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. 2025 Jan 15:565:120015.
doi: 10.1016/j.cca.2024.120015. Epub 2024 Oct 22.

Urine organic acid metabolomic profiling by gas chromatography mass spectrometry: Assessment of solvent extract evaporation parameters on the recovery of key diagnostic metabolites

Rachel S Carling  1 , Karolina Witek  2 , Erin C Emmett  2 , Claire Gallagher  3 , Stuart J Moat  4
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Free article

Urine organic acid metabolomic profiling by gas chromatography mass spectrometry: Assessment of solvent extract evaporation parameters on the recovery of key diagnostic metabolites

Rachel S Carling et al. Clin Chim Acta. .
Free article

Abstract

Background: Analysis of urinary organic acids (UOAs) by gas chromatography mass-spectrometry (GC-MS) is widely used in metabolomic studies. It is a complex test with many limitations and pitfalls yet there is limited evidence in the literature to support best practice. This study investigated the impact of drying down time and temperature on the recovery of 16 key analytes from solvent extracts.

Methods: Pooled urine specimens were enriched with organic acids. Urine aliquots (n = 3) were acidified and extracted into diethyl ether and ethyl acetate. Extracts were dried under nitrogen at ambient temperature (25 °C); 40 °C; 60 °C then left for 0; +5; +15 min. Dried extracts were derivatised with N,O,-bis-(trimethylsilyl)trifluoroacetamide prior to analysis by GC-MS. Urine specimens from individuals with biotinidase deficiency, maple syrup urine disease (MSUD) and ketotic hypoglycemia were analysed to demonstrate the potential clinical impact.

Results: Recovery of shorter chain hydroxycarboxylic acids decreased significantly when extracts were dried above 25 °C (mean recovery 89 % at 60 °C, p < 0.01) or left under nitrogen post-drying (mean recovery at ambient + 15 min, 40 °C + 15mins and 60 °C + 15mins was 56 %, 12 % and 2 %, respectively, p < 0.01). Whilst dicarboxylic acids/medium chain fatty acids were unaffected by temperature (mean recovery 100 %), prolonged drying reduced recovery (mean recovery 85 % at 60 °C + 15mins, p < 0.01).

Conclusions: Evaporation of solvent extracts with heat and/or prolonged drying under nitrogen results in significant losses of the shorter chain hydroxycarboxylic acids. The evaporation protocol must be carefully controlled to ensure accurate and reproducible results, preventing misdiagnoses and/or misinterpretation of results.

Keywords: Analysis; GC–MS; Inherited metabolic disease; Metabolomics; Organic acids.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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