We have developed a method to quantitate S2CP-cysteamine in urine using liquid chromatography with tandem mass spectrometry in positive ion mode using a synthesised deuterated analogue, d5-S2CP-cysteamine, as an internal standard. Chromatographic separation was achieved using a Discovery HS F5 column (5 cm x 2.1 mm, 5 μm) with a mobile phase consisting of 4 mM ammonium acetate, 4 mM heptafluorobutyric acid buffer (pH 3.25) and acetonitrile. For S2CP-cysteamine and d5-s2CP-cysteamine parent ions of mass 164 and 169 were detected respectively. Quantitation was carried out using fragmented daughter ions 119 for our analyte and 152 for the deuterated internal standard. The method was validated in spiked urine over the linearity range of 0.2-100 μmol/L. Elevated urinary levels of S2CP-cysteamine were demonstrated in two patients initially picked up because of increased hydroxy-C4-carnitine then diagnosed by enzymology and HIBCH gene analysis. The method may prove useful in monitoring treatment e.g. by valine restriction. Beta-ketothiolase deficiency is an inborn error of isoleucine and ketone body metabolism, caused by mutations in mitochondrial acetoacetyl-CoA thiolase gene (ACAT1). We have so far analyzed gene mutations in more than 80 ACAT1 deficient patients. We previously identified a homozygous deletion including exons 2-4 in one patient and a homozygous duplication including exons 8 and 9 in another patient. However, such gene alterations are harder to identify in the heterozygous state than in the homozygous state through cDNA analysis. Since there are patients in whom one mutation cannot be detected by our routine genomic and cDNA analyses, we hypothesized that they would have heterozygous intragenic deletion or insertion. Hence, we established an MLPA method to detect copy numbers in each exon in the ACAT1 gene and, mixing patients' and control cDNA samples, successfully detected a heterozygous deletion including exons 2- Meanwhile organic acid profile showed an increased peak of mevalonic acid (13616 μmol/mmol Creat) and the diagnosis of mevalonic aciduria was confirmed by enzymatic and molecular studies. The initial prednisolone course had favourable response, but not sustained in subsequent cycles. The evolution has been marked by failure to thrive, needing parenteral nutrition, bicytopenia, with regular transfusions and reactivation of CMV infection with interstitial pneumonia that impose non invasive ventilatory support until the death at the fourth month of life. Discussion: The manifestations of mevalonic aciduria are similar to congenital CMV infection and differential diagnosis is often an issue. In this patient, both disorders were confirmed with problematic therapeutic options and a poor prognosis. Background: Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an autosomal recessive disorder biochemically characterized by accumulation and predominant high urinary excretion of 3methylcrotonylglycine . Affected patients usually have neurologic dysfunction and brain abnormalities, whose pathogenesis is practically unknown.