Background and aimsIncreased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.MethodsThe assay was developed using liquid chromatography–tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.ResultsReliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.ConclusionsA liquid chromatography–tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (‟Newborn screening 2020”; Newborn Screening Center, University Hospital Heidelberg).

Background and aims Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine. Methods The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders. Results Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens. Conclusions A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.

Background Laboratory investigations of cerebrospinal fluid (CSF) are essential when suspecting an inborn error of metabolism (IEM) involving neurological features. Available tests are currently performed on different analytical platforms, requiring a large sample volume and long turnaround time, which often delays timely diagnosis. Therefore, it would be preferable to have an “one‐instrument” targeted multi‐metabolite approach. Method A liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) platform, based on two different methods for analysing 38 metabolites using positive and negative electrospray ionisation modes, was established. To allow for platform extension, both methods were designed to use the same CSF sample preparation procedure and to be run on the same separation column (ACE C18‐PFP). Results Assessment of the LC‐MS/MS platform methods was first made by analytical validation, followed by the establishment of literature‐based CSF cut‐off values and reference ranges, and by the measurement of available samples obtained from patients with confirmed diagnoses of aromatic l‐amino acid decarboxylase deficiency, guanidinoacetate methyltransferase deficiency, ornithine aminotransferase deficiency, cerebral folate deficiency and methylenetetrahydrofolate reductase deficiency. Conclusion An extendable targeted LC‐MS/MS platform was developed for the analysis of multiple metabolites in CSF, thereby distinguishing samples from patients with IEM from non‐IEM samples. Reference concentrations for several biomarkers in CSF are provided for the first time. By measurement on a single analytical platform, less sample volume is required (200 μL), diagnostic results are obtained faster, and preanalytical issues are reduced. Synopsis LC‐MS/MS platform for CSF analysis consisting of two differentially designed methods.