1094
INSTRUMENTATION
SPE methodologies for the separation of lipids
olid phase extraction (SPE) is a
sample preparation technique
that frequently is used for isolalion and fractionation of lipids. It is
rapidly replacing traditional sample
preparation techniques. e.g .. liquidliquid extraction, liquid-solid column
chromatography. and thin-layer chromatography (TLC). due to the ease of
use. speed. cost effectiveness, and
decreased requirements
for solvent
usage.
This ankle will describe briefly the
principles and general applications of
SPE for lipid analysis. In addition, the
reader is referred 10 two recent
reviews of the use and applications of
SPE for lipid separation (1.2) and to
S
resources provided
by $PE manufac-
turers (e.g., 3,4). The $PE separation
of oxygenated lipid metabolites (i.e.,
prostaglandins and leukotricnes), lipid
peroxidarion
products, and steroid
profiling, although important in a
complete lipid analysis, is beyond the
scope of this article.
Principles, applications of SPE
Small,
commercial
columns
prepncked with a variety of solid stationary phases were introduced in the
late 1970s. The earliest applications
of SPE for lipid analysis used silica,
bonded
aminopropyl.
and CI8
reverse-phase columns for the isolation and separation of various lipid
classes from foods and biological tissues (5-7). These phases continue to
be widely used for lipid analyses and
are discussed in greater detail in the
following sections. Alumina, macroporous carbon and ion exchange phases
also have found recent application for
the isolation and fractionation
of
lipids (8-12).
A number of solid phases are commercially available (Figure I), and
new SPE cartridges are continually
being introduced-an
indication of the
widespread interest and utilization of
SPE for sample preparation. Many of
the newer cartridges offer mixed mode
separation mechanisms (13,14) and
contain proprietary stationary phases
(15.16) which are designed for specific applications. Among the newer carINFORM. VOl. 7. no. 10 (October 1996)
....
-
This article was prepared by
Susan E. Ebeler and Jolm O.
Eheter: of the Department of
Viticulture and Enology,
University of California, Davis.
Davis, CA 95616.
rrtdges. restricted access media or
shielded hydrophobic
phases offer
potential advantages for the analysis
of biological samples because the
sample can be injected directly onto
the cartridge without fouling the stationary phase (16). Proteins and other
high molecular weight compounds are
unretained while smaller molecules
are retained by normal HPLC sorption
mechanisms. Several manufacturers
are preparing SPE-like cartridges with
these shielded hydrophobic phases.
These cartridges hold great potential
for lipid separations; however few literature reports of the application of
restricted access medi and other new
SPE sorbents for lipid analyses have
emerged.
SPE sorbents generally utilize relatively large particle and pore sizes
(30-60 mm, 60A), and nnalytes are
eluted at tow pressures. The sorberns
are typically packed in syringe-like
polyethylene or polypropylene tubes;
however. SPE disks are gaining popularity (16,17). With disks. the packing
is generally embedded or impregnated
into a PTFE or fiberglass network or
onto a membrane (17), a design that
prevents the channeling problems that
can occur with packed beds. In addition, the disks are thin and have a
large cross-sectional
area providing
faster now rates and shorter analysis
times (17). The number of phases currently available in the disk format is
limited, however (17).
Matrix solid-phase
dispersion
(MSPD) is another relatively new
technique which involves grinding
biological samples with CIS or other
bonded silica supports. transferring
J.D. EbeIer
the blend to a syringe barrel column,
and eluting with selected solvents or
solvent mixtures. This technique was
used to isolate and fractionate lipids
from Escherichia coli and Mycobacterium paratuberculosis
without the
need for separate lysis and fractionation steps (18). Mycobacteria typically
have thick cell walls which are hard to
disrupt without severe treatments such
as antibiotics. detergents, or sonication. Using MSPD. cells appeared to
be completely lysed and the cellular
components were well dispersed over
the C 18 panicles providing a homogeneous blend for subsequent lipid fractionation (18). The MSPD is a singlestep procedure that is chemically and
physically
mild and offers many
potential applications for the analysis
of lipids in a variety of matrices.
