Application of 13C stable isotope labeling liquid chromatography–multiple reaction monitoring–tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats

Abstract

The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C 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 (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C 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 (13)C-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 (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method.

Current understanding of transport and bioavailability of bioactive peptides derived from dairy proteins: a review

“…Even at low bioavailability, DBPs may also exert their bioactivities in vivo after oral administration. For instance, dipeptide Val-Tyr can be intact absorbed and counteract renal cell signalling mediated at 10 nM (Nakashima et al, 2011;Dias et al, 2017;Matsui, 2018). Moreover, IPP and VPP were detected in plasma at nM concentrations after oral administration and their accumulation had also been found in various other tissues (Ten Have et al, 2015;Nongonierma & FitzGerald, 2016).…”

Intestinal absorption of small peptides: a review

“…MS detection with liquid chromatographic (LC) separation (LC-MS) is a highly selective and sensitive assay, which can be used to quantify absorbed peptides in a small volume of plasma sample (>10 lL). Moreover, the use of isotopelabelled peptides allowed for selective analysis of absorbed peptides against the endogenous peptides (Nakashima et al, 2011;Fig. 2).…”

Isolation and Characterization of Peptides with Antihypertensive Activity in Foodstuffs

“…Multiple reaction monitoring (MRM) assays could be the technique of choice in these cases. Nevertheless, only one work in which antihypertensive peptides were quantified in rat plasma after their administration was found (Nakashima et al, 2011). To our knowledge, there is no work that had used this methodology to quantify antihypertensive peptides in foodstuffs.…”