Gas Chromatography-Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva.

Justyna Piechocka,Rafał Głowacki,Monika Wieczorek

doi:10.3390/ijms21239252

Justyna Piechocka, Rafał Głowacki + Show 1 more

Open Access

https://doi.org/10.3390/ijms21239252

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Abstract

Gas chromatography–mass spectrometry technique (GC-MS) is mainly recognized as a tool of first choice when volatile compounds are determined. Here, we provide the credible evidence that its application in analysis can be extended to non-volatile sulfur-containing compounds, to which methionine (Met), homocysteine (Hcy), homocysteine thiolactone (HTL), and cysteine (Cys) belong. To prove this point, the first method, based on GC-MS, for the identification and quantification of Met-related compounds in human saliva, has been elaborated. The assay involves simultaneous disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile (MeCN) deproteinization, followed by preconcentration by drying under vacuum and treatment of the residue with a derivatizing mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS). The validity of the method was demonstrated based upon US FDA recommendations. The assay linearity was observed over the range of 0.5–20 µmol L−1 for Met, Hcy, Cys, and 1–20 µmol L−1 for HTL in saliva. The limit of quantification (LOQ) equals 0.1 µmol L−1 for Met, Hcy, Cys, while its value for HTL was 0.05 µmol L−1. The method was successfully applied to saliva samples donated by apparently healthy volunteers (n = 10).

Highlights

Methionine (Met)-related compounds, such as homocysteine (Hcy), Hcy-thiolactone (HTL), and cysteine (Cys), have received continuing attention due to their physiological importance and considerable implications in so-called civilization diseases
Our efforts were predominantly attributed to the development of a highly effective analytical tool based on gas chromatography–mass spectrometry technique (GC-MS) for simultaneous determination of salivary Met, HTL, and total Cys and Hcy content as well as the application of the assay to real samples in order to confirm or exclude the performance of the method
The method allows simultaneous determination of a number of salivary biologically relevant compounds, namely, Met, total Cys, and Hcy as well as HTL in a single run. This is the first-ever report dealing with the presence of Met in the above-mentioned matrix as well as the first effective analytical tool enabling to determine Met in human saliva

Summary

Introduction

Methionine (Met)-related compounds, such as homocysteine (Hcy), Hcy-thiolactone (HTL), and cysteine (Cys), have received continuing attention due to their physiological importance and considerable implications in so-called civilization diseases. It seems to be essential to provide more robust and versatile platforms for a comprehensive assessment of Met and metabolically related compounds metabolome in order to increase knowledge about their role in the human body, in the near future. It seems to be essential to put efforts into developing new, simple, and low-price analytical tools facilitating large-scale screening of civilization diseases. Our efforts were predominantly attributed to the development of a highly effective analytical tool based on GC-MS for simultaneous determination of salivary Met, HTL, and total Cys and Hcy content as well as the application of the assay to real samples in order to confirm or exclude the performance of the method. The advantages, pitfalls, and limitations of the GC-MS assay are mentioned

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