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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.

Patients on haemodialysis have reduced carnitine, acylcarnitine and butyrobetaine plasma levels, which can be increased by supplementing carnitine. Propionylcarnitine, an important constituent of the acylcarnitine pool, can be removed by haemodialysis. Removal of potentially toxic acyl-groups may represent a mechanism for a beneficial effect of carnitine in these patients.

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Background. The current study was performed to investigate the kinetics of carnitine, individual acylcarnitines and butyrobetaine in patients on haemodialysis. Methods. Eight stable long-term haemodialysis patients were studied under basal conditions (no carnitine supplementation) and 3 weeks after intravenous supplementation with L-carnitine (10 or 20 mg/kg body weight) after each haemodialysis session. The kinetic studies included serial determinations of carnitine and metabolites just before, during or between haemodialysis sessions. Analysis was performed by liquid chromatography-tandem mass spectrometry. Results. Before haemodialysis, the plasma concentrations were (mmol/l) 15.1±0.6 (mean±SEM) for carnitine, 5.9±0.7 for acetylcarnitine, 0.66±0.04 for propionylcarnitine and 0.98±0.08 for butyrobetaine (basal conditions) or 142±23 for carnitine, 69±12 for acetylcarnitine, 6.0±1.1 for propionylcarnitine and 2.6±0.3 for butyrobetaine (carnitine 20 mg/kg). During haemodialysis, the plasma concentrations dropped by $80% for all compounds determined, with extraction coefficients ranging from 0.65 to 0.86. In patients supplemented with 20 mg/kg carnitine, the amount of carnitine removed by haemodialysis equalled 42% of the dose administered, consisting of 2.08 mmol carnitine, 1.03 mmol acetylcarnitine and 0.051 mmol propionylcarnitine. Between the haemodialysis sessions, carnitine, acylcarnitines and butyrobetaine reached apparent steady-state concentrations within 1 day both under basal conditions and after supplementation. Conclusions. Patients on haemodialysis have reduced carnitine, acylcarnitine and butyrobetaine plasma levels, which can be increased by supplementing carnitine. Propionylcarnitine, an important constituent of the acylcarnitine pool, can be removed by haemodialysis. Removal of potentially toxic acyl-groups may represent a mechanism for a beneficial effect of carnitine in these patients.