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.
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.
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).
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: firstname.lastname@example.org).