Silica sorbents. Many SPE lipid
analyses are performed following a
preliminary extraction of the sample
into chloroform or other similar nonpolar solvents, making silica a logical
sorbern choice for further lipid separation and purification steps. The polar
snanot groups adsorb polar compounds, and analyte retention and elution are related directly to solvent
polarity. Ion-exchange effects. due to
the mildly acidic nature of the silica
particles, also influence separations on
silica-based sorbenrs. Silica adsorbs
varying amounts of water, which may
affect separations. The columns may
require either drying prior to analysis
or the use of solvents with controlledmoisture contents (19).
In an early application. silica SPE
[continued on pag~ 1096,
1096
INSTRUMENTATION
Mod.
Pb.le
Struct1lH
Silica
• Si-OH
1
I
I
Cy~'
• SI-C3HeCN
I
I
- Si-CaHeOCH2CH2
I
I
I
"'"
alal
Aminopropyl··
I
• Si.C3HeNH2
I
I
- SI-C1SH31
Cl1 or ODS
I
I
C,
- Si.CaH17
I
1
C,
- SI-C
I
,.H
""",I
I
.SI·O
BemenesulfODlle
- ~i.CI'\-O-SOI-
PropylsulfOlWC
- Si-C3HeS03'
~
""'""
• May abo be used II • R:w:rsc phase
•• May also be used asan ioo e~
page 1094)
columns were used to isolate phosphatidylcholine
(PC) and phosphatidylelhanolamine
(PE) from the
nonpolar lipids of chocolate (5).
Recoveries for PC ranged from 90 10
99% with excellent precision (I to
7.8% coefficient of variation). Early
studies also showed that silica SPE
was an excellent sample preparation
technique for isolating microgram and
nanogram quantities of phospholipids
and neutral lipids from limited
amounts
of biological
samples
(20-22).
In a comparison of SPE and traditional silica column chromatography.
INFORM. Vol. 7. no. 10 (OCtober
1996)
I
+
- Si-C3He N (CH~3
phase
Agure 1. Some eommen;:~lIy
(continued/rom
I
available SPE solid phases
Juaneda and Rocquelin (23) found no
quantitative differences in recovery or
reproducibility between the two methods for the separation of nonphosphorous lipids and phospholipids from rat
heart, liver, and kidney tissues. However, the SPE procedure effectively
separated the two fractions in approximately five minutes, compared to several hours required for the traditional
column procedure. Similarly, Sebedio
et al. (24) found that SPE and a standardized IUPAC-AOAC silica column
chromatographic procedure gave similar results for the fractionation of nonpolar and polar lipids from commercial frying oils. Roemen and van der
Vusse (25) felt that the SPE separation
of the free fatty acids from the polar
lipids in heart tissue extracts was more
effective than traditional column chromatographic techniques. Using silica
SPE, no hydrolysis of fatty acids from
the phosphoglycerides or other esterified fatty acid classes occurred, a
problem previously
reported with
ether silica column chromatographic
methods. This advantage may be due
to the much shorter separation time of
the SPE method.
More complex lipid separations are
also possible on silica SPE columns.
The number of fractions obtained and
their chemical
compositions
are
dependent on the solvent systems
used. Hamilton and Comai (26,27)
developed an SPE procedure which
was effective in isolating cholesterol
ester, triglyceride (TG), free fatty acid
(FFA), cholesterol, and acidic PE and
phosphatldylinositcl and neutral (PC.
sphingomyelin, lyso-PC) phospholipid
fractions
from human serum, rat
serum and liver, and cultured smooth
muscle cells. Using different elution
solvents, Ingalls et al. (28) isolated
similar fractions from plasma with
recoveries of 80-91 %.
lanero and Burghardt (29) reported
that MTBE-based solvent mixtures
utilized with the procedures of Hamilton and Comai were effective in separating PE from the choline-containing
phospholipids but resulted in loss of
approximately
60% of the biologic
activity of platelet-activating
factor
(PAF), an important choline phospholipid. These authors showed that
sequential
elution
of rat heart
extracts-first
with chloroform/acetic
acid,
followed
by
methanol/chloroform, and finally with
methanollchloroform/water-resulted
in excellent separation of neutral nonphosphorous lipids from PE and PC,
while PAF maintained full biologic
activity.
