Stable isotope dilution analysis of n‐hexanoylglycine, 3‐phenylpropionylglycine and suberylglycine in human urine using chemical ionization gas chromatography/mass spectrometry selected ion monitoring

We describe a gas chromatographic/mass spectrometric method for the accurate determination of n‐hexanoylglycine, 3‐phenylpropionylglycine and suberylglycine in urine for the diagnosis of hereditary medium‐chain acyl‐CoA dehydrogenase (MCAD) deficiency. These acylglycines had previously been detected in urine from patients with MCAD deficiency, but their diagnostic values were unknown because of a lack of appropriate analytical methods. n‐Hexanoyl(1,2‐¹³C)glycine, 3‐phenylpropionyl(2‐¹³C, ¹⁵N)glycine and suberyl(2‐¹³C, ¹⁵N)glycine were synthesized and used as internal standards. Ammonia chemical ionization was utilized to generate intense [M + H]⁺ ions for selected‐ion monitoring quantification. The whole procedure is fast and can be performed by a low‐resolution gas chromatographic/mass spectrometric system, giving accurate results over a range of three orders of magnitude (0.0167–16.7 μg/ml). The results from the analyses of 54 urine samples from 21 MCAD‐deficient patients and various control samples using this method established that n‐hexanoyglycine and 3‐phenylpropionylglycine were highly diagnostic for this disease, while suberylglycine was found less specific.