3.1 Initial GC/MS measurements and study design
GC/MS analysis of silylated aliquots of the saponified PSO batches
(section 2.3) indicated that tocochromanols contributed with
~3% to the total peak area (Fig. 2 , range c).
GC/MS chromatograms of the saponified batches did not differ from
analytically saponified PSO (data not shown). The most abundant peaks
(GC elution range 19-20 min, Fig. 2 , range b) showed GC/MS
spectra similar to squalene (five poorly separated terpenoid peaks withm/z 69 as the base peak, followed by m/z 81, m/z 95
as well as m/z 121 and m/z 137 at about the same intensity
(Hammann et al., 2019)). The shorter retention time range of 10-14 min
(Fig. 2 , range a) featured less prominent isoprenoid compounds
with GC/MS spectra similar to phytol (base peak at m/z 143
(Vetter et al., 2012)) or farnesol (base peak m/z 69, andm/z 81, m/z 93 and m/z 135 (Lee et al., 2007)).
Phytol and the other isoprenoids most likely originated from saponified
chromophores like protochlorophyll and protopheophytin (Fruhwirth and
Hermetter, 2008). In agreement with literature reports (Fruhwirth and
Hermetter, 2008), PSO mainly featured Δ7-sterols (Fig. 2 , range
d) and the pattern was dominated by α-spinasterol
(∆7,22-stigmastadienol, Fig. 2 ) which was confirmed by
M+ at m/z 484 (two db) and the lack ofm/z 129 (which indicates absence of a db on C-5 (Goad and
Akihisa, 1997)), and presence of m/z 213 and m/z 255
(characteristic for one db in the B-ring, most likely on C-7) andm/z 229 (db on C-25 of the side chain).
The tocochromanol pattern, i.e. γ-T (89.3% of total tocochromanol area)
followed by 7.3% α-T, 2.5% γ-T1 and 0.9% α-T1 (Fig. 2 , range
c), agreed well with the one reported by Butinar et al. (2011). The high
abundance of squalene and other compounds hindered the detection of
further, less abundant tocochromanols in the PSO extract. Compared to
that, Butinar et al. also detected low amounts of β-T, δ-T and γ-T3 in
PSO (Butinar et al., 2011). The GC/MS spectrum of silylated γ-T1 was
similar to the one of silylated γ-T except for the shift of the
silylated M+ to m/z 486 (γ-T1) instead ofm/z 488 (γ-T). Additionally, the allylic ion at m/z 69 was
visible and produced evidence that the db in the side chain was located
at C-11´ (Müller et al., 2020; Fiorentino et al, 2009).
On average, each 40 g batch of PSO (section 2.3) provided
~0.22 g unsaponifiable extract (~0.55%
yield). Accordingly, four batches of ~40 g PSO were
combined in order to reach the maximum sample load of the CCC system
(0.8-1.0 g sample). Based on an estimated tocochromanol contribution of
~3% to the total peak area in saponified and silylated
PSO, ~30 mg tocochromanols were expected in 1 g
saponified PSO. Assuming a contribution of 2.5% to the tocochromanols
(see above), the amount of γ-T1 was estimated at ~0.75
mg in 1 g saponified PSO extract. Hence, eleven CCC runs were carried
out in order to obtain amounts of >5 mg γ-T1.