Development of an On -Line LC-GC Method

On-Line
for Rapid Analysis of γ--Oryzanol
Oryzanol in Rice Lipids
A. Miller, Th. Frenzel, H.-G. Schmarr, K.-H. Engel
Lehrstuhl für Allgemeine Lebensmitteltechnologie, Technische Universität München,
Am Forum 2, D-85350 Freising-Weihenstephan, Germany

Introduction On-line LC-GC analysis of γ-oryzanol in rice

γ-Oryzanol was first isolated from Rice lipids were extracted from ground rough rice using n-hexane/iso-
rice bran oil in the early 1950s [1] propanol (1:1; v/v) under sonication. After evaporation of the solvent and
and was revealed to be a mixture redilution in n-hexane, rice lipids were subjected to on-line LC-GC. The fact
of phytosteryl ferulates com- O that GC analysis did not require a derivatisation of the phenolic group of the
prising cycloartenyl ferulate, 24- CH3O
O
ferulates allowed the isolation of γ-oryzanol from rice lipids (e.g. triglycerides)
methylenecycloartanyl ferulate by normal-phase liquid chromatography (Fig. 3A). γ-Oryzanol was detected at
HO
and campesteryl ferulate (Fig. 1) the specific wavelength of 290nm whereas other lipids were monitored at
as major components [2]. Fig. 1. Campesteryl ferulate 200nm. Separation of the on-line transferred γ-oryzanol into its major
constituents campesteryl ferulate, β-sitosteryl ferulate, cycloartenyl ferulate
In addition to its technological usefulness, e.g. stabilization of vegetable oils and 24-methylenecycloartanyl ferulate was achieved by capillary GC analysis
at frying temperature, physiological properties, such as superoxide dismutase- (Fig. 3B). Identification of the ferulates was based on the data obtained from a
like antioxidative activity and hypocholesterolemic effects have been reported commercial γ-oryzanol preparation by (i) LC-ESI-MS investigation and (ii)
for γ-oryzanol [3-5]. Depending on the compositions of the γ-oryzanol separation of the mixture by semipreparative RP-HPLC, transesterification of
preparations, different effects in hyperlipidemic rats were observed [6]. The the esters and subsequent GC/MS analysis of the liberated sterol moieties.
three major components of γ-oryzanol were shown to differ significantly in
their antioxidative activities against cholesterol oxidation [7]. These studies
demonstrate the need for appropriate analytical methods to differentiate the A
individual components of γ-oryzanol in rice and rice bran oil. triglycerides γ-oryzanol
Previously reported analytical approaches for separation of individual
constituents by RP-HPLC as well as by capillary GC were time-consuming
because of additional purification by low-pressure column chromatography
followed by preparative HPLC [2] or liquid-liquid fractionation followed by
preparative TLC [8]. The objective of this study was to develop a method for
rapid analysis of γ-oryzanol in rice lipids by on-line coupling liquid
chromatographic preseparation with capillary gas chromatography (on-line
LC-GC).
1

On-line LC-GC instrumentation

B (mV) 16
4
waste solvent 3
vapour exit FID
injection
12.8

UV 9.6
detector
uncoated coated analytical 6.4 1
pump LC column pre-column pre-column column
2
hydrogen
3.2
HPLC loop-type interface GC
0
0 7.2 14.4 21.6 28.8 36.0
Fig. 2. On-line LC-GC instrumentation (min)

Fig. 3. Analysis of γ-oryzanol in a rough rice sample by on-line LC-GC

On-line LC-GC was performed with a commercial instrument (Dualchrom
Separation on normal-phase LC with indication of the transfer window
3000, Thermo Finnigan). γ-Oryzanol was separated from other rice lipids by
containing γ-oryzanol; UV detection at 200nm (blue) and 290nm (red) (3A).
liquid chromatography on a Eurospher Si 100-5 (25cm x 2mm i.d., Knauer)
column using n-hexane with 5% tert-butyl methyl ether and 0.5% iso-propanol GC chromatogram after on-line transfer; (1) campesteryl ferulate, (2) β-
as mobile phase. The eluent was monitored by UV detection at 200nm and sitosteryl ferulate, (3) cycloartenyl ferulate, (4) 24-methylenecycloartanyl
290nm, respectively. γ-Oryzanol was transferred on-line from LC to GC via a ferulate (3B)
loop-type interface (Fig. 2). The valve was switched when the γ-oryzanol
containing fraction was in the loop, so that γ-oryzanol was transferred to GC
The described approach drastically reduces the time required for analysis of
by the carrier gas. Concurrent eluent evaporation was used as the transfer
γ-oryzanol in complex matrices. The GC separation of the transferred LC-
technique, since the components of γ-oryzanol exhibit only low volatility [9,
fraction is not yet optimised. Ongoing work focuses on further improvement in
10]. GC separation was performed on a trifluoropropylmethyl polysiloxane
order to be able to detect additional minor constituents of γ-oryzanol [2].
column (Rtx-200, 27m x 0.25mm i.d., 0.10µm film thickness) connected in 3
series with an uncoated phenylsilylated fused silica capillary (3m x 0.53mm
i.d.) and a coated pre-column (3m x 0.25mm i.d.) having the same coating as
the analytical column. Solvent vapour was released during transfer by an Summary
early solvent vapour exit, which was installed between the coated pre-column
and the separation column and switched to a restrictor leaving a small purge The developed on-line LC-GC method provides a rapid and effective isolation
flow during analysis after transfer. Hydrogen was used as carrier gas with an of γ-oryzanol from rice lipids by means of normal-phase liquid chromatography
inlet pressure behind the flow regulator (1.9 ml/min at 140°C) of 250kPa. After and the separation of its major constituents campesteryl ferulate, β-sitosteryl
holding the transfer temperature of 140°C for 5min, temperature was ferulate, cycloartenyl ferulate and 24-methylenecycloartanyl ferulate by means
programmed to 310°C at 15°/min, and after holding for 5min to 340°C at of capillary gas chromatographic analysis of the on-line transferred LC-
2.5°/min, which was held for 3min. fraction.
2 4

Literature
[1] R. Kaneko and T. Tsuchiya, J. Chem. Soc. JPN, 1954, 57, 526 [6] S. Nakayama, A. Manabe, J. Suzuki, K. Sakamoto, T. Inagaki, Japan J. Pharmacol., 1987, 44, 135
[2] Z. Xu and J. S. Godber, J. Agric. Food Chem., 1999, 47 (7), 2724 [7] Z. Xu, N. Hua, J. S. Godber, J. Agric. Food Chem., 2001, 49 (4), 2077
[3] C. Gertz, S. Klostermann, S. P. Kochar, Eur. J. Lipid Sci. Technol., 2000, 102, 543 [8] R. P. Evershed, N. Spooner, M. C. Prescott, L. J. Goad, J. Chromatogr. A, 1988, 440, 23
[4] S. J. Kim, D. Han, K. D. Moon, J. S. Rhee, Biosci. Biotech. Biochem., 1995, 5, 822 [9] K. Grob, J.-M. Stoll, HRC & CC, 1986, 9, 518
[5] G. S. Seetharamaia, N. Chandrasekhara, J. Food Sci. Technol., 1993, 30 (4), 249 [10] K. Grob, H.-G. Schmarr, A. Mosandl, HRC & CC, 1989, 12, 375 5