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. 2023 Feb 14;95(6):3187-3194.
doi: 10.1021/acs.analchem.2c03098. Epub 2023 Feb 1.

Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids

Matthew B Kilgore  1 , Dimitrios Platis  2 , Timothy Lim  1 , Samantha Isenberg  1 , C Austin Pickens  1 , Carla Cuthbert  1 , Konstantinos Petritis  1
Affiliations

Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids

Matthew B Kilgore et al. Anal Chem. .

Abstract

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
HILIC method LC–MS/MS profile for labeled and unlabeled second-tier analytes of interest in high enrichment QC materials. Unlabeled analytes have gray traces and labeled analytes have black traces. Abbreviations: Percent mobile phase B (%B).
Figure 2
Figure 2
Heat map showing the effect of the extraction solvent composition on the extracted biomarker peak area fold change relative to the mean peak area of each analyte. Solvents tested include either equal percentages of MeOH/ACN/water (MeOH/ACN) or variable percentages of MeOH/water (MeOH) and ACN/water (ACN), 100/0, 90/10, 80/20, and 70/30, with or without 0.1% FA. Above 1 (mean peak area) is red, below 1 is blue, and 1 is white. The material extracted was the NSQAP QC material lot# D1814.
Figure 3
Figure 3
Heat map showing the effects of varying extraction conditions on the extracted biomarker peak area fold change relative to the mean peak area of each analyte. Conditions tested include DTT or TCEP reduction, room temperature or 45 °C extraction, variable FA at (0, 0.05, 0.1, or 0.2%), or variable oxalic acid (Oxalic acid at 1, 3, or 9 mM) using 0.1% FA. In addition, 3 mM oxalic acid with no FA was tested (*). All conditions use 80% ACN with 20% water. Above 1 is red, below 1 is blue, and 1 is white. The material extracted was the NSQAP QC material lot# D1814.
Figure 4
Figure 4
Extracted ion chromatograms of biomarkers for adrenoleukodystrophy (ALD) and maple syrup urine disease (MSUD) in a (A) normal specimen, (B) ALD-positive DBS enriched with LPC 24:0 and LPC 26:0 produced for ALD proficiency testing, and (C) MSUD-positive DBS enriched with Leu, aIle, and Ile produced for MSUD quality control.
See this image and copyright information in PMC

References

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