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The pyridinium crosslinks are important biomarkers of mature hard tissue collagen degradation. This paper describes an isocratic ion-paired reversed-phase high-performance chromatographic assay using narrow-bore columns and high-sensitivity fluorescence detection to enable for the first time the determination of pyridinium crosslinks in both serum and synovial fluid samples. Extracted freeze-dried acid hydrolysates were re-suspended in 20 mM pentafluoropropionic acid (PFPA). Separations were carried out using an Exsil 100 5-μm ODS2 column (100 mm × 2.1 mm I.D.) eluted with 10 mM PFPA in water at 0.15 ml/min and detected using a Jasco 821-FP detector (xenon lamp: excitation 290 nm, emission 400 nm). Fluorescent response was linear from 269 to 8620 fmol for pyridinoline (Pyr) and 85 to 2710 fmol for deoxypyridinoline (dPyr). The limits of detection were 28 and 57 fmol, respectively. The coefficient of variation for extraction and analysis of normal serum was 7.96% for Pyr and 6.30% for dPyr (n = 6). The mean ± S.D. concentration of Pyr in normal serum was 3.26 ± 0.83 nM.

Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be

Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.

A rapid and sensitive assay for serum cytidine deaminase has been developed utilising ion-pair reversed-phase high-performance liquid chromatography. The addition of 1-octanesulphonic acid (OSA) caused the retention of cytidine and uridine to reverse and uridine, the minor component in the assay, to elute first. Cytidine, uridine and allopurinol (internal standard) were separated on a 5-micron Hypersil ODS column using 100 mM ammonium acetate with 1% (v/v) methanol and 1 mM OSA adjusted to pH 5.0. Detection was at 262 nm. Peak areas were linear from 7 pmol to 6 nmol injected (r = 0.99). Intra-assay variation was 7.8% (n = 10) and the correlation with a colorimetric assay was r = 0.78 (p less than 0.001).