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Development of Flow Injection Analysis Method for the Second-Tier Estimation of Succinylacetone in Dried Blood Spot of Newborn Screening

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Abstract

Tyrosinemia type 1 (TYR1) is a devastating aminoacidopathy, leading to mortality without medical intervention. Although, detection and quantification of tyrosine in dried blood spot (DBS) is possible, but being a non-specific marker for TYR1 and its frequent association with transient neonatal tyrosinemia limits its applicability. Despite, Succinylacetone (SUAC) being a pathognomonic marker for TYR1, but not often detectable by routine newborn screening (NBS). We envisaged to determine SUAC in DBS by an in-house flow injection analysis method on a liquid chromatography/tandem mass spectrometry (LC–MS/MS). Succinylacetone was eluted from the residual 3.2 mm DBS of primary NBS by an extraction solution containing acetonitrile–water–formic acid mixture containing stable-isotope labelled internal standard (IS) for SUAC and hydrazine. Detection and quantification was performed by the mass spectrometer using multiple reaction monitoring mode at m/z 155.1 → 109.1 for SUAC and m/z 160.1 → 114.1 for the SUAC IS. The assay was linear over a calibration range of 0.122–117.434 µmol/L. The Intra-day and Inter-day precision and accuracy for the assay was determined at two different levels of SUAC (2.542 µmol/L and 14.641 µmol/L), which showed a coefficient of variation of (6.91% and 12.65%) and (8.57% and 12.27%) respectively. The accuracy also ranged between 101.2 and 103.87%.This method provided the necessary sensitivity, precision, accuracy, recovery and linearity and hence, has the potential to reduce the false positive, false negative results which significantly minimise the cost involved in the screening and follow up of TYR1 patients.

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Acknowledgements

The Authors are grateful to Professor Dr. B.K. Thelma (University of Delhi-South Campus, Delhi) and Mr. Chandrashekar (Sciex Pvt. Ltd, Gurugram, India), who has monitored my progress and offered me with invaluable learning opportunities. My acknowledgement would not be complete without thanking my team mates whose small and big contributions and helpful scientific discussion was the driving force for this study.

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Authors and Affiliations

  1. Department of Paediatrics, Paediatrics Research and Genetic Lab, Maulana Azad Medical College (University of Delhi) and Associated Lok Nayak Hospital, New Delhi, 110002, India

    Bijo Varughese, Sunil Kumar Polipalli & Seema Kapoor

  2. Perkin Elmer Pvt. Ltd, Mumbai, Maharashtra, India

    Dnyanoba Madrewar

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  1. Bijo Varughese
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  2. Dnyanoba Madrewar
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Correspondence to Bijo Varughese.

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Ethical Approval

Ethical clearance approval obtained before the start of the study from institutional ethics committee, MAMC (F. No./11/IEC/MAMC/2011/317/2015).

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Informed consents are not applicable. Residual samples after completion of the requested laboratory analysis were anonymized and used for the study. Study results were reported to sample anonymizer who conveyed the reports to the treating clinician.

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Varughese, B., Madrewar, D., Polipalli, S.K. et al. Development of Flow Injection Analysis Method for the Second-Tier Estimation of Succinylacetone in Dried Blood Spot of Newborn Screening. Ind J Clin Biochem 37, 40–50 (2022). https://doi.org/10.1007/s12291-020-00944-z

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