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

• Published in: Clinica chimica acta Vol. 565; p. 120015
• Main Authors: Carling, Rachel S., Witek, Karolina, Emmett, Erin C, Gallagher, Claire, Moat, Stuart J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22.10.2024
• Subjects: Analysis; GC–MS; Inherited metabolic disease; Metabolomics; Organic acids; Metabolomics; Inherited metabolic disease; ERNDIM; IMD; GC–MS; Analysis; BSTFA; SIL; IQC; UOAs; GC; Organic acids
• DOI: 10.1016/j.cca.2024.120015
• ISSN: 0009-8981; 1873-3492

•Analysis of urine organic acids by GC–MS is widely used in clinical laboratories and increasingly, in metabolomics.•Whilst this powerful technique can detect a wide range of compounds, it is not optimised for individual analytes.•Without careful control (time/temperature) of the solvent evaporation step, hydroxycarboxylic acids can be lost.•Potential for missed diagnosis of IMDs by clinical labs, and the collection of inaccurate data during metabolic studies. 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. 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. 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). 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.