Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy

Background: Pre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C₂₀-C₂₆ lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening. Methods: LPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA-MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C₂₀, C₂₂, C₂₄, C₂₆) and the internal standard (d₄-C₂₆-LPC). Analysis time is 1.5min per sample. Results: The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C₂₀-LPC, 4.4-18.6% for C₂₂-LPC and 4.5-14.3% for C₂₄-LPC. Limits of detection were determined for C₂₀-LPC (LOD=0.03μg/mL), C₂₂-LPC (0.03μg/mL), C₂₄-LPC (0.03μg/mL) and C₂₆-LPC (0.01μg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified. Conclusion: Analysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC-MS/MS.