Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)­HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes

Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry1

An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)2(C18:1)2 versus (C18:2)3(C18:0)1. These compounds were then analyzed by HPLC-MS3 to examine specific diac ylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined

Probing peroxisomal β-oxidation and the labelling of acetyl-CoA proxies with [1-(13)C]octanoate and [3-(13)C]octanoate in the perfused rat liver

We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal β-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the (13)C-labelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio <1.0) or in both mitochondria and peroxisomes (ratio >1.0). The goals of the present study were to test, in rat livers perfused with [1-(13)C]octanoate or [3-(13)C]octanoate, (i) whether peroxisomal β-oxidation contributes acetyl groups for malonyl-CoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, β-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal β-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1+2 of β-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle