Identification and quantification of important steryl esters in aspen wood

Article abstract of DOI:10.1007/s11746-000-0066-9

Steryl esters make up a major portion of the total lipids in aspen wood, and contribute significantly to pitch deposit problems during pulping. Fungal treatment of aspen is an attractive method for removing these compounds because it is inexpensive and environmentally acceptable; however, the mechanism of steryl ester removal remains unclear. Identification of the steryl esters will lead to a better understanding of how they are removed by fungi. The steryl ester fraction from aspen wood was obtained by acetone extraction then further purified by silica gel column chromatography and argentation-silica gel column chromatography. This led to the isolation of three major fractions: fraction I, fraction II, and fraction III. The major steryl esters of fractions I and II were identified by gas chromatography, gas chromatography-mass spectroscopy, and proton nuclear magnetic resonance analysis of the intact fraction as well as sterol and fatty acid moieties obtained after base hydrolysis. Identification of the steryl esters was carried out by mass spectra comparisons with steryl ester standards synthesized in the laboratory and comparison with mass spectra libraries (Wiley and NIST) by mass fragmentography. Fraction I contained primarily the palmitate, stearate, and eicosanoate esters of α- and β-amyrin. Fraction II consisted mainly of the palmitate, stearate, and eicosanoate esters of tirucalla-7,24-dien-3β-ol and lupeol.

Full text of DOI:10.1007/s11746-000-0066-9

Identification and Quantification of Important Steryl
Esters in Aspen Wood
Alessio N. Serreqi, Robert Leone, Luis F. Del Rio, Shirley Mei,
Marc Fernandez, and Colette Breuil*
Chair of Forest Products Biotechnology, Department of Wood Science, Faculty of Forestry, University of British Columbia,
Vancouver, British Columbia, Canada, V6T 1Z4,
ABSTRACT: Steryl esters make up a major portion of the total saponifiables to unsaponifiables, such as sterols, SE, and W in
lipids in aspen wood, and contribute significantly to pitch de- aspen wood, relative to softwoods, has been reported as con-
posit problems during pulping. Fungal treatment of aspen is an
attractive method for removing these compounds because it is
inexpensive and environmentally acceptable; however, the
mechanism of steryl ester removal remains unclear. Identifica-
whole logs, usually 12 mon of seasoning is required, while for
tion of the steryl esters will lead to a better understanding of how
wood chips in piles, seasoning may take 2 mon. The long stor-
they are removed by fungi. The steryl ester fraction from aspen
wood was obtained by acetone extraction then further purified
by silica gel column chromatography and argentation-silica gel
tributing substantially to the frequent pitch problems during
kraft pulping (4). Seasoning of aspen logs or wood chips is
currently the method of choice to reduce pitch deposition. For
age times and management of the wood make this method te-
dious and expensive. Also, appreciable loss of brightness and
pulp yield is observed due to sap stain and decay fungi.
Treating wood with fungi that consume the lipophilic ex-
column chromatography. This led to the isolation of three major
fractions: fraction I, fraction II, and fraction III. The major steryl
esters of fractions I and II were identified by gas chromatogra- tractives has been studied as a way of accelerating seasoning
phy, gas chromatography–mass spectroscopy, and proton nu- and reducing pitch deposition (5–8). Fungal treatment of
clear magnetic resonance analysis of the intact fraction as well
as sterol and fatty acid moieties obtained after base hydrolysis.
Identification of the steryl esters was carried out by mass spectra
comparisons with steryl ester standards synthesized in the labo-
ratory and comparison with mass spectra libraries (Wiley and
and W (9,10). In complementary work, a series of fungi were
NIST) by mass fragmentography. Fraction I contained primarily
the palmitate, stearate, and eicosanoate esters of α- and β-
wood is an attractive method for removing extractives be-
cause it is environmentally benign and relatively inexpensive.
Recent work on fungal treatment of aspen chips with Car-
tapip® showed that the fungi did not significantly reduce SE
tested for their ability to consume the problematic steryl es-
ters and waxes of aspen in liquid media (11). Several fungi
amyrin. Fraction II consisted mainly of the palmitate, stearate,
were identified that could potentially be used to biotreat aspen
wood. To better understand the mechanism of extractive re-
moval by fungal treatment, it is important to identify the indi-
and eicosanoate esters of tirucalla-7,24-dien-3β-ol and lupeol.
