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Multicenter Evaluation of a Novel Nanoparticle Immunoassay for 5-Fluorouracil on the Olympus AU400 Analyzer

Beumer, Jan H PhD, PharmD*; Boisdron-Celle, M PhD; Clarke, William PhD; Courtney, Jodi B BS§; Egorin, Merrill J MD*; Gamelin, Erick MD, PhD; Harney, Rebecca L BS§; Hammett-Stabler, Catherine PhD; Lepp, Sandy PhD; Li, Yunying PhD§; Lundell, Gregory D BS§; McMillin, Gwen PhD#; Milano, Gerard PhD**; Salamone, Salvatore J PhD§

doi: 10.1097/FTD.0b013e3181b9b8c0
Original Article

Background: 5-Fluorouracil (5-FU) is the most widely used chemotherapy drug, primarily against gastrointestinal, head and neck, and breast cancers. 5-FU has large pharmacokinetic variability resulting in unexpected toxicity or ineffective treatment. Therapeutic drug management of 5-FU minimizes toxicity and improves outcome. A nanoparticle-based immunoassay was developed to provide oncologists with a rapid, cost-effective tool for determining 5-FU plasma concentrations.

Methods: Monoclonal antibodies, bound to nanoparticles, were used to develop an immunoassay for the Olympus AU400. Assay precision, linearity, calibration stability, and limit of detection were run at multiple centers; interference, cross-reactivity, lower limit of quantitation and recovery at 1 center. Clinical samples collected from 4 cancer centers were analyzed for 5-FU concentrations by liquid chromatography-tandem mass spectrometry and compared with the immunoassay results.

Results: With calibrators from 0 to 1800 ng/mL 5-FU and autodilution, concentrations up to 9000 ng/mL could be determined. Time to first result was 10 minutes, and 400 samples per hour could be quantitated from a standard curve stored for >30 days. Imprecision across all laboratories was <5%, and the assay was linear upon dilution over the entire range. Cross-reactivities for dihydro-5-FU, uracil, capecitabine, and tegafur were <1%, 9.9%, 0.05%, and 0.23%, respectively. The limit of detection was 52 ng/mL with a lower limit of quantitation of 86 ng/mL. Assay results of clinical samples (93-1774 ng/mL) correlated with liquid chromatography-tandem mass spectrometry results: (R = 0.9860, slope 1.035, intercept 10.87 ng/mL).

Conclusions: This novel immunoassay is suitable for quantitating 5-FU plasma concentrations with advantages of speed, small sample size, minimal sample pretreatment, and application on automated instrumentation. These advantages enable efficient therapeutic drug management of 5-FU in clinical practice.

From the *University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; †Paul Papin Cancer Center, Département de Biopathologie des tumeurs, Angers, France; ‡Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland; §Research and Development, Saladax Biomedical, Bethlehem, Pennsylvania; ¶University of North Carolina, Pathology and Laboratory Medicine, Chapel Hill, North Carolina; ∥Technical Operations and Technical Support, Olympus Instruments, Irving, Texas; #Toxicology, ARUP Laboratories, Salt Lake City, Utah; and **Centre Antoine Lacassagne, Nice, France.

Received for publication April 6, 2009; accepted July 28, 2009.

Supported by P30-CA47904, NCI (J.H.B., M.J.E.). The sponsor was responsible for the supply of all study materials, protocol design, and data analysis.

Correspondence to: Salvatore J. Salamone, PhD, Saladax Biomedical Inc, 116 Research Drive, Bethlehem, Pennsylvania 18015 (e-mail: ssalamone@saladax.com).

© 2009 Lippincott Williams & Wilkins, Inc.