Quantitative acylcarnitine determination by UHPLC-MS/MS — Going beyond tandem MS acylcarnitine “profiles”

Abstract

Tandem MS “profiling” of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine “profiling” also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or β-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay’s ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS “profiles”. We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

Acylcarnitines as markers of exercise‐associated fuel partitioning, xenometabolism, and potential signals to muscle afferent neurons

“…The details of the HPLC‐MS acylcarnitine metabolite detection and analysis are presented elsewhere (Minkler et al . a , b ). Briefly, to 10 μl of sample (plus internal standards) was added organic solvents to precipitate salts and proteins.…”

Succinyl-CoA synthetase ( SUCLA2 ) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion

“…SM samples were frozen in liquid nitrogen, shipped on dry ice to CIDEM, and stored at −80°C until analysis. Samples from both siblings were processed and acylcarnitines analyzed using UHPLC-MS/MS as previously described [13, 14]. …”

Mitochondrial Metabolism in Aging Heart

“…In fact, rates of replacement and half-lives of the two populations in vivo is a key question relevant to the aged heart, and can be addressed by stable isotope approaches and the analysis of mitochondrial proteins and phospholipids. 204 It is critical to understand the activation of mitochondrial removal mechanisms in vivo in the aged wild-type heart, both at baseline and in disease states. Transgenic models provide key insights into mechanisms of removal, but may be less informative in identifying the pathway of activation for removal of dysfunctional mitochondria in aging with or without superimposed disease.…”