Paper no. J9405 in JAOCS 77, 413–418 (April 2000).
KEY WORDS: Aspen, argentation-silica gel chromatography, vidual components of the SE and W.
gas chromatography, gas chromatography–mass spectrometry,
pitch analysis, Populus tremuloides Michx, sterols, steryl esters,
triterpenes.
The free sterols and triterpene alcohols, free fatty acids,
combined sterols and triterpene alcohols, and combined fatty
acids in SE from aspen wood have previously been identified
(
12). This analysis, however, gives little information on the
composition of the intact SE. To determine the structure of
In aspen-pulping processes, pitch depositions continue to be a the intact SE of aspen wood, the SE fraction was further re-
serious problem (1,2). Pitch problems are primarily caused by solved by chromatographic techniques. The fractions were
lipophilic wood extractives, commonly called pitch or wood analyzed by gas chromatography (GC) and GC–mass spec-
resins, which consist mainly of fatty and resin acids, glyc- trometry (GC–MS), and the components were identified by
erides, terpene alcohols, sterols, steryl esters, and waxes (1,3). comparison with SE standards synthesized in the laboratory.
The roles of triglycerides, fatty acids, and resin acids in initi- Further identification of the SE was done by mass spectra
ating pitch deposits are well documented, while the role of comparisons with the SE standards and comparison with mass
triterpene alcohols, sterols, steryl esters (SE), and waxes (W) spectra in the Wiley and NIST libraries.
in pitch formation is poorly understood. The low ratio of
MATERIALS AND METHODS
*To whom correspondence should be addressed at Department of Wood Sci-
ence, Faculty of Forestry, University of British Columbia, 4041-2424 Main
Mall, Vancouver, British Columbia, Canada, V6T 1Z4.
E-mail: breuil@interchg.ubc.ca
Reagents. Analytical thin-layer chromatography (TLC) plates
(Whatman silica gel G, F-254, 250-mm layer) were purchased
Copyright © 2000 by AOCS Press
413
JAOCS, Vol. 77, no. 4 (2000)

4
14
A.N. SERREQI ET AL.
from Fisher Scientific (Nepean, ON). Spots were visualized
Fractionation of fraction II. An analytical TLC plate was
by staining with phosphomolybdic acid in EtOH/H SO (4:1) impregnated with 2% silver nitrate in methanol then air dried
2
4
and heating. Column chromatography was carried out on sil- and activated in an oven at 105°C for approximately 15 min.
ica gel 200–400 mesh, 60Å purchased from Aldrich (Milwau- Five milligrams of fraction II dissolved in 0.5 mL of ethyl
kee, WI). Silver nitrate and lupeol were purchased from ether was applied to the plate with a glass pipette. The plate
Sigma Chemical Co. (St. Louis, MO); α-amyrin was pur- was then eluted with 60:40 hexane/toluene in a large devel-
chased from ICN Pharmaceuticals Inc. (Costa Mesa, CA). All opment tank. Visualization was carried out by cutting a strip
fatty acid standards were purchased from Aldrich. All sol- from the plate and staining with phosphomolybdic acid. The
vents used were analytical grade and obtained from Fisher two major bands observed on the strip were used to locate the
Scientific. All gases used were chromatographic grade and bands on the plate. The bands were scraped off and extracted
purchased from Praxair (Vancouver, BC).
with ethyl ether (3 × 3 mL). The solutions were filtered into
Synthesis of SE standards. SE were prepared according to vials and dried under a stream of N . The samples were fur-
2
the procedure of Hassner and Alexanian (13). In a typical reac- ther purified on a short silica gel column eluted with 80:20
tion, α-amyrin (4.5 mg, 11 µmol) was dissolved in methylene hexane/toluene and dried under N , then under vacuum to
2
chloride (1.0 mL) and treated with N,N′-dicyclohexylcarbodi- give fraction II 1 and 2.
imide (9.0 mg, 44 µmol), palmitic acid (16.0 mg, 63 µmol), and
Hydrolysis of steryl esters. The eluted SE (15–25 mg) were
catalytic amounts of 4-dimethylaminopyridine (a few crystals). refluxed in 0.5 N potassium hydroxide in 90% ethanol (5 mL)
The reaction was stirred at room temperature for 18 h and the until the reaction was complete (5–6 h), as verified by TLC.
