Flow injection ionization-tandem mass spectrometry-based estimation of a panel of lysophosphatidylcholines in dried blood spots for screening of X-linked adrenoleukodystrophy

Abstract

Background: Elevated blood C26:0 lysophosphatidylcholine (LPC) is a diagnostic marker for X-linked adrenoleukodystrophy (X-ALD). Our aim was to develop a flow injection ionization-tandem mass spectrometry (FIA-MS/MS) method for estimating a panel of LPCs (C20:0-C26:0-LPCs) in dried blood spots (DBS) and to determine the sensitivity and specificity of this method for high-throughput screening for X-ALD.

Methods: LPCs (C20:0-C26:0) were extracted from 3.2 mm DBS in a 96-well plate, spiked with isotopically-labelled internal standard (C26:0-d4-LPC) and measured by FIA-MS/MS in electrospray ionization (ESI)-positive, multiple reaction monitoring (MRM) mode using a triple quadrupole, tandem mass spectrometer. The sensitivity and specificity of the FIA-MS/MS method for screening of X-ALD was determined. The FIA-MS/MS method was compared with the LC-MS/MS method for estimating LPC concentrations.

Results: Elevated C26:0 and C24:0-LPCs were 100% sensitive for identification of X-ALD. However, specificity was only 78.33% for C26:0 and 98.33% for C24:0-LPCs. Sensitivity for C22:0 and C20:0 LPCs were 89.29%, 78.33% and specificity, 67.86% and 73.33%, respectively. The FIA-MS/MS method showed good concordance with the LC-MS/MS method.

Conclusion: The FIA-MS/MS method for estimating C26:0 and C24:0-LPCs in DBS is suitable for first-tier screening of newborns for X-ALD. Second-tier confirmatory testing is required to screen positive cases.

Keywords: Dried blood spots; Flow injection ionization-tandem mass spectrometry; Lysophosphatidylcholines; Screening; X-linked adrenoleukodystrophy.