Acylcarnitines detecting

This in fact is what happened. Tandem MS has clearly been shown to be the only technology to screen for disorders of fatty acid oxidation and could also detect many disorders of organic acid metabolism. Tandem MS has the ability to detect both compound classes (amino acids and acylcarnitines) and after demonstrating that both classes could be prepared in the same manner, the MS/MS analysis of blood spots for newborn screening applications was born. 

A simple and efficient alternative to the traditional UV detection of amino acids and related compounds is nowadays represented by the evaporative light scattering (ELS) detector, which allows the direct chromatographic separation, with no need for preliminary derivatization. In the field of glycopeptides-based CSPs, it was applied for the first time in the chromatographic resolution of carnitine and 0-acylcarnitine enantiomers on a TE CSP [61]. The considered compounds are nonvolatile solids and gave optimal ELS response under a variety of experimental conditions (buffered and unbuffered mobile phases, flow-rates from 0.5 to 1.5 mL/min, different kind and... 

Calculation of the molecular weights of the -butyl esters of acylcarnitines, expressed as (M+H)+ molecular ions, allows the designation of each peak (Table 3.2.1). Quantitation software provided by the instrument vendor (i.e., Chemoview for SCIEX instruments) allows calculation of quantitative and semiquantitative concentrations for these acylcarnitines by comparison of the detected abundance of each acylcarnitine versus that of a designated internal standard with a known concentration. The results for each sample are further compared to age-appropriate reference ranges (Table 3.2.2). 

Formiminoglutamate (FIGLU), a marker for glutamate formimino-transferase deficiency, was recently also shown to be detectable by acylcarnitine analysis represented as a peak with m/z 287 (Fig. 3.2.3d) [64]. In poorly resolved acylcarnitine profiles, this peak may be confused with iso-/butyrylcarnitine (m/z 288). To avoid the incorrect interpretation of acylcarnitine profiles, we recommend performing the analysis in product scan mode as opposed to multiple reaction monitoring (MRM) mode. For example, the FIGLU peak at m/z 287 would not have been correctly identified in MRM mode because the transition of 287 to 85 is typically not selected. However, the 288/85 transition would reveal abnormal results, but in fact not represent either butyryl- or isobutyrylcarnitine, but another FIGLU related ion species. 

Schmidt-Sommerfeld E, Penn D, Duran M, et al (1992) Detection and quantitation of acylcarnitines in plasma and blood spots from patients with inborn errors of fatty acid oxidation. Prog Clin Biol Res 375 355-362... 

Gibson KM, Bennett MJ, Naylor EW, Morton DH (1998) 3-Methylcrotonyl-coenzyme a carboxylase deficiency in amish/mennonite adults identified by detection of increased acylcarnitines in blood spots of their children. J Pediatr 132 519-523... 

This extract was derivatized to make butyl esters of the acylcarnitines and analyzed using precursors of a 85-Da scan. The common fragment of acylcarnitine butyl esters is an ion at m/z 85 and all acylcarnitines share this common product. The spectra show the precursor ions or molecular ions that were being scanned at the time, detected at m/z 85 for both the internal standards and endogenous acylcarnitines. To the left, or lower mass values, the metabolites of interest are labeled as, for example, , C8, C16. Those acylcarnitines that are key to the detection of MCAD deficiency are underlined. 

Diagnosis of mitochondrial myopathies is initially clinical, involving phenotypic (observable expression of characters and traits) evaluation, followed by laboratory evaluation. If an mtDNA mutation is detected, diagnosis is relatively straightforward in the absence of an mtDNA mutation, diagnosis becomes difficult. Laboratory studies may include blood plasma or cerebral spinal fluid measurement for lactic acid, ketone bodies, plasma acylcarnitines and organic acids in the urine. If they are abnormal, a muscle biopsy is performed. 

Figure 55-3 shows a flow chart for the postmortem evaluation of sudden and unexpected death that is centered on the analysis of acylcarnitines in blood and bile spots. Blood and bile could be conveniently collected on a single filter paper card, one identical to those used for newborn screening, which can be shipped at room temperature once properly dried. Both specimens should be collected to detect patients who may have only mild blood abnormalities when carnitine is depleted (Figure 55-4). In cases with a higher level of suspicion, an effort should be made to collect and freeze a specimen of liver and a skin biopsy, which could be analyzed only if needed to reduce cell culture workload. ...

Detection of C6-C10 acylcarnitines in neonatal blood spots by MS/MS is the basis for newborn screening of MCAD deficiency. Identification of numerous patients by newborn screening has led also to the discovery of patients... 

A free-standing liquid junction interface was coupled to a flat edge glass CE microchip for the analysis of small molecules (drugs, metabolites). It was demonstrated for the detection of recovered carnitine, acylcarnitines, imipramine, and desipramine spiked into urine or plasma at 5-500 p,g/mL level. Separations were typically performed in < 1 min and intra-assay precisions ranged from 4.1% to 7.3% Relative Standard Deviation RSD. A similar device, but fabricated in polymeric Zeonor material, was demonstrated for the analysis of carnitine standards. ... 

When palmitate is used as substrate the addition of equimolar amounts of DTA stimulates the oxidation 3 fold (Fig. 2A). With the shorter laurate as substrate (Fig. 2B) the effect decreases and is no longer detectable with octanoate (Fig. 2C). This pattern of responsiveness suggests that 3-thia fatty acids affect the mitochondrial CPT system, since oxidation of long fatty acids, in contrast to short ones, is dependent on the transport of acylcarnitines across the mitochondrial inner membrane. 

Analysis of carnitine and acylcarnitines by MS/MS is now considered routine in those laboratories that possess the appropriate technology. The ionization techniques commonly used with MS/MS are fast atom (or fast ion) bombardment and electrospray. Both are sufficiently sensitive to detect abnormally elevated concentrations of specific metabolites in all types of specimen, although the latter is much more widespread and is the more sensitive of the two, especially for long-chain acylcarnitines. The method is quantitative or at least semi-quantitative for most analytes, and uses stable isotope-labeled forms of the analytes as internal standards. The acylcarnitines are analyzed simultaneously in the positive ion mode as their methyl or butyl esters using a precursor ion scan function [14, 23] that detects the parent (molecular) ions. Free carnitine and total carnitine are determined by assaying the same specimen before and after alkaline hydrolysis, without derivatization, using Hs-carnitine as internal standard [17]. The value for acylcarnitine is determined by difference. This value includes the contribution of short, medium and long-chain acylcarnitines. Analysis time for each method is approximately 2 min. 

---