The peroxisome is a cellular organelle that plays an important role in a number of functions, including the beta-oxidation of very long chain fatty acids (VLCFA). Peroxisomal disorders are characterized by impaired, reduced or total absence of peroxisomes in cells, which can result in the accumulation of VLCFAs, such as tetracosanoic and hexacosanoic acids, in plasma. Some variants of these disorders are characterized by the accumulation of phytanic acid.
Up to now, quantification of VLCFA has been done by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS). These methods are often time-consuming and quite demanding in terms of sample preparation. Moreover, due to the lack of appropriate standards, quantification of VLCFAs is frequently based only on the calculation of significant ratios such as C 26:0/C 22:0 and C 24:0/C 22:0.
Recently, D.W.Johnson proposed a new procedure for the rapid analysis of VLCFAs based on tandem mass spectrometry(1). This approach targets all the VLCFA forms, i.e. free forms of VLCFA as well as forms that are incorporated into phospholipids and ester forms. The method does not, however, distinguish isobaric forms, because the measurements are done in flow-injection mode.
This application note describes a method for the quantitation of VLCFAs, using a simple and robust LC/MS/MS hardware configuration. The method includes a chromatographic step and a non-isotopicallylabelled internal standard. The resulting approach has the potential of greater specificity, lower detection limits, and a broader dynamic range than previously suggested strategies, for a wide variety of fatty acids.
Sensitive and selective Multiple Reaction Monitoring (MRM) scan function for linear quantitative analysis with wide dynamic range
Patented LINAC collision cell for multi-component analysis at reduced MRM dwell times, maintaining sensitivity and preventing cross talk. This reduces significantly the occurrence of false positive results.
Rugged and reliable triple quadrupole tandem MS system for maximum uptime
Compatibility with the broadest range of ionization sources for the analysis of a wide range of compounds.
Samples were prepared according to the procedure described by Johnson(1) with some modifications. The hardware configuration included an API 3000TM LC/MS/MS System Triple Quadrupole Mass Spectrometer equipped with a TurboIonSpray source. The source operates in positive ion mode at a voltage of +5500 volts and with a turbo gas flow of 8 L/min of air heated at 300 degrees C (nominal heating-gun temperature).
MRM measurements were made using declustering potential (DP) and collision energy (CE) values as optimized automatically by the software for each of the analytes (DP value at 40 V; CE between 30 and 45 eV for the singly charged species measured here).
Chromatography was performed through an Agilent 1100 LC Binary Pump, and separation was accomplished via a Kromasil C8 5μm, 2 x 50 mm column housed in an Agilent 1100 Column Oven at 60C. 3 μL of the reconstituted sample extract were injected into the column using an Agilent 1100 Wellplate Autosampler. All hardware set-up details, solution and reagents requirements, and operating parameters were stored on CD for quick transfer of the proposed methodology.
Results and Discussion
The results of this research study are summarized in the Figures below.
As shown in Figure 1, chromatographic separation and identification of the derivatized pristanic, phytanic, docosanoic, tetracosanoic, and hexacosanoic VLCFAs, and the derivatized internal standards were obtained within 10 minutes of the chromatographic run in gradient mode.
Calibration curves were established for all the compounds in the range of 0.1 to 10 μM with good linearity. As seen in Figure 2, all analytes exhibited correlation coefficients of r = 0.9992 or better (pristanic acid, r = 0.9992; phytanic acid, r = 0.9998; all others r > 0.9999).
Figure 3 shows a typical trace obtained with the proposed protocol on an actual patient sample. Several of the VLCFAs are easily recognized (docosanoic, C 22:0; tetracosanoic, C 24:0; and hexacosanoic, C 26:0). Other signals, which appear through the same MRM transition but at different chromatographic retention times, account for isobaric forms.
The chromatographic peak appearing at 3 minutes through the transition of phytanic acid does not collimate with the peak in Figure 1 appearing at 2.4 minutes and, therefore, must be assigned to an isomer of phytanic acid (C 20:0)
. In Figure 4 the traces shown in Figure 3 are magnified, making it easier to recognize the true phytanic acid, appearing at the expected retention time (RT = 2.4 min.). An isomer of pristanic acid can also be seen, overlapping the phytanic acid peak. The true pristanic acid (indicated by arrow) appears at very low concentration, as expected in a normal sample. Since the measurements included other MRM transitions, signals from several unsaturated-VLCFAs are also evident such as C 22:1 (two adjacent isomers) and C 24:1.
Based on these results, the proposed method proved highly effective in characterizing any single VLCFA due to the chromatographic separation. In addition, the method demonstrated high sensitivity and selectivity for very low physiological concentration of VLCFAs and did not appear susceptible to contamination from external sources. (During sample preparation, detergents used for glassware washing can contaminate samples, resulting in spurious peaks. The chromatographic process used in this method helped to prevent false attributions of VLCFA.) Finally, the method presented in this study was cost-effective, as a result of performing calibration using a homologue as an internal standard rather than using isotopically-labelled standards.
1D.W. Johnson, J. Inherit. Metab. Dis.(2000); 23:475-486.
Applied Biosystems/MDS SCIEX aknowledges our collaborators, Hlne Belva-Besnet and Bruno Casetta, and Maryse Gravier, Yves Tavet, Isabelle Cuvelier and Didier Olichon from Laboratoire PASTEUR-CERBA, Cergy (France) for providing the method, samples and data for this application note.