resulting ester was purified by solid-phase extraction (SPE) The solutions were then acidified to pH 2.0 with 1 N HCl and
(
14) to give a white amorphous solid (5.0 mg, 7.5 µmol, 68% concentrated by rotary evaporation at 50°C. The saponified
yield). The purity of the synthetic ester was determined by ana- residues were dissolved in chloroform and run through an an-
1
lytical TLC (R = 0.86, hexane/EtOAc, 9:1); H nuclear mag- hydrous magnesium sulfate column. The solutions were then
f
netic resonance (NMR) (CDCl , 200 MHz) δ 0.78, 0.85, 0.89, dried under a stream of N gas and fractionated by SPE ac-
3
2
0
.96, 0.99, 1.05, 1.23, 2.27 (2H, t, J = 7 Hz), 4.49 (1H, m), 5.10 cording to the method of Chen et al. (14) to give a sterol frac-
−
1
(
1H, t, J = 3.6 Hz); IR νmax/cm 2930, 1740, 1660, 1450, tion and a fatty acid fraction.
380, 1250, 1160; mass spectral data in Table 1. Detection of the components in the SE fraction by GC. The
Lupeol palmitate was synthesized in the same manner as Hewlett-Packard (Palo Alto, CA) HP 5890 Series II gas chro-
1
described above. The product was obtained as a white amor- matograph was equipped with a flame-ionization detector
phous solid (4.7 mg, 7.1 µmol, 95% yield). The purity of the (FID) and an HP 7673 automatic sampler. The SE were sepa-
synthetic ester was determined by analytical TLC (R = 0.86, rated on a DB-5 fused-silica capillary column (15 m × 0.25
f
1
hexane/EtOAc, 9:1); H NMR (CDCl , 200 MHz) δ 0.77, mm i.d., 0.25-µm film, J&W Scientific, Folsom, CA). The
3
0
4
1
.82, 0.85, 0.92, 1.23, 2.27 (2H, t, J = 7 Hz), 4.45 (1H, m), temperature program started at 50°C, increased at 10°C/min
−
1
.55 (1H, br s), 4.66 (1H, br s); IR νmax/cm 2924, 1728, to 325°C, and then was held for 45 min. The total running
640, 1468, 1382, 1180; mass spectral data in Table 1. time was 65 min. Helium was used as the carrier gas at a flow
A list of all prepared SE standards and their mass spectra rate of 30 cm/s. The injection volume was 2.0 µL. The inlet
data are given in Table 1. temperature was set at 330°C and the FID at 330°C. All the
Extraction and fractionation of SE. The aspen wood was samples analyzed by GC were injected at a concentration of
from a 68-year-old tree in northern Alberta. The freshly cut 1.0 mg/mL. The equipment, data collection, and processing
wood was reduced to small blocks and immediately frozen at were controlled by Hewlett-Packard HP 3365 Series II soft-
−
20°C. The moisture content of the wood, determined gravi- ware. Relative retention times (RRT) and concentrations of
metrically after drying at 105°C overnight, was 28.4%. The GC peaks were calculated relative to the internal standard,
wood chips were ground to fine sawdust powder before ace- cholesteryl myristate, which was added before GC analysis.
tone extraction in a Soxhlet apparatus (18 h, 5–6 cycles/h). A response factor of 0.64 was determined for cholesteryl
After extraction, the acetone was dried over magnesium sul- myristate against α-amyrin palmitate. This value was used to
fate, filtered, then concentrated by rotary evaporation and determine the component concentrations in fraction I. A re-
dried under vacuum to give the acetone extractives of aspen sponse factor of 0.76 was determined against lupeol palmi-
[54 ± 1 g/kg oven-dried (OD) wood]. The SE/W fraction was tate and this value was used in determining concentrations of
obtained by applying the total extractives on a silica gel col- fraction II components.
umn eluted with hexane/ether (50:1). The purified fractions
Detection of components in SE fraction by GC–MS. A Var-
were pooled, concentrated, and vacuum-dried to yield the ian (Palo Alto) 3800 GC was coupled to a Varian Saturn 2000
SE/W (3.1 ± 0.3 g/kg OD wood). The SE/W fraction was fur- MS (ion trap detector). The GC was equipped with a J&W
ther purified by 10% argentation-silica gel column chroma- DB-XLBitd column (10 m × 0.25 mm diameter, 0.25-m film
tography (~0.4 g/mg of residue) eluted with a hexane/toluene thickness), and a deactivated 1078 fritted splitter inlet sleeve
gradient from 4:1 to 1:4, then 100% ethyl acetate. The com- loaded on a 1079 injector. The MS electron multiplier volt-
ponents were separated by their polarity and degree of satura- age was set at 2,150 V, with an ionization time of 25,000 µs,
tion into three fractions referred to as fractions I, II, and III.
