Analyses of iminodipeptides containing C-terminal proline or hydroxyproline in biological samples by liquid chromatography mass spectrometry

Analyses of authentic iminodipeptides containing an N-terminal proline (Pro-X) or C-terminal proline (X-Pro), and iminodipeptides in the urine and serum of patients with prolidase deficiency, have been demonstrated using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. The separation of iminodipeptides was carried out on a reversed phase high-performance liquid chromatographic column using 0.1% aqueous trifluoroacetic acid-methanol (75:25, v/v) as mobile phase. Very intense protonated molecular ions [M+H](+) of various synthetic iminodipeptides, Pro-Gly, Gly-Pro, Pro-Ala, Ala-Pro, Pro-Val, Val-Pro, Pro-Leu and Leu-Pro, were observed. Pro-Gly (Pro-X) and Gly-Pro (X-Pro) have the same protonated molecular ion (m/z 173), but the peaks of these compounds on the mass chromatograms were clearly distinguished by the differences of the retention times and mass spectra. The quasimolecular ions [M+H](+) of various iminodipeptides were observed in the urine samples of patients with prolidase deficiency, and Gly-Pro, Ala-Pro, Val-Pro, Leu-Pro, Ile-Pro, Ser-Pro, Thr-Pro, Glu-Pro, Asp-Pro, His-Pro, Lys-Pro, Pro-Pro and Tyr-Pro as iminodipeptides containing proline with C-terminal residues and Glu-Hyp, Pro-Hyp, Ile-Hyp and Gly-Hyp as iminodipeptides containing hydroxyproline with C-terminal residues were identified in the urine of patients with prolidase deficiency. The quasi-molecular ions [H+H](+) of various iminodipeptides containing with C-terminal proline and hydroxyproline also were observed in the sera of patients with prolidase deficiency, using selected ion monitoring. The quasi-molecular ions [M+H](+) of iminodipeptides containing C-terminal proline were not observed in the sera of normal subjects or the patients' mother, but the latter contained various iminodipeptides containing C-terminal hydroxyproline. This method proved useful for the determination of iminodipeptides in the sera of patients with prolidase deficiency. (C) 1997 Elsevier Science B.V.