(LiposamTM), respectively from the interesterification between tricaprylin and
EPAEE after a 12-h reaction time. A solvent gradient program was applied (for
details see Han et al., 1999a) which allowed the separation of TAG containing
EPA. This depends on the number of EPA molecules incorporated in the glycerol
backbone and their isomeric distribution. The molecular weight of each peak
was determined by HPLC followed by (atmospheric-pressure chemical ioniza-
tion)-mass spectroscopy (APCI/MS) assay (as shown in Table 2), together with their
peak number. The compounds which were eluted as peaks 3 and 4 had the same
molecular mass; thereby the two compounds were isomers of TAG-A. This identi-
fication was further confirmed by the observation of fragment ions analyzed by
HPLC-APCI/MS, as shown in Figure 1. The identification of peaks 3 and 4 as
TAG-A3 and TAG-A1 (and/or TAG-A2) (see Table 2), respectively, was made
on the regioselectivities of the enzymes used. Only peak 4 appeared in the reaction
using 1,3–specific lipase, whereas peaks 3 and 4 were observed utilizing the non-
specific lipase. Thus it was concluded that peak 4 corresponds to TAG-A (TAG-A1
and/or TAG-A2) containing EPA atsn-1 (or 3)-position, and peak 3 shows thesn-2
positional isomer (TAG-A3).
The gradient of hexane/2-propanol/acetonitrile as mobile phase was also success-
fully applied to monitor the transesterification reaction between docosahexaenoic
acid ethyl ester (DHAEE) and tricaprylin [Equation (2)] (as shown in Figure 2),
although the acetonitrile content of solvent A and the mobile phase gradient pro-
gram were slightly different (Iwasaki et al., 1999).
DHAEE + C 8 C 8 C 8 R(DHA)C 8 C 8 +C 8 EE (2)
Again, only peak 4 appeared with 1,3-specific lipase and both peaks 3 and 4 were
obtained with non-specific lipase. Each peak identification is shown in Table 3.
The following points should be noted with regard to the silver ion HPLC for
Equations (1) and (2):
* Separation of positional isomers is critically affected by the acetonitrile content of
solvent A so that it must be optimized for each pair of sTAG.
* One isomer with an unsaturated FA atsn-2 position elutes faster than the other
with unsaturated FA atsn-1 orsn-3 position.
* (DHA)C 8 C 8 elutes later than (EPA)C 8 C 8.
* The stereoisomers, TAG-A1 and TAG-A2 (see Table 2), and (DHA)C 8 C 8 and
C 8 C 8 (DHA) (Table 2) cannot be separated. The positional isomers, TAG-B (Ta-
ble 1), and [(DHA)C 8 (DHA), (DHA)(DHA)C 8 and C 8 (DHA)(DHA)] (Table 3),
cannot be separated, either.
Silver-ion HPLC was proven to be useful in the analysis of the TAG positional com-
position of more complicated mixtures. The lipase-catalyzed acidolysis of a single
cell oil (SCO) of high DHA (and docosapentaenoic acid, DPA) content with caprylic
acid (C 8 ) was also investigated (Iwasaki et al., 1999) according to Equation (3).
SCO + C 8 RTAG containing one DHA (and DPA) and two C 8 + Others (3)
154 9 Lipase-Catalyzed Synthesis of Structured Triacylglycerols
