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 C-20-C-26 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 (C-20, C-22, C-24, C-26) and the internal standard (d(4)-C-26-LPC). Analysis time is 1.5 min per sample. Results: The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C20-LPC, 4.418.6% for C-22-LPC and 4.5-143% for C-24-LPC. Limits of detection were determined for C-20-LPC CLOD = 0.03 mu g/mL), C-22-LPC (0.03 mu g/mL), C-24-LPC (0.03 mu g/mL) and C-26-LPC (0.01 mu g/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. (C) 2014 Elsevier Inc. All rights reserved.