Bonded aminopropyl sorbents. The
surface silanol groups of silica can be
reacted with siloxane derivatives to
form bonded phases. A number of
bonded phases are available (Table I)
and normally are classified as nonpolar (reverse phase), polar (normal
phase), or ion exchange. The bonded
phases can be either monomeric or
polymeric depending on the number
1097
A typica. ~Id phase axtrKtlon e.rtridge.
The c.rtrldgoI .hown (;OIIQin. 100 mg of •
CHI,...,.,... phase eorbenL
of layers of the bonding reagent on the
silica surface (I). The choice of
monomeric or polymeric phases is
dependent on the specific application
(1,13),
AminopropyJ bonded phases are
polar and are therefore classified as
normal-phase
sorbents (Table I).
Hydrogen bonding to the primary
amine results in a stronger interaction
with analytes having polar functional
groups than with nonpolar analytes.
The amino group also can impart
weak anion exchange properties so
that separations
based
on ion
exchange mechanisms are possible.
Because of their polarity, aminopropyl columns have found wide
applications for lipid analyses. A basic
method for the rapid separation of
individual
neutral and polar lipid
classes using bonded aminopropyl
SPE cartridges
was developed by
Kaluzny et al, (7), and modifications
of this method continue to be widely
used (30-32). Using the separation
scheme described by Kaluzny, neutral
lipids are eluted and subsequently separated on a second column with solvents of increasing polarity. FFA and
phospholipids
bind to the column
mainly via ionic interactions. These
ionic interactions
are removed by
changing the pH of the elution solvents. "Piggy-backing"
two aminopropyl columns, so that eluant from
one column was placed onto another
column and further fractionated,
yielded complete separation of a crude
lipid extract into eight fractions: neutral lipids,
FFA, phospholipids,
cholesterol (or other sterol) esters,
TO, cholesterol
(or other sterols),
For inforrnotion
diglycerides,
and monoglycerides.
Recovery of the lipid class standards
was 96-101% with less than 2% contamination between fractions.
Kaluzny et al. (7) compared this
SPE procedure to a standard preparative TLC separation for the fractionation of bovine adipose tissue extracts.
Average recovery for all lipid classes
was approximately 100% for the SPE
procedure compared to 80% for the
TLC procedure. Similarly. Prasad et
el. (33) reported SPE recoveries of
greater than 90% for arachidonate
compared to less than 70% recovery
by TLC procedures; other saturated
and monosaturated
fatty acids also
showed losses in the TLC procedure.
but these losses were not as significant
as those for arachidonate.
Other
authors (34). however. have reported
inconsistent TO recoveries (80-90%)
with the SPE procedure of Kaluzny et
al. When used to isolate FFA from
dairy products, aminopropyl columns
seemed to avoid many of the problems
previously reponed with other column
chromatographic procedures. including hydrolysis of glycerides and interference from lactic acid (8,35). In a
recent application,
aminopropyl
columns were used to concentrate
polyunsaturated
fatty acid methyl
circle '114
INFORM. Voi. 7. no. 10 (October
1996)
1098
INSTRUMENTATION
esters from the saturated and monoun-
saturated esters in fish oil (36). This
may prove a useful alternative to crystallization, urea adduct fractionation,
and fractional distillation methods for
concentration of polyunsaturated fatty
acid methyl esters .
Prieto et al . (37) developed a
Derived Irom . ..
method for the separation of neutral
lipids. glycolipids,
and phospholipids from wheat flour using a combination of silica and aminopropyJ
bonded-phase columns. Wheal flour
lipid extracts were placed on a silica
SPE column and eluted with solvent
combinations of increasing polarity
Coconut Oil (Whole CUI and Froctionaled)
Canola Oil (Oleic Acid)
Linseed Oil (Linolenic Acid)
Soya (Stearic Acid)
Sunllower Oil (Oleic Acid)
Twin Rivers
Technologies
"Uncompromising
Customer Satisfaction"
780 Washington Street. Quincy. Massachusetts.
Tel (617) 472-9200 Fax (617) 472-5460
For InformatIon circle *125
INFORM. Vol. 7. no. 10 (October
1990)
02169
to separate steryl esters, TO, FFA,
diglycerides, monoglycerides, monogalactosylmonoand monogatacrosyldiglycerides
(MOMO
and
MODO), digalactosylmonoand
digalactosyldiglycerides
(DGMG
and DODO), and PC and Iyso-PC.
