Strategy for the Isolation, Derivatization, Chromatographic Separation, and Detection of Carnitine and Acylcarnitines

Abstract

A strategy for detection of carnitine and acylcarnitines is introduced. This versatile system has four components: (1) isolation by protein precipitation/desalting and cation-exchange solid-phase extraction, (2) derivatization of carnitine and acylcarnitines with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase chromatography using a single non-end-capped C8 column, and (4) detection of carnitine and acylcarnitine pentafluorophenacyl esters using an ion trap mass spectrometer. Recovery of carnitine and acylcarnitines from the isolation procedure is 77-85%. Derivatization is rapid and complete with no evidence of acylcarnitine hydrolysis. Sequential ion-exchange/reversed-phase HPLC results in separation of reagent byproducts from derivatized carnitine and acylcarnitines, followed by reversed-phase separation of carnitine and acylcarnitine pentafluorophenacyl esters. Detection by MS/MS is highly selective, with carnitine pentafluorophenacyl ester yielding a strong product ion at m/z 311 and acylcarnitine pentafluorophenacyl ester fragmentation yielding two product ions: (1) loss of m/z 59 and (2) generation of an ion at m/z 293. To demonstrate this analytical strategy, phosphate buffered serum albumin was spiked with carnitine and 15 acylcarnitines and analyzed using the described protein precipitation/desalting and cation-exchange solid-phase extraction isolation, derivatization with pentafluorophenacyl trifluoromethanesulfonate, chromatography using the sequential ion-exchange/reversed-phase chromatography HPLC system, and detection by MS and MS/MS. Successful application of this strategy to the quantification of carnitine and acetylcarnitine in rat liver is shown.

Metabolite Profiling of Bendamustine in Urine of Cancer Patients after Administration of [14C]Bendamustine

“…This quarternary ammonium compound can be endogenously formed and is required for transport of long-chain fatty acids across intracellular membranes, which is facilitated by oxidation of the fatty acids into acylcarnitines (Strijbis et al, 2010). The suggestion of Met17 being a carnitine conjugate of bendamustine is supported by the most abundant product ion at m/z 424.1624, because the loss of 59.0729 Da (loss of trimethylamine) is characteristic for carnitine and acylcarnitine conjugates (Minkler et al, 2005). Although the mass spectra give no definite answer on the site of the conjugation, it is hypothesized that Met17 may be monohydrolyzed bendamustine, conjugated with carnitine at the butyric acid moiety to form an acylcarnitine derivative.…”

Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue

“…Recovery of acyl-coenzyme A esters from tissue specimens is often disappointing, with documented recoveries between 30 and 60% [1,5,6]. A general procedure for the isolation of a wide range of acyl-coenzyme A esters, with good documented recoveries from tissues, is presently unavailable.Despite their widely different polarities, we have shown that acylcarnitines (short-, medium-, and long-chain) can be isolated, in a single fraction, from biological samples using organic solvent extraction followed by ion-exchange solid phase extraction (SPE) [7]. This is a highly selective approach, since it combines two orthogonal procedures [8] of isolation: organic solvent extraction and ion-exchange.…”

“…Despite their widely different polarities, we have shown that acylcarnitines (short-, medium-, and long-chain) can be isolated, in a single fraction, from biological samples using organic solvent extraction followed by ion-exchange solid phase extraction (SPE) [7]. This is a highly selective approach, since it combines two orthogonal procedures [8] of isolation: organic solvent extraction and ion-exchange.…”

Necrotizing Enterocolitis and Respiratory Distress Syndrome as First Clinical Presentation of Mitochondrial Trifunctional Protein Deficiency

“…NBS acylcarnitine profiling was performed as described by Chace et al (2003). Plasma acylcarnitine profiling was performed as described by Minkler et al (2005). In addition, keto-acylcarnitine profiling was performed by incubating 50 mL of plasma with 5 mL of MOX Reagent (Pierce, Rockford, IL, USA) containing 2% methoxyamineÁHCl in pyridine.…”

“…The mixture was left to stand at room temperature for 2 h to allow the formation of methoxime-derivatives of all keto-containing substances. Following this incubation, the samples were treated in the usual manner for the isolation and derivatization of acylcarnitines, which were analyzed as their butylester derivatives (Minkler et al 2005).…”