Investigation of the effects of storage and freezing on mixes of heavy-labeled metabolite and amino acid standards

Abstract

Rationale: High-throughput metabolomics has now made it possible for small/medium-sized laboratories to analyze thousands of samples/year from the most diverse biological matrices including biofluids, cell and tissue extracts. In large-scale metabolomics studies, stable-isotope-labeled standards are increasingly used to normalize for matrix effects and control for technical reproducibility (e.g. extraction efficiency, chromatographic retention times and mass spectrometry signal stability). However, it is currently unknown how stable mixes of commercially available standards are following repeated freeze/thaw cycles or prolonged storage of aliquots.

Methods: Standard mixes for ¹³ C, ¹⁵ N or deuterated isotopologues of amino acids and key metabolites from the central carbon and nitrogen pathways (e.g. glycolysis, Krebs cycle, redox homeostasis, purines) were either repeatedly frozen/thawed for up to 10 cycles or diluted into aliquots prior to frozen storage for up to 42 days. Samples were characterized by ultra-high-pressure liquid chromatography/mass spectrometry to determine the stability of the aliquoted standards upon freezing/thawing or prolonged storage.

Results: Metabolite standards were stable over up to 10 freeze/thaw cycles, with the exception of adenosine and glutathione, showing technical variability across aliquots in a freeze/thaw-cycle-independent fashion. Storage for up to 42 days of mixes of commercially available standards did not significantly affect the stability of amino acid or metabolite standards for the first 2 weeks, while progressive degradation (statistically significant for fumarate) was observed after 3 weeks.

Conclusions: Refrigerated or frozen preservation for at least 2 weeks of aliquoted heavy-labeled standard mixes for metabolomics analysis is a feasible and time-/resource-saving strategy for standard metabolomics laboratories.