N -acyl-phosphatidy
lethanolami ne
(NAPE)
and N-acyl-Iysophosphatidyethanolamine
(NALPE),
which coeluted on the silica column,
were placed on an aminopropyl SPE
column and completely
separated
with
mixtures
of
ch Ioroform/met
ha nolla mmoni u m
hydroxide. Cross-contamination
of
the phospholipid fractions, as determined by HPLC, was less than 14%.
Recovery
of the phospholipids
ranged from 81-87%. Separation of
the lipid classes
using only an
aminopropyl column was also evaluated; however, the monoglycerides
and monogalactosylglyceride
fractions, as well as the PC and LPC
fractions, were poorly separated on
this column.
C 18 bonded-phase
sorbents.
Reverse-phase
separations on C 18
sorbcnts. while not as common as normal-phase separations, also have been
employed for lipid fractionations.
Lipids are retained selectively by the
nonpolar CI8 sortent: therefore polar,
non lipid components can be easily
removed and the lipids can be fractionated by eluting with increasingly
nonpolar solvents.
Reverse-phase
separations may be particularly suited
for isolation of lipids dissolved or dispersed in aqueous or other polar solvents, eliminating the need for a preliminary extraction into an organic
solvent. For example, Pempkowiak
(38) isolated short- and long-chain
fatty acids directly from distilled
water and sea water using CI8 SPE
columns. Salts present in sea water
did not influence the separation efficiency. Using CI8 SPE, PC, cerebrosides. sulfatides,
and gangliosides
were isolated from polar precursors
and enzymes in aqueous, in vitro
biosynthetic mixtures (39). The procedure was effective in removing watersoluble precursors without loss of the
lipid products and was easier and
more effective than Folch extractions
1099
or other similar procedures.
Fatty
acyl-CoA esters also have been isolated successfully using CIS cartridges
(40-43), however the recent development of oligonucleotide
SPE cartridges may provide a more rapid and
highly specific technique for isolation
of these compounds (44).
Argentation
$PE. Argentation
chromatography may be used to separate lipid materials on the basis of the
number, type, and position of the
unsaturated centers. Briefly, this type
of chromatography takes advantage of
the fact that compounds
with
ethylenic or acetylenic bonds interact
weakly with silver ions. The nature of
the interaction
is not completely
understood but appears to involve
overlap of the olefin p-orbitals and the
d- and s-orbital of the silver ion. Steric factors affect the ease of overlap,
and the double bond remains essentially intact during cornplexing (45).
Argentation
chromatography
involves impregnating a sorbent, usua\ly silica, with silver ions, generally
in the form of aqueous silver nitrate.
The method has been applied to column chromatography,
TLC. and
HPLC separations
of unsaturated
lipids. Silver ion or argentation chromatography
has been extensively
reviewed (45,46).
Christie (47) showed that small
columns, packed with a bonded benzenesulfonate ion-exchange medium,
could be loaded with silver ions;
importantly the amount of silver needed was far less than required for traditional TLC or column techniques. The
silver-ion SPE cartridges achieved
excellent
separation
of fatly acid
methyl esters (FAMEs), cholesterol
esters, and TO based on the degree of
unsaturation in the fatty acid moiety.
In addition, the fractions were not
contaminated with silver ions (47-52).
Although silver ion SPE cartridges
currently must be impregnated by the
analyst, their potential usefulness
eventually may lead to their commercial availability.
