MSACL 2019 EU Abstract

Method Development of Amino Acid Analysis in a Dried Blood Spot for the Second-Tier Test in the Newborn Screening Program

Zahra Talebpour (1), Maryamossadat Mousavi (1), Farideh Haghighi (1,2)
(1) Department of Chemistry, Faculty of Physics and Chemistry, Alzahra University, Vanak, Tehran, 19938-93973, Iran. (2) BioMEMS and Bioinspired Microfluidic Laboratory, Department of Mechanical and Manufacturing Engineering, University of Calgary, Alberta, Canada

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Zahra Talebpour (Presenter) Department of chemistry, Alzahra University >> POSTER (PDF)

Presenter Bio: Zahra Talebpour received her B.Sc. degree in Applied Chemistry
and M. Sc. degree in Analytical Chemistry from the Sharif University of Technology, Tehran, Iran and Ph.D. degree in Analytical Chemistry from the Tarbiat Moddares University, Tehran, Iran. Her current research interests include miniaturized sample preparation techniques, inborn errors of metabolism, and chemometrics.

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Alzahra University Conflict mitigation NOT REQUIRED.

Introduction: Metabolic disorders are caused by the accumulation of metabolites in the body which lead to irreversible physiological effects. The first-tier screening procedure for these disorders is typically performed on dried blood spot (DBS) samples by the MS/MS system. When a newborn screen is found to be positive, second-tier tests using plasma samples are provided to reduce false-positive and false-negative results. To prevent resampling and provide comfort for newborns, it is more convenient to use the same DBS sample taken in the first stage of screening. Objective: The objective of this study was development of a method based on the DBS instead of plasma analyses for each aminoacidemia disorder that could potentially use as second-tier tests. Methods: To optimize the extraction conditions of 19 naturally occurring amino acids from DBS, the central composite design and response surface methodology were used to demonstrate the influence of effective factors on the responses. Also, four different types of validation were compared, and the best validation method was selected.
Results: The best conditions for extraction of each amino acid related to the PKU and MSUD biomarkers were selected based on the response surface equations. Then the effect of the actual sample matrix and extraction efficiency on the responses have been measured based on the slop ratio of four different calibration curves. By comparison of their slop ratios, matrix free calibration method has been selected to continue the study. The intra- and inter-day precisions of all amino acids were below 11%. Mean recoveries from spiked DBS samples were among 78% and 111%. The lower limit of detection and lower limit of quantification for all amino acids were obtained between 0.02-0.10 and 0.06-0.33 µmol/L respectively. The biomarkers of PKU and MSUD were quantified among 148 participants. Finally, the results of HPLC-PDA with MS/MS as NBS standard protocol has compared and a good correlation between the results has been observed.
Conclusion: The study results provide evidence that the optimized methods are reliable not only as second-tier tests but also as affected children’s follow-up methods.