running in electron ionization (EI) mode, with transfer line,
JAOCS, Vol. 77, no. 4 (2000)

STERYL ESTERS IN ASPEN WOOD
415
SCHEME 1
trap, and manifold temperatures of 350, 250, and 50°C, re- RESULTS AND DISCUSSION
spectively. Samples were injected via a Varian 8200 auto-
sampler fitted with a 10-µL syringe. The SE/W fractions and Synthesis of SE standards. A series of standard SE was syn-
the synthetic SE standards were dissolved in hexane or chlo- thesized by combining commercially available sterols and
roform at various concentrations. Subsequently, 1–5 µL of triterpenes with the major fatty acids using dicyclohexylcar-
these solutions was injected onto the GC–MS with a split of bodiimide as a coupling agent (Scheme 1, Table 1). These
1
:60, and injector temperature of 300°C. A continuous flow standards were then used to identify the intact SE through
rate of 1.6 mL/min of helium was used with a temperature both spiking experiments and by comparing their GC–MS
profile starting at 200°C for 1 min followed by a 15°C/min fragmentation patterns. This approach was necessary because
ramp to 300°C, then finally a 2°C /min ramp to 325°C which the mass of most SE was greater than the maximum de-
was held for 48 min.
tectable mass of the GC–MS system.
Identification of SE. The SE from the three fractions of
Purification and identification of SE in aspen wood. After
aspen wood were identified by mass spectra comparison with Soxhlet extraction of ground aspen wood with acetone,
those in the NIST and Wiley libraries and, when possible, by medium-pressure silica gel column chromatography was used
comparisons of mass spectra of standards.
to separate the wood extractives based on the component po-
TABLE 1
Electron Impact (70 eV) Mass Spectra Profiles of Synthesized Steryl Ester Standards
Steryl ester
Molecular weight
636
Mass spectral data (% relative intensity)
α-Amyrin myristate
55(23), 69(20), 81(22), 95(35), 107(30), 122(40), 135(38), 147(52), 161(41), 175(24), 189(87),
2
03(68), 218(100), 231(10), 270(12), 409(11)
55(16), 69(16), 81(18), 95(29), 107(23), 122(33), 135(35), 147(38), 161(38), 175(21), 189(79),
03(64), 218(100), 231(11), 270(12), 409(10)
55(22), 69(18), 81(21), 95(29), 107(28), 122(35), 135(33), 147(45), 161(38), 176(22), 190(30),
03(23), 218(100), 231(11), 271(8), 410(11)
55(24), 69(22), 81(18), 95(29), 107(28), 121(34), 135(30), 147(39), 161(39), 17622(22), 190(28),
04(68), 218(100), 231(10), 271(9), 410(11)
55(23), 67(34), 81(34), 95(54), 109(44), 121(42), 135(37), 147(31), 161(31), 175(32), 189(100),
03(43), 216(20), 229(17), 257(9), 297(14), 393(14), 410(12)
55(23), 57(23), 67(32), 81(35), 95(56), 109(42), 121(45), 135(37), 147(33), 161(34), 175(34),
89(100), 203(49), 216(22), 229(18), 257(10), 297(12), 410(14)
55(19), 57(19), 67(26), 81(35), 95(46), 109(36), 121(38), 135(16), 147(33), 161(30), 175(26),
89(100), 203(49), 216(20), 229(16), 257(11), 297(14), 394(14), 410(12)
55(22), 57(21), 67(34), 81(34), 95(55), 109(41), 121(42), 135(35), 147(34), 161(32), 175(31),
89(100), 203(24), 216(20), 229(16), 257(11), 297(12), 394(15), 410(14)
α-Amyrin palmitate
α-Amyrin-stearate
α-Amyrin eicosanoate
Lupeol myristate
664
692
720
636
664
692
720
2
2
2
2
Lupeol palmitate
Lupeol stearate
1
1
Lupeol eicosanoate
1
JAOCS, Vol. 77, no. 4 (2000)

4
16
A.N. SERREQI ET AL.
larities. The nonpolar SE were eluted with a hexane/ether lupeol and an unidentified compound similar to lanosterol.