Potential problems/disadvantages
or
SPE
When utilizing SPE techniques, care
must be taken to avoid sample overload and subsequent analyte break-
Solid phase extraction vacuum manifold. Stlverel cartridge. can be analyzed at one time and
fractions can be collected In teat tubes placed In the manifold chambe.r A range 01 SPE
cartridge sizes are COInmen:I.lly .v.lI.ble.a ahown In the lower·lell portlon of the photograph.
through. This problem can be minimized with careful attention to sorbent conditioning, flow rates through
the columns, and the choice of solvents for dissolving and eluting the
analytes. Most manufacturers provide
guidelines
for optimizing
these
parameters.
A recent evaluation of lot-to-lot
reproducibilities in mixed-mode solidphase extraction columns found no significant differences between the tested
lots for the analysis of drugs from
blood (53); however. lot-to-lot variability in commercial SPE cartridges has
been observed (54). Depending on the
application, single lots of sorbcru from
a single supplier may he necessary for
a particular analysis.
Contaminant's extracted from the
plastic cartridges and frits or from the
sorbent
are a common
problem
(25,33,55,56).
In some cases these
contaminants can interfere with subsequent analyses or with the biological
activity of the analytes (57). These
interferences
can be minimized by
using glass columns, stainless-steel
frits (25,33), and by rinsing the cartridges with a variety of solvents prior
to adding the sample (38.47).
Summary
SPE provides a flexible and selective
technique for the fractionation, purification. and concentration
of lipids.
When compared to traditional methods such as column chromatography,
TLC, and liquid-liquid
extractions,
SPE cartridges can offer improved
recoveries and separation efficiencies
with significant cost and time savings.
Usc of SPE disks may decrease sample preparation times even further.
SPE methods for the isolation and
fractionation of lipids continue to utilize mainly silica, aminopropyl. and
C 18 sorbents; however, the development of new phases offers the lipid
chemist exciting opporruniues for the
continuing
evolution of improved
sample preparation protocols.
References
I. Wachob.
G.D., Solid phase
extraction of lipids, in Analyses
of Fats, Oils and Lipoproteins,
edited by E.G. Perkins, American
Oil Chemist's
Society, Champaign,
Illinois,
1991,
pp.
122-137.
2. Ebeler, S.E., and T. Shibamoto.
Overview and Recent DevelopINFORM. Vol. 7. no. 10 (October 1996)
1100
INSTRUMENTATION
meets in Solid-phase Extraction
for Separation of Lipid Classes,
in Lipid Chromatographic Analysis, edited by T. Shlbamoto, Marcel Dekker Inc., New York, New
York. 1994. pp. 1-49.
13.
3. Products Catalog and Reference
Guide. 1995-1996. J&W Scientific. Folsom. California.
4. Solid Phase Extraction Applications Guide and Bibliography. 6th
Edn .. Millipore Corporation, Bedford. Massachusetts. 1995.
5. Hurst, W.J., and R.A. Martin Jr.,
The HPLC Separation and Quantiunion of Lecithin in Chocolate,
J.
Am.
Oil
Chern
Soc.
57,307-310 (1980).
6. Williams,
M.A"
and R.H.
14.
15.
McCluer. The Use of Sep-Pek
7.
8.
9.
10.
11.
12.
C 18 Cartridges During the Isolation of Oangliosides,
J. Neurochem. 35( I):266-269 (1980).
Kaluzny. M.A .. L.A. Duncan,
M.V. Merrill and D.E. Epps,
Rapid Separation of Lipid Classes
in High Yield and Purity Using
Bonded Phase Columns. J. Lipid
Res. 26:135-144 (1985).
de Jong, C .. and H.T. Badings,
Determination
of Free Fatty
Acids in Milk and Cheese. Procedures for Extraction, Clean Up.
and Capillary
Gas Chromatographic Analysis. JHRC & cc.
13,94-98 (1990).
Hutta, M., E. Simunicova,
D.
Kaniansky,
J.Tkacova
and J.
Brtko, lsotachophorctic Derermination of Short-Chain Fatty Acids
in Drinking Water after SolidPhase Extraction with a Carbonaceous Sorbent, J. Chromatogr.
470:223-233 (1989).
