The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using 13C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting 13C-labeled peptides in blood sample with a C18 cartridge, the extract was applied to a 13C monoisotopic transition LC-MRM-MS/MS system with d-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The 13C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three 13C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each 13C- labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.
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