A new study published in Analytical Chemistry has revealed that the proposed liquid chromatography−mass spectrometry (LC−MS/MS) method may simultaneously detect second-tier biomarkers for newborn screening (NBS) disorders, including Pompe disease and select fatty acid oxidation disorders.

The Newborn Screening program is a public health initiative that examines newborn babies for different health problems immediately after birth. This screening process employs multiple techniques, such as physical examinations, pulse oximetry, immunoassays, molecular assays, and mass spectrometry.

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There are existing challenges associated with the adoption of second-tier screening. First, they only screen for one or very few disorders requiring laboratories to maintain multiple assays simultaneously, the study authors noted.

Second, the reflex rate to second-tier screening for some disorders is low. These associated challenges often lead laboratories to not use the second-tier screening for such disorders, batch enough specimens before performing the assay, or outsource the second-tier screening to other/reference labs, they added.

The study developed, optimized, and analytically validated a novel hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) second-tier NBS assay. The system multiplexed a diverse set of 19 second-tier biomarkers to screen various disorders such as aminoacidopathies, organic acid disorders, fatty acid oxidation disorders, Pompe disease, and adrenoleukodystrophy.

The research team created a dried-blood spot (DBS) for method validation in the following manner. First, O+-packed red blood cells stored in CPDA-1 were washed with saline three times. After that, hematocrit was adjusted to 50% using a hormone-depleted serum.

Finally, it was freeze-lysed for at least 1 week at −20°C. Moreover, Imtakt Intrada amino acid column 150 × 2 mm, 3 μm, in association with Waters Acquity UPLC BEH C18 2.1 × 5 mm, 1.7 μm VanGuard Pre-Column was used to perform the chromatographic separation.

“To the best of our knowledge, this is the highest multiplexed second-tier NBS assay to date in terms of both metabolites and disorders screened,” the researchers highlighted.

The proposed novel method has an advantage in the sense that it can multiplex several adrenoleukodystrophy, biomarkers, primarily LPC C26:0, which, when tested using traditional (first-tier) NBS methods, often result in a high number of false positive results that require further screening. Thereby increasing the test specificity and drastically reducing false positive referrals.

“This assay will allow NBS laboratories to perform second-tier screening daily, eliminating previously encountered delays associated with low presumptive positive specimen quantities that needed to be analyzed with assays that were performed only once or twice per week,” the authors added.


Kilgore, MB, Platis, D, Lim, T, et al. Development of a universal second-tier newborn screening LC–MS/MS method for amino acids, lysophosphatidylcholines, and organic acids. Analytical Chemistry. doi:10.1021/acs.analchem.2c03098