-
A Study of Polyethoxylated Alkylphenols by Packed Column
SupercriticalFluid ChromatographyB.J. Hoffman and L.T. Taylor
..........................................................................................................61
An Isocratic Liquid Chromatographic Method with Diode-Array
Detection forthe Simultaneous Determination of -Tocopherol,
Retinol, andFive Carotenoids in Human SerumS. Gueghuen, B. Herbeth,
G. Siest, and P. Leroy
............................................................................69
A Procedure for Sampling and Analysis of Air for Energetics
andRelated CompoundsM.A. Hable, J.B. Sutphin, C.G. Oliver, R.M.
McKenzie, E.F. Gordon, and R.W. Bishop..................77
Displacement Study on a Vancomycin-Based Stationary Phase
UsingN-Acetyl-D-Alanine as a Competing AgentI. Slama, C. Ravelet,
A. Villet, A. Ravel, C. Grosset, and E.
Peyrin..................................................83
Influence of Hydrolysis, Purification, and Calibration Method
onFurosine Determination Using Ion-Pair Reversed-Phase
High-PerformanceLiquid ChromatographyM.A. Serrano, G. Castillo,
M.M. Muoz, and A. Hernndez
..........................................................87
The Use of Nonendcapped C18 Columns in the Cleanup of
Clenbuterol and aNew Adrenergic Agonist from Bovine Liver by Gas
ChromatographyTandemMass Spectrometry AnalysisM. Fiori, C. Cartoni,
B. Bocca, and G. Brambilla
...........................................................................92
Simultaneous High-Performance Liquid Chromatographic
Determination ofParacetamol, Phenylephrine HCl, and
Chlorpheniramine Maleate inPharmaceutical Dosage FormsH. Senyuva
and T.
zden..............................................................................................................97
Evaluation of Select Variables in the Ion Chromatographic
Determination ofF, Cl, Br, NO3, SO42, and PO43 in Serum SamplesZ.
Benzo, A. Escalona, J. Salas, C. Gmez, M. Quintal, E. Marcano, F.
Ruiz, A. Garaboto,and F.
Bartoli...............................................................................................................................101
Temperature Effect on Peak Width and Column Efficiency in
SubcriticalWater ChromatographyY. Yang, L.J. Lamm, P. He, and T.
Kondo......................................................................................107
A High-Pressure Liquid ChromatographicTandem Mass
SpectrometricMethod for the Determination of Ethambutol in Human
Plasma,Bronchoalveolar Lavage Fluid, and Alveolar CellsJ.E. Conte,
Jr., E. Lin, Y. Zhao, and E. Zurlinden
...........................................................................113
Departments
Cover: Photomicrograph of MoS2 by Michael W. Davidson, National
Magnetic Field Laboratory, Florida State University atTallahassee.
Microscopes provided by Nikon Instrument Group, Melville, NY.
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Contents
C hroma t o gra p h i c S c i enc eJOURNAL OF Vol. 40, No. 2
FEBRUARY 2002Preston Publications
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61
Alkylphenol polyethoxylates (APEs) are a widely used group
ofnonionic surfactants in commercial production. Characterization
ofthe composition of APE mixtures can be exploited for
thedetermination of their most effective uses. In this study
samplemixtures contain nonylphenol polyethoxylates and
octylphenolpolyethoxylates. The separation of individual
alkylphenols byethoxylate units is performed by supercritical fluid
chromatography(SFC)-UV as well as normal-phase high-performance
liquidchromatographic (HPLC)-UV employing packed columns.
Thestationary phase and column length are varied in the SFC setup
toproduce the most favorable separation conditions.
Additionally,combinations of packed columns of different stationary
phases aretested. The combination of a diol and a cyano column is
found toproduce optimal results. An advantage of using packed
columnsinstead of capillary columns is the ability to inject large
amounts ofsample and thus collect eluted fractions. In this regard,
fractionsfrom SFC runs are collected and analyzed by flow
injectionanalysiselectrospray ionizationmass spectroscopy in order
topositively identify the composition of the fractions. In
comparingthe separation of APE mixtures by SFC and HPLC, it is
found thatSFC provides shorter retention times with similar
resolution. Inaddition, less solvent waste is produced using
SFC.
Introduction
Alkylphenol polyethoxylates (APEs) are referred to as
nonionicsurfactants. Since the mid 1940s, APEs have been used
commer-cially for their surfactant ability. The term surfactant
includessurface-active compounds characterized by their ability to
con-centrate at surfaces and form micelles in solution (1). They
havebeen used in a wide variety of applications including
industrialprocess aids, dispensing agents in paper and pulp
production,emulsifying agents in latex paints and pesticide
formulations,flotation agents, industrial cleaners (metal surfaces,
textile pro-cessing, and food industry), and household cleaners
(1). Thesecompounds are commercially available as oligomeric
mixtureswith varying ethoxylate chain lengths as well as varying
alkylsizes. Certain APEs have been determined to be estrogenic in
fish,birds, and mammals (2).
