Dried Urine Spots for Quantification of C0 (Free) Carnitine Excretion: A Validated Method for Follow-Up and Confirmatory Testing of Primary Carnitine Deficiency

Abstract

Primary carnitine deficiency (PCD), caused by Organic Cation Transporter Novel 2 (OCTN2) deficiency, is detected by newborn screening (NBS) through low blood free carnitine (C0) concentrations. Confirmation of the diagnosis may be supported by demonstrating increased urinary C0 excretion, but urine collection in newborns is technically challenging. We developed and validated a dried urine spot (DUS) method for quantifying urinary C0 excretion as a practical, non-invasive alternative to conventional liquid urine sampling. This approach has the potential to allow parents to obtain urine at home by placing filter paper cards between the baby’s skin and a dry diaper, simplifying sample collection. C0 carnitine and creatinine were quantified in 6 mm DUS punches by flow injection analysis–tandem mass spectrometry (FIA–MS/MS) following butylation. The method was analytically validated for sensitivity, precision, stability, and agreement with liquid urine measurements. The method demonstrated excellent analytical performance, with lower limits of detection (LLOD)/lower limits of quantification (LLOQ) values of 0.02/0.07 µmol/L for C0 and 0.01/0.04 mmol/L for creatinine. Normalization of C0 to creatinine reduced variability related to sampling and punch location. Both analytes remained stable in DUS for at least 10 days at room temperature and after 11 freeze–thaw cycles. C0/creatinine obtained from DUS showed excellent agreement with liquid urine (R2 > 0.9967), although concentrations were consistently lower, requiring a correction factor of 1.27 to estimate concentrations equivalent to liquid urine. DUS provides a reliable and accurate alternative to liquid urine for measuring C0 excretion in newborns. This simple, home-based sampling approach facilitates non-invasive urine collection and has the potential to support follow-up and confirmatory testing of PCD.

The authors' abstract, as published at the source. International Journal of Neonatal Screening, 2026 · DOI ↗