Microderivatization of 4-[18O]hydroxyproline and quantitation with a benchtop mass spectrometer

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [18O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [18O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph-mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of 18O incorporation.