.4% and 112.1%, respectively. The method was linear (coefficient of linearity, 0.998–0.999) over the full range of concentrations as- sayed, from 0.78125 mg/L (limit of quantitation) to 100 mg/L for both drugs. The Sy.x values were equal to 0.25 6 0.10 and 0.29 6 0.18 mg/L for daptomycin and linezolid, respectively. Precision values were lower than 20% over the entire range of calibration standard, and accuracy was within the range of 80% to 120% for all concentrations. The present method proved to be sensitive and specific to measure daptomycin and linezolid plasma concentrations in patients affected by severe Gram-positive infections, allowing therapeutic drug monitoring in those patients at major risk of severe adverse events. Key Words: linezolid, daptomycin, therapeutic drug monitoring, high-performance liquid chromatography (Ther Drug Monit 2010;32:200–205) INTRODUCTION Severe infections resulting from Gram-positive bacteria remain one of the most important factors for mortality in hospital settings. 1 The early diagnosis and identification of causative microorganisms, together with their sensitivity spectrum to antibacterial agents, may lead to the choice of optimal drugs and schedules with superior rates of cure. However, the effectiveness of antibacterial chemotherapy suffers from the occurrence of bacterial strains that are less sensitive or frankly resistant to pharmacologic agents. 2,3 Thus, the introduction of newer molecules such as linezolid (an oxazolidinone) and then daptomycin (a lipopeptide), has represented a step forward in the treatment of severe Gram- positive infections. 4 Linezolid is effective in the treatment of lower-tract respiratory and soft tissues infections 5 achieving higher cure rates than teicoplanin. 6 The oxazolidinone acts through the inhibition of protein synthesis, interfering with both fMet- tRNA binding to the P site on the 30S ribosomal subunit initiation complex and peptide bond formation with the trans- location of the peptide chain from A to P site. 7 Daptomycin has been registered for right-sided endocarditis, with or without bacteremia, and skin and soft tissue infections. 8 As a result of its chemical structure, the lipopeptide is able to penetrate bacterial cell membranes leading to membrane disruption, even if interference with bacterial transcriptome and inhibition of cell wall have been postulated as other putative mechanisms of action. 9 The use of both drugs has been associated with toxi- cities, and high plasma concentrations and long periods of administration are considered risk factors for the occurrence of adverse drug reactions. 10,11 At the same time, despite their innovative mechanisms of action, low plasma and tissue concentrations may lead to resistant strain selection, 12,13 hence compromising the effectiveness of the treatment. Furthermore, recent studies are suggesting that standard doses of linezolid Received for publication July 31, 2009; accepted January 13, 2010. From the *Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy; †Unit of Infectious Diseases; and ‡Intensive Care Units, University Hospital, Pisa, Italy. Correspondence: Antonello Di Paolo, MD, PhD, Division of Pharmacology and Chemotherapy, Department of Internal Medicine, Via Roma 55, 56126 Pisa, Italy (e-mail: a.dipaolo@ao-pisa.toscana.it). Copyright Ó 2010 by Lippincott Williams & Wilkins 200 Ther Drug Monit  Volume 32, Number 2, April 2010