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 gl