Quantitative Determination of 2-Oxo-Imidazole-Containing Dipeptides by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Abstract

2-Oxo-imidazole-containing dipeptides (2-oxo-IDPs), novel imidazole-containing dipeptide (IDP) derivatives, exhibit a much higher antioxidant capacity than that of IDPs. However, quantitative methods have only been developed for IDPs, and methods for the quantitative analysis of 2-oxo-IDPs are needed. In this study, we developed methods for the quantitative analysis of 2-oxo-IDPs by high-performance liquid chromatography with online electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) coupled with a stable isotope dilution method. First, we prepared stable isotope-labeled IDP and 2-oxo-IDP standards for MS analyses. Next, using these standards, we established highly sensitive, selective, and absolute quantitative analysis methods for five IDPs and five 2-oxo-IDPs by HPLC-ESI-MS/MS, achieving a limit of detection in the fmol range. Finally, we applied the method to various types of meat, such as beef, pork, chicken, and whale meat, demonstrating the detection of both IDPs and 2-oxo-IDPs. Furthermore, we provide the first evidence for the endogenous production of 2-oxo-balenine in meats. The methods developed in this study enable the detection of trace levels of 2-oxo-IDPs in biological samples and could be helpful for understanding the biological relevance of 2-oxo-IDPs.

Keywords: 2-oxo-imidazole-containing dipeptides; anserine; balenine; carnosine; homoanserine; homocarnosine; imidazole-containing dipeptides; mass spectrometry; meat; stable isotope dilution method.

Figure 1

MS/MS spectra of IDPs. IDPs were synthesized by the carbodiimide coupling reaction and subjected to HPLC-ESI-MS/MS. MS/MS spectra of balenine (A), homocarnosine (C), homoanserine (D), carnosine (E), and anserine (F) are shown, respectively. Product ions of balenine were assigned as shown in (B).

Figure 2

MS/MS spectra of 2-oxo-IDPs. 2-Oxo-IDPs were prepared by the copper/ascorbate oxidation system and subjected to HPLC-ESI-MS/MS. MS/MS spectra of 2-oxo-balenine (A), 2-oxo-homocarnosine (C), 2-oxo-homoanserine (D), 2-oxo-carnosine (E), and 2-oxo-anserine (F) are shown. The assignment of the product ions of 2-oxo-balenine were conducted as shown in (B).

Figure 3

Antioxidant capacity of 2-oxo-IDPs and IDPs. Antioxidant capacity of 2-oxo-IDPs and IDPs were evaluated by DPPH radical scavenging assay. Data are expressed as Trolox equivalent antioxidant capacity (TEAC): μmol Trolox equivalent (TE) per mmol samples. Data are presented as means ± standard deviation (SD) (n > 6). **** p < 0.0001 versus the corresponding IDPs, compared using unpaired Student’s t test.

Figure 4

Separation of several IDPs and 2-oxo-IDPs using HPLC-ESI-MS/MS. A standard mixture of IDPs and 2-oxo-IDPs (1 µM each) was subjected to HPLC-ESI-MS/MS using an Intrada Amino Acid column. (A) Representative MS/MS chromatograms of IDPs. Open triangles indicate each IDP, and the gray triangle indicates balenine detected in MRM for anserine. (B) Representative MS/MS chromatograms of 2-oxo-IDPs. Filled triangles indicate each 2-oxo-IDP, and gray triangle indicates 2-oxo-balenine detected in MRM for 2-oxo-anserine.

Figure 5

HPLC-ESI-MS/MS analysis of IDPs and 2-oxo-IDPs in beef. Meat samples were homogenized in 80% acetonitrile/water containing stable isotope-labeled standards, and extracted IDPs and 2-oxo-IDPs were subjected to HPLC-ESI-MS/MS analysis. (A) Representative chromatograms of endogenous IDPs (upper trace), and spiked isotope-labeled IDPs (lower trace) are shown. Open triangles indicate each IDP. (B) Representative chromatograms of endogenous 2-oxo-IDPs (upper trace), and spiked isotope-labeled 2-oxo-IDPs (lower trace) are shown. Filled triangles indicate each 2-oxo-IDP.