Salari, H., Solid-Phase Extraction
and Reversed-Phase High-Performance Liquid Chromatographic
Technique for Isolation and Estimarion of Platelet Activating Factor in Plasma, Ibid. 382:89-98
(1986).
Salari. H., Comparative Study of
Solid-Phase
and Liquid-Phase
Extraction Techniques for Isolation of Phospholipids from Plasma. [bid. 419: 103-1 II (1987).
Coene J., E. Van den Eeckhcut, P.
Herdewijn and P. Sandra, Gas
Chromatographic
Determination
INFORM, Vol. 7.
no.
10 (October 1996)
16.
17.
18.
19.
20.
21.
22.
of Alkyl Iysophospholipids After
Solid-Phase Extraction from Cell
Culture Media, Ibid. 612:21-26
(1993).
Hcrack, J., and R.E. Majors, Perspectives from the Leading Edge
in Solid-Phase Extraction, LOGC
11(2P4. 76. 79-80. 82. 84. 86.
88.90(1993).
Mills, M.S., E.M Thurman and
M.J. Pedersen, Application
of
Mixed-Mode.
Solid-Phase
Extraction in Environmental and
Clinical
Chemistry,
J. Chromatogr, 629:22-21 (1993).
Majors, R.E., New Chromatography Columns and Accessories at
the 1995 Pittsburgh Conference,
Part II. LC/GC. 13(4):290,
292-293.
295-296.
298-301
(1995).
Majors, R.E. , New Chromatography Columns and Accessories at
the 1996 Pittsburgh Conference,
Pan II, Ibid. 14(4):278,280,
282-289.292-293
(1996).
Majors, R.E., New Approaches to
Sample
Preparation,
Ibid.
13(2),82. 84. 87-88. 90. 92. 94
(1995).
Barker, S.A., A.R. Long and M.E.
Hines II, Disruption and Fractionation of Biological Materials by
Matrix Solid-Phase Dispersion. J.
Chromatogr. 629:23-34 (1993).
Blunk, H.-C .. and H. Steinhart.
Separation of Phospholipids
in
Bovine Tissue with Disposable
Silica Gel Extraction Columns, Z.
Lebensm.
Unters.
Forsch.
190,123-125 (1990).
Hedegaard, E., and B. Jensen,
Nunc-scale Densitometric Quantitation of Phospholipids, J. Chrornatogr. 225:450--454 (1981).
Bocckino, S.B .• P.E Blackmore
and J.H. Exton, Stimulation of
1,2-Diacylglycerol Accumulation
in Hepatocytes by Vasopressin.
Epinephrine. and Angiotensin U,
J.
BioI.
Chern.
260(26),14201-14207 (1985).
Yandrasitz, J.R., G. Berry and S.
Segal, High-Performance Liquid
Chromatography
of Phospholipids with UV Detection: Optimization of Separations on Silica,
J. Chromatogr.
225:319-328
(1981).
23. Juuneda. P., and G. Rocquelin.
Rapid and Convenient Separation
of Phospholipids and Nonphosphorus Lipids from Rat Heart
Using Silica Cartridges, Lipids
20,<U}..41(1985).
24. Sebedlo, J.L.. Ch. Septier and A.
Grandgirard,
Fractionation
of
Commercial Frying Oil Samples
Using Sep-Pak Cartridges,
J.
.Am.
Oil
Chern.
Soc.
63,1541-1543 (1986).
25. Roemen. Th.H.M" and GJ. van
der Vusse, G. J. , Application of
Silica Gel Column Chromatography in the Assessment of NonEsterified Farry Acids and Phosphoglycerides in Myocardial lissue, J. Chrornatogr. 344:304-308
(1985).
26. Hamilton, J.G., and K. Cornai,
Separation of Neutral Lipids and
Free Fatty Acids by High-Performance Liquid Chromatography
Using Low Wavelength Uuraviolet Detection,
1. Lipid Res.
25(10), 1142-1148 (1984).
27. Hamilton, J.G., and K. Com ai,
Rapid Separation
of Neutral
Lipids. Free Fatty Acids and
Polar Lipids Using Prepacked Silica Sep-Pak Columns,
Lipids
23:1146-1149 (1988).
