“…They measured the enzymatic activity of GALC by quantitatively analyzing the enzymatic product using an internal standard with nearly identical ESI‐MS/MS ionization efficiencies (Li, Brockmann, et al, 2004). Several “multiplex” MS/MS assays for common LSD enzymes (Cho et al, 2016; Elliott et al, 2016; Supriya et al, 2020) were later developed using enzyme‐specific artificial substrate, as well as additional internal standards to quantify the reaction product by selected reaction monitoring (SRM) in tandem MS; 5‐plex LSD's (Li, Scott, et al, 2004), 6‐plex LSD's (Ribas et al, 2017), 8‐plex LSD's (Chien et al, 2020) and the most recent 18‐plex LSD's screening (Hong et al, 2020). The challenges of applying tandem MS/MS procedures for the detection of LSDs were related to (i) the availability of synthetic enzymatic substrates, which were initially taken from those used in fluorimetry; (ii) the need to remove contaminants and reduce the complexity of the blood sample before injection into the mass spectrometer by using conventional solid and/or liquid‐phase chromatography or ultra‐performance liquid chromatography (UPLC); and (iii) Developing screening algorithms to determine specific enzymatic cut‐off values that serve as criteria for determining whether to proceed with second‐tier measurements or to refer the patient for alternative diagnostic evaluations, such as genetic testing and/or comprehensive pediatric physical examinations immediately (Burlina et al, 2019; Kemper et al, 2017).…”