Research note: Compound-specific isotopic analysis in ³⁴S-labelled chicken tissues using high resolution gas...

Many food supplements include sulphur (S)-containing additives such as methionine or synthetic compounds like 2‑hydroxy-S-methyl-thiobutyric acid (HMTBA). However, monitoring the metabolic use efficiency of S-containing additive is challenging, and requires specific methods, based on isotopic labelling. The most direct route is the utilisation of ³⁴S-enriched material and subsequent measurement of ³⁴S-abundance in tissues. While this can be carried out routinely using elemental analysis coupled with isotope ratio mass spectrometry (IRMS) for bulk S from raw tissue material, there is currently no IRMS-based method adapted to compound-specific isotopic analysis for sulphur. Here, we present a method based on gas chromatography coupled to high resolution mass spectrometry (GC-MS) to measure ³⁴S-abundance in both free and protein-bound S containing amino acids. This method elaborates on metabolomics based on GC-MS analysis of trimethylsilylated extracts. Specific ion fragments comprising a sulphur atom could be identified and their isotopic pattern was used to compute % ³⁴S. The high resolution was useful to avoid the confounding effect of natural carbon (¹³C₂) or (¹⁸O) isotopologues but required a correction for silicium (Si) isotope because the mass excess of ³⁰Si (+ 1.9968 a.m.u.) was close to that of ³⁴S (+ 1.9957 a.m.u.) and therefore the ³⁰Si and ³⁴S isotopologues could not be separated. This technique was applied to broilers fed with ³⁴S-labelled methionine and showed that ³⁴S could be easily traced in different organs. ³⁴S-methionine redistributed mostly to homocysteine with little ³⁴S-enrichment in cysteine and taurine, due to the isotopic dilution by food cysteine supply. The results show that our method can be implemented to follow the metabolic incorporation of S-containing additives such as methionine in broilers.