28. Ingalls, S.T., M.S. Kriaris, Y. Xu,
D.W. DeWuf, Ke-Y Tsemg and
C.L. Hoppel, Method for Isolation of Non-Esterified Fatty Acids
and Several Other Classes of
Plasma Lipids by Column Chromatography
on Silica Gel, J.
Chromatogr. 619:9-19 (1993).
29. Janero, D.R., and C. Burghardt,
Solid-Phase Extraction on Silica
Cartridges as an Aid to PlateletActivating Factor Enrichment and
Analysis, Ibid. 526: 11-24 (1990).
30. Przybylski.
R., and N.A.M.
Eskin, Two Simplified Approaches to the Analysis
of Aereal
Lipids. Food Chern. 51:231-235
(1994).
31. Caboni, M.R., S. Menoua and G.
Lercker, High-Performance
liquid Chromatography
Separation
and Light-Scattering Detection of
Phospholipids from Cooked Beef,
J. Chromatogr.
A. 683:59-65
(1994).
(continued on page 1102 )
1102
INSTRUMENTATION
(cominuedfrom page 1/00)
32. Grav. H.J .. O.K. Asiedu and R.K.
Berge. Gas Chromatographic
Measurement of 3· and 4-Thia
Fatty Acids Incorporated
into
Various Classes of Rat Liver
Lipids During Feeding Expertments.
J. Chromalogr.
B.
658, 1-10 (1994)
33. Prasad, M.R .• R.M. Jones, H.S.
Young. L.B. Kaplinsky and O.K.
Das. Analysis of Tissue-Free
Fatty Acids Isolated by Aminopropyl Bonded-Phase Columns. 1.
Chromarcgr.
428:221-228
(1988).
34. Nurmela, K.V.V., and L.T. Salama, Quantitative Analysis of TO
by High-Performance
Liquid
Chromatography Using Non-Linear Gradient Elution and Flame
Ionization
Detection.
Ibid.
435,,39-,48
(1988).
35. de Jong, C., K. Palma and R.
Neeter,
Sample
Preparation
Before Capillary Gas-Chromatographic Estimation of Free Fatty
Acids in Fermented Dairy Products, Netherlands Milk & Dairy J.
48,,5,-,56
(1994).
36. Wilson. R.. R.J. Henderson. I.C.
Burkow and J.R. Sargent, The
Enrichment of n-S Polyunsaturated Fatty Acids Using Aminopropyl Solid Phase Extraction
Columns,
Lipids
28:51-54
(1993).
37. Prieto, J.A., A. Ebri and C. Collar, Optimized Separation of Nonpolar and Polar Lipid Classes
from Wheat Flour by Solid-Phase
Extraction,
J. Am. Oil Chern.
Soc. 69,387-391 (1992).
38. Pempkowiak, J., CI8 ReversedPhase Trace Enrichment of Shortand Long-Chain
(C2·C8-C20)
39.
40.
41.
42.
43.
44.
45.
46.
SCORES 100% IN SPECIAliZED TECHNICAL
SERVICES FOR CONSUMER PIIOOUCTS.
47.
Boston • Atlanta
Contact Ttida Baresst, TEL (617) 328-7600 FAX(617) 77Q.<1)57
For information circle '118
INFORM. Vol. 7. no. 10 (October 1996)
48.
Fatty Acids from Dilute Aqueous
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Figlewicz, D.A., C.E. Nolan, l.N.
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26,140-144 (1985).
Styrene. S. and G. Glad. Acyl
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in
Microsomes of Developing Soya
Bean Cotyledons and Its Role in
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16,298-305(1981).
Stymne, S .• A.K. Stoban and G.
Glad, The Role of the Acyl-CoA
Pool in the Synthesis of Polyunsaturated 18-Carbon Fatty Acids
and Triacylglycerol Production in
the Microsomes of Developing
Safflower Seeds, Biochim. Hiephys. Acta. 752: 198-208 (1983).
