Artefacts in organic acid analysis: occurrence and origin of partially trimethylsilylated 3-hydroxy-3-methyl carboxylic acids

Previous reports of patients with 3-hydroxy-3-methylglutaric aciduria have described the occurrence of di-trimethylsilyl (TMS) and tri-TMS derivatives of 3-hydroxy-3-methylglutaric acid on analysis using gas chromatography an d mass spectrometry, leading to difficulty in quantification and ambiguity in diagnosis. We have extracted organic acids From the urine of patients wi th 3-hydroxy-3-methylglutaric aciduria using a variety of procedures. Solve nt extraction combined with hydrochloric acid/sodium chloride resulted in p roduction of both di-TMS and tri-TMS derivatives of 3-hydroxy-3-methylgluta ric acid and also mono-TMS and di-TMS derivatives of 3-hydroxyisovaleric ac id. The effects were not abolished by heating. Use of sulphate-based reagen ts minimised artefact formation and use of DEAE-Sephadex anion exchange ext raction resulted in single fully trimethylsilylated derivatives. Artefact f ormation during use of chloride-based reagents was abolished by pyridine ad ded prior to trimethylsilylation. Chloride ions form adducts with hydroxyl groups in these 3-hydroxy-3-methyl carboxylic acids that prevent complete t rimethylsilylation, Chloride-based reagents should be avoided in the solven t extraction of organic acids from physiological fluids or, if used, pre-tr eatment of the dried extract with pyridine is essential to avoid partial tr imethylsilylation of 3-hydroxy-3-methyl carboxylic acids. (C) 2000 Elsevier Science B.V. All rights reserved.