Abstract
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 and mass spectrometry, leading to difficulty in quantification and ambiguity in diagnosis. We have extracted organic acids from the urine of patients with 3-hydroxy-3-methylglutaric aciduria using a variety of procedures. Solvent extraction combined with hydrochloric acid/sodium chloride resulted in production of both di-TMS and tri-TMS derivatives of 3-hydroxy-3-methylglutaric acid and also mono-TMS and di-TMS derivatives of 3-hydroxyisovaleric acid. The effects were not abolished by heating. Use of sulphate-based reagents minimised artefact formation and use of DEAE–Sephadex anion exchange extraction resulted in single fully trimethylsilylated derivatives. Artefact formation during use of chloride-based reagents was abolished by pyridine added prior to trimethylsilylation. Chloride ions form adducts with hydroxyl groups in these 3-hydroxy-3-methyl carboxylic acids that prevent complete trimethylsilylation. Chloride-based reagents should be avoided in the solvent extraction of organic acids from physiological fluids or, if used, pre-treatment of the dried extract with pyridine is essential to avoid partial trimethylsilylation of 3-hydroxy-3-methyl carboxylic acids.
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