mixture and recovered in the first several fractions. The SE The structure of the unknown triterpene was determined
were then purified further by argentation-silica gel chroma- through further purification of fraction II by argentation thin-
tography, which separated, on the basis of their polarity and layer chromatography. This led to the isolation of two sub-
degree and mode of unsaturation, into three fractions: frac- fractions: fraction II-1 and fraction II-2. Fraction II-1 was
tions I, II, III (Scheme 2). Fraction I was base-hydrolyzed and base-hydrolyzed and the resulting triterpene alcohol was iso-
the resulting sterols and fatty acids were analyzed by GC–MS lated and analyzed by proton NMR and identified as tirucalla-
according to the method of Peng et al. (12). The primary fatty 7,24-dien-3β-ol by comparison to literature data (15). Lupeol
acids were identified as palmitic, stearic, and eicosanoic acid, ester standards were synthesized and compared to the com-
while the main triterpenes were α- and β-amyrin. From this ponents in unhydrolyzed fraction II. Through GC spiking ex-
information, the α-amyrin ester standards were synthesized periments and comparison of the mass spectra data, the pri-
and compared to the components in unhydrolyzed fraction I. mary lupeol esters in fraction II were identified.
Through GC spiking experiments and comparison of the mass
Concentration and composition of SE in aspen wood. In
spectra fragmentation data, the primary α-amyrin esters in aspen wood, the SE/W were present at 3.1 ± 0.3 g/kg OD
fraction I were identified. The very similar mass spectra frag- wood. Fraction I accounted for 28 ± 2% of the total SE/W
mentation pattern of the other components led to their β- fraction. The eight major identified components of this frac-
amyrin ester assignment.
tion represented 67% of the material balance (Table 2). The
Fraction II was analyzed in the same manner as described palmitate esters were the most abundant and α-amyrin palmi-
above. The primary fatty acids were identified as palmitic, tate alone accounted for 33% of this fraction. Fraction II rep-
stearic, and eicosanoic acids, while the main triterpenes were resents 35 ± 7% of the total SE/W fraction. From this frac-
SCHEME 2
JAOCS, Vol. 77, no. 4 (2000)

STERYL ESTERS IN ASPEN WOOD
417
TABLE 2
Steryl Esters Present in Fraction I
Composition (%)
of total steryl esters
Content (mg/kg freeze-dried wood)
in fraction I
Steryl ester
RRta
β-Amyrin myristate
α-Amyrin myristate
β-Amyrin palmitate
α-Amyrin palmitate
β-Amyrin stearate
α-Amyrin stearate
β-Amyrin eicosanoate
α-Amyrin eicosanoate
a
1.11
1.15
1.29
1.34
1.50
1.56
1.80
1.89
0.08
0.2
5.6
9.5
1.0
1.6
0.4
0.7
3
5
170
290
29
48
12
22
Relative retention times (RR ) of steryl esters on DB5 capillary column (cholesteryl myristate: 1.00).
t
TABLE 3
Steryl Esters Present in Fraction II-1 and Fraction II-2
Composition (%)
of total
steryl esters
Content (mg/kg
freeze-dried wood)
in fraction I
Steryl ester
RRta
Tirucalla-7,24-dien-3β-yl palmitate
Lupeol palmitate
Tirucalla-7,24-dien-3β-yl stearate
Lupeol stearate
Tirucalla-7,24-dien-3β-yl eicosanoate
Lupeol eicosanoate
1.26
1.33
1.49
1.55
1.75
1.87
4.2
11.1
0.9
2.8
0.2
129
338
27
85
5
1.0
30
a
RR of steryl esters on DB5 capillary column (cholesteryl myristate: 1.00). See Table 2 for abbrevia-
t
tion.
tion, six components were identified that made up 61% of the
material (Table 3). Again, the palmitate esters were the most
abundant, and lupeol palmitate accounted for 33% of the frac-
tion. It also was the most abundant SE present in aspen, ac-
counting for 11% of the total.
Fraction III represents 37 ± 8% of the total SE/W fraction.
At present, the components in this fraction appear to be pri-
marily steryl linoleate esters. Unfortunately, GC analysis of
this fraction gave a complex mixture of unresolved and broad
peaks. Therefore, more research is needed to conclusively
identify the SE in this fraction. The components that have
been identified and quantified account for 1.2 g/kg OD wood
or 39% of the SE/W fraction. The identification of SE in
aspen should enable a better understanding of the mechanism
of their removal during seasoning or biotreatment.