APEs contain two main molecular regions: the polyethoxylate(POE)
chain (EO) is polar and thus hydrophilic and the alkyl-phenol is
the hydrophobic area. The hydrophilic nature of the EOis attributed
to the hydration of the ether-linked oxygen atoms(3). A technical
synthesis of APEs start with phenol, which is alky-lated by
trimethylpentane and thus produces octylphenol (OP), orby nonene
isomers, which forms nonylphenol (NP) in an acid-cat-alyzed
process. Ethoxylation is performed by using KOHethanolas a catalyst
with a known ratio of ethylene oxide to thealkylphenol (1). The
reaction results in an oligomeric mixture ofthe alkylphenol
containing an EO chain of varying lengths.
The separation and identification of the components of an
APEmixture can be useful for the determination of their most
effec-tive applications. Several different types of chromatography
havebeen studied previously in efforts to achieve better
separationconditions. Gas chromatography (GC) coupled with flame
ioniza-tion detection as well as mass spectrometry (MS) has been
used inthe analysis of APEs (4). Isomers of each oligomer tend to
be sep-arated into clusters by GC. Usually, it is necessary to
derivatizesamples containing APEs for analysis by GC, because the
com-pounds are not very volatile. GC poorly separates higher
molec-ular-weight oligomers because of their lower volatility.
High-performance liquid chromatography (HPLC) has beenused to
separate APEs of higher mass oligomers. Both reversed-phase (3) and
normal-phase (57) chromatographic separationshave been performed on
solutions containing APEs. Eacholigomer is separated by an
ethoxylate unit, and isomers of eacholigomer tend to coelute.
Recently, Gundersen used a graphiticcarbon column in research to
separate isomers of individualethoxylated alkylphenols by HPLC (8).
Ferguson et al. usedreversed-phase HPLCelectrospray ionization
(ESI)MS to ana-lyze APEs and their metabolites in aquatic
environments (9).Normal-phase HPLCESIMS was used by Shang et al. to
quanti-tate NPEOs in marine sediment (10).
In addition to traditional forms of chromatography,
supercrit-ical fluid chromatography (SFC) has been employed for APE
sep-aration. SFC has advantages over both HPLC and GC. SFC
canoperate at lower temperatures than GC, allowing samples that
arethermally labile to be analyzed. Supercritical fluids have
densitiessimilar to liquids and diffusivities similar to gases.
These qualitiesallow large molecular-weight molecules that are not
volatile to be
Abstract
A Study of Polyethoxylated Alkylphenols by PackedColumn
Supercritical Fluid Chromatography
Brian J. Hoffman and Larry T. TaylorVirginia Tech, Department of
Chemistry, Blacksburg, VA 24061-0212
Reproduction (photocopying) of editorial content of this journal
is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
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Journal of Chromatographic Science, Vol. 40, February 2002
62
separated by SFC similar to HPLC but with shorter retentiontimes
because of the physical properties of supercritical fluids.This
reduces solvent waste and decreases the total analysis
time.Capillary-column SFC using flame ionization detection
(11,12)has been used to separate both NPEO and OPEO. Because
asample is generally destroyed by this method, it is not possible
todirectly determine analyte identity. Peak identity can be
surmisedby comparing retention times of samples with other APE
mix-tures that contain a large fraction of a known single oligomer.
Adisadvantage associated with capillary columns is the inability
toinject large sample volumes, which precludes
semipreparativefraction collection.
In addition, OPEO mixtures have been separated on packed-column
SFC using reversed-phase (13,14) and normal-phase(15,16) packing
material. Both Takeuchi and Saito and Giorgettieet al. used C18
packed columns to separate OPEO samples bySFC. Takeuchi and Saito
found that a microcolumn (1.0 500mm) had the best separation
performance, but a semimicro-column (1.7 250 mm) produced the best
results. A conventionalcolumn (6.0 250 mm) was used in their
research for preparativepurposes. Packed-column SFC allows larger
amounts of sampleto be injected into the system for the
semipreparative collectionof analyte fractions. Giorgettie et al.
studied mixed mobile phasesusing the addition of a modifier in
order to make their mobilephase more polar. They used pressure
programming and a modi-fier additon to produce optimum separations.
Highly efficientseparations were produced under constant modifier
concentra-tion and pressure programming.
The object of this study was to compare the ability of
normal-phase packed columns to separate APEs on an SFC
system.Individual packed columns as well as stacked packed columns
ofdifferent stationary phases were used in the SFC
experiments.Additional goals of this study were to identify the
componentsthat gave rise to the chromatographic peaks in hopes of
pro-ducing individual ethoxylated alkylphenol standards.
Fractionsthat contain a single ethoxylate compound could later be
used asstandards for quantitating APEs in a variety of
applications. Acomparison of the ability of SFC and HPLC to
separate APEsusing normal-phase packed columns was also
studied.
