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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. [ABSTRACT FROM AUTHOR]

• Validated UPLC-ESI-MS/MS method for quantification of B 6 vitamers in human CSF. • New simple protein precipitation using acetonitrile and minimal CSF amount of 50 µL. • Applicable for diagnostic analysis of pyridoxal 5́-phosphate and pyridoxal. • >150 CSF samples for reference range determination. Vitamin B 6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B 6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B 6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories. Until today, vitamin B 6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B 6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B 6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory. [ABSTRACT FROM AUTHOR]