Elsevier

Analytical Biochemistry

Volume 440, Issue 2, 15 September 2013, Pages 158-165
Analytical Biochemistry

Stable deuterium internal standard for the isotope-dilution LC–MS/MS analysis of elastin degradation

https://doi.org/10.1016/j.ab.2013.05.014Get rights and content

Abstract

Chemical synthesis of the deuterium isotope desmosine-d4 has been achieved. This isotopic compound possesses all four deuterium atoms at the alkanyl carbons of the alkyl amino acid substitution in the desmosine molecule and is stable toward acid hydrolysis; this is required in the measurement of two crosslinking molecules, desmosine and isodesmosine, as biomarkers of elastic tissue degradation. The degradation of elastin occurs in several widely prevalent diseases. The synthesized desmosine-d4 is used as the internal standard to develop an accurate and sensitive isotope-dilution liquid chromatography–tandem mass spectrometry analysis, which can serve as a generalized method for an accurate analysis of desmosine and isodesmosine as biomarkers in many types of biological tissues involving elastin degradation.

Section snippets

Reagent and materials

(1) For the chemical synthesis of desmosine-d4: deuterium gas (99.9% atom%) was purchased from Sigma (St. Louis, MO, USA). All reactions were conducted with magnetic stirring using dry solvents unless otherwise indicated. CD3OD for the reactions was purchased from Kanto Chemicals (Tokyo, Japan). All reagents were obtained from commercial suppliers and used without further purification unless otherwise stated. Analytical thin-layer chromatography was performed on silica gel 60 F254 plates

Chemical synthesis of the deuterium isotope, desmosine-d4

As described in Fig. 1, desmosine-d4 is prepared by deuteration of the 4-alkynyl desmosine derivative compound 1, which we have synthesized according to the reactions previously developed in our laboratory [38], [39]. Newly synthesized desmosine-d4 possesses all four deuterium atoms at the stable alkane carbons, which will be stable toward acidic condition. The synthesized deutereo-desmosine consists of four deutereo-isotopomers, desmosine-d4 (50.53%), -d3 (38.93%), -d2 (10.10%), and -d1

Discussion

We have chemically synthesized a stable deuterated isotope, desmosine-d4, that possesses all four deuterium atoms at the alkanyl carbons of the alkyl amino acid substitution in the desmosine molecule and is stable toward acid hydrolysis; this is required in the measurement of two crosslinking DES and IDS molecules as biomarkers of elastic tissue degradation. Our recent achievement in the total synthesis of desmosine molecule [38], [39] enables us to synthesize this stable desmosine-d4 that can

Acknowledgments

This work was supported by funds from James P. Mara Center for Lung Diseases, the Flight Attendant Medical Research Institute, the Charles A. Mastronardi Foundation, the Ned Doyle Foundation, the Alpha-1 Foundation, and by funds from Ethel Kennedy, John Kennedy, and Judith Sulzberger. This work is also supported by funds (to T.U.) from a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI Grant 22710224); the Shimadzu

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