A method that measures 12 analytes in urine and reflects possible exposure to pesticides was developed. The sample preparation involves enzyme hydrolysis and solvent extraction through the use of laboratory robotics, followed by phase-transfer catalysis derivatization and silica cleanup. Samples are analyzed by capillary gas chromatography and tandem mass spectrometry using an isotope dilution technique with 13C-labeled internal standards. The limit of detection is 1 microgram/L (1 part per billion) for most analytes, and most analytes have a linear response up to 100 micrograms/L. The precision of the method is reflected in the variation observed in quality control materials over 33 months; the variation averaged 17% for these analytes. On the basis of the detectable analyte levels of unspiked urine samples collected from unexposed volunteers, this method can be used to measure the low levels necessary for establishing reference range values of the selected pesticides or metabolites.
Traditionally, visible fluorophores have been used as labels in DNA sequencing. They absorb and fluoresce in a region of the spectrum that is susceptible to biological interferences in sequencing samples. The increased noise level due to autofluorescence of glass, solvent, or impurities can greatly reduce the potential sensitivity of the analysis. In an attempt to increase the sensitivity, we have investigated the use of near-infrared (near-IR) fluorophores as labels in DNA sequencing. Near-IR fluorophores possess spectral properties which are observed between 700 and 1200 nm, a region with characteristically little interference by biomolecules. Therefore, the detection of near-IR dyes is not limited by noise levels, but rather by detector capabilities. Near-IR dyes are also suitable for selective excitation with commercially available laser diodes which can further enhance the observed fluorescence signal. We have covalently linked functionalized near-IR dyes to modified DNA oligomers for use in DNA sequencing. We report the synthesis and chromatographic purification of near-IR-labeled DNA oligomers. We further discuss the inherent properties of the conjugates and their use in DNA sequencing procedures.Fluorescent labels are being used increasingly in bioanalytical applications.1-6 They have been attached successfully to a variety of biomolecules such as enzymes, antibodies, and nucleic acids for use in immunoassay, flow cytometry, and sequencing
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