Determination of carnitine and acylcarnitines in plasma by high‐performance liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

Abstract

A high-performance liquid chromatography/mass spectrometry method was developed for the determination of carnitine, its biosynthetic precursor butyrobetaine, and eight acylcarnitines in plasma. The procedure includes a solid-phase extraction for carnitine and short- and medium-chain acylcarnitines, and a liquid-liquid extraction for protein-bound long-chain acylcarnitines, followed by separation on a reversed-phase column in the presence of a volatile ion-pairing reagent. Detection was achieved using an ion-trap mass spectrometer run in the tandem mass spectrometry (MS/MS) mode. The choice of the matrix for calibrators, used for quantification of these endogenous compounds, was also investigated. Validation was performed for standard quality controls diluted with 4% bovine serum albumin solution and for spiked plasma quality control samples at concentrations between 0.5 and 80 micromol/L, depending on the compound. Intra- and inter-day precisions for the determination of carnitine were below 3.4% and accuracies were between 95.2 and 109.0%. Application of the method to the diagnosis of pathological acylcarnitine profiles of metabolic disorders in a patient suffering from methylmalonic aciduria is presented. The method allows quantification of carnitine, butyrobetaine, acetylcarnitine and propionylcarnitine, and semiquantitative analysis of medium- and long-chain acylcarnitines. In contrast with other methods, no derivatization step is needed.

Reversal of muscle insulin resistance by weight reduction in young, lean, insulin-resistant offspring of parents with type 2 diabetes

“…Plasma concentrations of IL-6 and TNF-α were measured using Quantine High Sensitivity kits (R&D Systems) and total adiponectin concentrations were measured using a double-antibody RIA kit (Linco). Plasma concentrations of acylcarnitines were measured by LC-MS/ MS, as previously described (47). Plasma amino acid concentrations were measured as the trifluoro-acetamide n-butyl ester derivatives (1), using GC-MS (HP 5973MSD; Hewlett-Packard).…”

In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes

“…In a subsequent purifi cation step, cation exchange solid-phase extraction using silica gel was applied; this has previously been shown to be effective to suffi ciently isolate acylcarnitines from urine and plasma (29)(30)(31)(32). The relatively apolar ratio of 6.5 parts acetonitrile-methanol (4:1, v/v) to 1 part sample volume resulted in suffi cient retention (96.0% to 99.5%) of acylcarnitines to the silica-gel columns.…”

L-Carnitine Treatment in Incident Hemodialysis Patients

“…Randomized, controlled studies and a meta-analysis suggested that L-carnitine supplementation might have a positive effect on response to EPO in longterm hemodialysis patients (17)(18)(19)(20)(21). Each of these studies included around 30 patients or fewer, highly selected for probable rHuEPO resistance secondary to carnitine deficiency, defined by a free carnitine level ,30 mmol/L (or total ,40 mmol/L) (22). These results led to the recommendation of L-carnitine treatment in hemodialysis patients for symptoms potentially related to carnitine deficiency, such as hyporesponsive rHuEPO-dependent anemia (23).…”