Finally, in the most recent note on this topic, Chen et al.
3 argued that OAHFAs, originally described in our recent papers
12,13 and ARVO presentations (Butovich IA, et al.
IOVS 2009;50:ARVO E-Abstract 2545; Butovich IA.
IOVS 2010;51:ARVO E-Abstract 4154; Wojtowicz JC, et al.
IOVS 2010;51:ARVO E-Abstract 4160; Eule JC, et al.
IOVS 2010;51:ARVO E-Abstract 4159), could be the in situ-generated fragments of other lipids (e.g., OAHFA-St). In fact, our previous
12 and current (
Fig. 1) HPLC-MS experiments clearly demonstrated that this hypothesis is not true. It seems that Chen et al.
3 overlooked the fact that the retention times of free OAHFA reported in our papers
12 were much shorter than those of even the shortest CE found in meibum.
2,7,11 Thus, OAHFA-St could not be the MS precursors of these much more polar lipids. Importantly, OAHFA-St, whose existence was initially proposed by Nicolaides et al.,
4 were recently evaluated by HPLC-MS in human, gorilla, and canine meibum (Butovich IA.
IOVS 2010;51:ARVO E-Abstract 4154; Wojtowicz JC et al.
IOVS 2010;51:ARVO E-Abstract 4160; Eule JC et al.
IOVS 2010;51:ARVO E-Abstract 4159)
2 in our experiments and in the parallel SG-MS experiments by Chen et al.
1,3 In concordance with our earlier reports, in our current HPLC-MS experiments, the pool of free OAHFAs (
Fig. 1A, group 1) was spatially separated from the pool of related, but more hydrophobic compounds, most likely esterified (e)-OAHFA (
Fig. 1A, group 2 of HPLC peaks). Anions of OAHFA were detected and monitored in negative ion mode MS experiments (NIM) as (M–H)
− species (
Fig. 1B). Note that the MS spectra of both pools of OAHFA are very similar. Fragmentation patterns of the OAHFA ions found in peak groups 1 and 2 were demonstrated to be similar.