Stymne, S., and A.K. Stobart,
Involvement of Acyl Exchange
Between Acyl-CoA and Phosphatidylcholine
in the Remodelling of Phosphatidylcholine
in
Microsomal Preparations of Rat
Lung, Ibid. 837:239-250 (1985).
Molaparast-Saless. E. E. Shrago,
T.L. Spennetta,
S. Douatetlo.
L.M. Kneeland, S.H. Nellis and
A.J. Liedtke, Determination
of
Individual
Long-Chain
Fatty
Acyl-CoA Esters in Heart and
Skeletal
Muscle,
Lipids
2N90-492
(1988).
Deutsch,
J., E. Grange,
S.1.
Rapoport and A.D. Purdon, Isolation and Quantitation of LongChain Acyl Coenzyme A Esters
in Brain Tissue by Solid-Phase
Extraction,
Anal.
Biochem.
2B21-323
(1994).
Morris, L.J., Separation of Lipids
by Silver Ion Chromatography, J.
Lipid Res. 7:717-732 (1966).
Analysis of Triglycerides,
C.
Litchfield, Academic Press, New
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Christie, W.W., Silver Ion Chromatography Using Solid-Phase
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a Bonded-Sulfonic Acid Phase, J.
Lipid Res. 30: 147 [-1473 (1989).
Ulbenh, E, and E. Achs, Argenta.
1103
lion Chromatography
of Fatty
Acid Methyl Esters Using SilverLoaded Solid-Phase Extraction
Columns.
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504,202-206 (1990).
49. Hoving. E.B .• FAJ. Muskiet and
W. W. Christie.
Separation
of
Cholesterol Esters by Silver Ion
Chromatography Using High-Performance Liquid Chromatography or Solid-Phase
Extraction
Columns Packed with a Bonded
Sulfonic
Acid Phase,
Ibid.
565,102-110 (1991).
50. Augustyn. O.P.H .. D. Ferreira and
J.L.
Kock.
Differentiation
Between
Yeast Species.
and
Strains Within a Species. by Cellular Fatty Acid Analysis. 4. Saccharomyces sensu stricto. Henseniaspora. Saccharomycodes
and
Wickerhamiella,
System. App!.
Microbial. 14:324-334 (1991).
51. Augustyn, O.P.H., J.L.F. Kock
and D. Ferreira, Differentiation
Between
Yeast Species,
and
Strains Within a Species, by Cellular Fatty Acid Analysis. 5. A
Feasible
Technique?
Ibid.
15:105-115 (1992).
52. Kemppinen,
A .• and P. Kalo.
Fractionation of the Triacylglycerols of Lipase-Modified
Buller
Oil, J. Am. Oil Chern. Soc.
70: 1203-1207 (1993).
53. Chen, X.-H., J.-P. Franke, J. Wijsbeek and R.A. de Zeeuw, Study
of Lot-to-Lot Reproducibilities of
Bond Elut Certify and Clean
Screen DAU Mixed-Mode SolidPhase Extraction Columns in the
Extraction of Drugs from Whole
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Chromatogr.
617:147-151 (1993).
54. Shreedhara Murthy, R.S., and L.J.
Crane, Characterization of Cyano
Bonded Silica Phases from SolidPhase Extraction Columns. Correlation of Surface Chemistry
with Chromatographic Behavior,
Ibid. 542:205-220 (1991).
55. Junk, G.A., M.J. Avery and J.J.
Richard, Interferences in SolidPhase Extraction Using C 18 Bonded Porous Silica Cartridges. Anal.
Chern. 60: 1347-1350 (1988).
56. Crowley, X.w., V. Murugaiah, A.
Nairn and R.W. Giese, Masking
as a Mechanism for Evaporative
Loss of Trace Analyte, Especially
after Solid-Phase Extraction, J.
Chromatogr.
A., 699:395-402
(1995).
57. Nickmilder, M.J., D. Larinne. A.
Hassoun and P.E. Wallemacq,
Inhibitory Effects of Solvent and
Solid-Phase Extraction Residues
on Mixed Lymphocyte Cultures,
JPM, 32(1),31-33 (1994).
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