Quantitative analysis of a synthetic peptide, NR58-3.14.3, in serum by LC-MS with inclusion of a diastereomer as internal standard

A method for quantifying an intramolecularly linked all-D-amino acid peptide, NR58-3.14.3, in rat serum by LC-MS using selected ion monitoring with inclusion of a diastereomer as internal standard was developed. The reproducible quantitation of multiply charged compounds by LC-MS using single ion or selective reaction monitoring is often a challenge as the intensity ratio of the ions in a series of different charge states can vary. Good precision was obtained in the selected ion monitoring mode by integrating the summed ion currents of the singly, doubly, and triply charged molecular ions. Since stable isotope analogs are costly and integration of residual unlabeled material can be of concern, a diastereomer of NR58-3.14.3, NR58-3.14.5, was used as internal standard. The diastereomers mere indistinguishable by electrospray MS, but fully separated by reversed-phase LC. Consequently, interference due to isotopic impurities or coelution was not encountered. The calibration plot was linear throughout a concentration range of 0.2 to 200.0 mu g/ml (r(2) = 0.9996). Intraday precision of the standards analyzed was less than 12% RSD over the calibration range and the accuracy within +/-11% RE. Serum pharmacokinetics were in good agreement with the pharmacokinetic profiles of small, ionic, and polar molecules. (C) 2000 Academic Press.