Aspen Causes More Pitch Problems Than Softwoods in Kraft
Pulping, Tappi J. 78:143–149 (1995).
3
. Sjöström, E., Wood Chemistry: Fundamentals and Applications,
Academic Press Inc., San Diego, 1993, pp. 90–113.
. Sitholé, B.B., J.L. Sullivan, and L.H. Allen, Identification and
Quantification of Acetone Extractives of Wood and Bark by Ion
Exchange and Capillary GC with a Spreadsheet Program, Holz-
forsch. 46:409–416 (1992).
. Blanchette, R.A., R.L. Farrell, T.A. Burnes, P.A. Wendler, W.
Zimmerman, T.S. Brush, and R.A. Snyder, Biological Control
of Pitch in Pulp and Paper Production by Ophiostoma piliferum,
Tappi J. 75:102–106 (1992).
6. Brush, T.S., R.L. Farrell, and C. Ho, Biodegradation of Wood
Extractives from Southern Yellow Pine by Ophiostoma pil-
iferum, Ibid. 77:155–159 (1994).
. Fisher, K., M. Akhtar, R.A. Blanchette, T.A. Burnes, K. Mess-
ner, and T.K. Kirk, Reduction of Resin Content in Wood Chips
During Experimental Biological Pulping Processes, Holzforsch.
4
5
7
4
8:285–290 (1994).
8
. Chen, T., Z. Wang, Y. Gao, C. Breuil, and J.V. Hatton, Wood
Extractives and Pitch Problems: Analysis and Partial Removal
by Biological Treatment, Appita 47:463–466 (1994).
ACKNOWLEDGMENTS
This work was supported by a grant from the National Science and
Engineering Research Council. The authors would like to thank Drs.
Paul Watson and Bruce Sitholé of PAPRICAN for their valuable
advice.
9. Rocheleau, M.J., B.B. Sitholé, L.H. Allen, S. Iverson, R. Far-
rell, and Y. Noel, Fungal Treatment of Aspen Chips for Wood
Resin Reduction: A Laboratory Evaluation. J. Pulp Paper Sci.
2
4:37–42 (1998).
1
0. Rocheleau, M.J., B.B. Sitholé, L.H. Allen, and Y. Noel, Fungal
Treatment of Aspen Chips for Wood Resin Reduction: Effect on
Aged Aspen Wood Chips at Room Temperature and at 5°C,
Holzforsch. 53:16–20 (1999).
REFERENCES
1
. Allen, L.H., B.B. Sitholé, J.M. Macleod, C.L. Lapointe, and F.J.
McPhee, The Importance of Seasoning and Barking in the Kraft
Pulping of Aspen, J. Pulp Paper Sci. 17:J85–J91 (1991).
11. Leone, R., and C. Breuil, Filamentous Fungi Can Degrade
Aspen Steryl Esters and Waxes, Intl. Biodet. Biodeg.
41:133–137 (1998).
2
. Chen, T., Z. Wang, Y. Zhou, C. Breuil, O.K. Aschim, E. Yee,
and L. Nadeau, Using Solid-Phase Extraction to Assess Why
JAOCS, Vol. 77, no. 4 (2000)

4
18
A.N. SERREQI ET AL.
1
2. Peng, J., R. Leone, A.N. Serreqi, and C. Breuil, Are Aspen 15. Itoh, T., T. Tamura, and T. Matsumoto, Proton Magnetic Reso-
Sterols and Steryl Esters Changed Structurally by Kraft Pulping
and Bleaching? Tappi J. 82:204–211 (1999).
3. Hassner, A., and V. Alexanian, Direct Room Temperature Es-
terification of Carboxylic Acids, Tetrahedron Lett. 46:
nance Spectra of Tetracyclic Triterpene Alcohols Under the In-
fluence of a Lanthanide Shift Reagent, Steroids 27:275–285
(1976).
1
4
475–4478 (1978).
1
4. Chen, T., C. Breuil, S. Carrière, and J.V. Hatton, Solid-Phase
Extraction Can Rapidly Separate Lipid Classes from Acetone
Extracts of Wood and Pulp, Tappi J. 77:235–240 (1994).
[Received September 24, 1999; accepted January 25, 2000]
JAOCS, Vol. 77, no. 4 (2000)