Experimental
Packed-column SFCA Berger (Newark, DE) SFC system was used in
the SFC anal-
ysis. A Berger autosampler with a 10-L injection loop was
usedfor conventional sample analysis, and a 75-L injection loop
wasused for the injection of semipreparative samples.
SFC-gradecarbon dioxide (Air Products and Chemicals, Inc.,
Allentown, PA)was used with methanol (Burdick & Jackson,
Muskegon, MI) as amodifier. The mobile phase flow rate was 2.0
mL/min. The oventemperature was set at 60C, and the outlet pressure
was kept at120 atm. Absorbance was read at 225 nm by a
diode-arraydetector. The detection wavelength was determined by
finding themaximum absorbance of an individual APE sample by
obtainingits UVvis spectrum. Supelcosil LC-Diol, Supelcosil
LC-CN(Supelco, Bellefonte, PA), and Spherisorb NH2 (Waters,
Millford,
MA) columns were used for the chromatographic separation ofthe
APE mixtures. All columns measured 4.6 250 mm with a 5-m particle
size. A diol bonded silica guard column was used.
Normal-phase HPLCFor HPLC analysis, a Hewlett-Packard (Little
Falls, DE) 1050
Series HPLC system was used with a variable wavelength
detector(reading 225 nm) and an inline vacuum degasser. Injections
weremade manually with a Rheodyne (Rohnert Park, CA)
injectorequipped with a 20-L injection loop. Data were collected
andchromatograms were processed by MassLynx software
(FisionsInstruments, Altricham, U.K.). A Supelcosil LC-Diol column
(4.6 250 mm, 5 m) was used for the chromatographic separation ofthe
APE mixtures.
Flow injection analysisMSA Fisions Instruments VG Platform MS
was used for the mass
analysis of collected sample fractions. All samples were
analyzedunder positive ESI. A syringe pump (Harvard Apparatus,
SouthNatick, MA) supplied an 80:20 methanolwater mobile phase tothe
probe. Samples were injected by a Rheodyne injectorequipped with a
20-L injection loop. Nitrogen was used as boththe drying and sheath
gas. Data were collected and analyzed byMassLynx software.
Alkylphenol samplesPOE-(4)-NP (ChemService, West Chester, PA)
and Triton N-101
(Sigma-Aldrich, Milwaukee, WI) were used as NPEO
mixtures.POE-(5)-tert-OP (ChemService) was used as an OPEO
mixture.All of the samples that were analyzed by SFC were dissolved
inmethanol, and samples analyzed by normal-phase HPLC weredissolved
in hexane. The Triton N-101 sample that was used forHPLC was
dissolved in 9:1 hexaneacetone in order to increasesolubility. HPLC
samples were prepared at approximately 1.0mg/mL, and SFC samples
were prepared at approximately 2.0-mg/mL concentrations.
Semipreparative SFCA tee was placed inline between the column
and diode-array
detector of the SFC system, splitting effluent approximately
75%to the collection and 25% to the detector. Eluent was
divertedusing a portion of fused-silica capillary tubing. Fractions
werecollected in preweighed 16-mL collection vials. Absorbance
wasmonitored, and fractions were collected manually between
min-imum absorbance values. POE-(4)-NP and POE-(5)-tert-OP
wereseparated in this fashion. Fractions were evaporated by
nitrogenblow-down on a hot plate. The remaining residue was
weighed.The fractions were then diluted to 10.0 mL with
methanol.Fractions were analyzed by SFC-UV followed by flow
injectionanalysis (FIA)ESIMS for purity.
FIAESIMS methodSFC-collected fractions were evaporated by
nitrogen blow-
down and weighed. Collected fractions were then dissolved
inmethanol. Optimal MS settings were found by injecting each
frac-tion and tuning the instrument. Fractions were then
reinjected,and mass-spectral data were recorded and analyzed. The
sourcetemperature was set at 100C. ESI nebulizing gas flow was set
at
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Journal of Chromatographic Science, Vol. 40, February 2002
63
20 L/h, and the drying gas flow was 300 L/h. Samples
wererecorded in full-scan mode from m/z 200 to 700. The cone
voltageranged from 52 to 75 V, and the high voltage lens and ESI
capil-lary voltage were kept at 0.88 and 3.46 kV, respectively.
HPLC methodHexane and isopropanol were used as the mobile phase.
A linear
gradient was used starting with 100% hexane and then changingto
70:30 hexaneisopropanol over 30 min. From t = 30 to 35 min,the
mobile phase was returned to 100% hexane and held for 5 min in
order to equilibrate. POE-(4)-NP and POE-(5)-tert-OPwere separated
in this fashion.
Results and Discussion
APEs are complex mixtures that provide moderate challengesfor
chromatographic techniques. Our research studied how thetotal
column length, stationary phase, and column stacking orderof
different stationary phases affect the SFC separation of
ethoxy-late units in APE mixtures. Our goal was to find a setup
that pro-duced the best separation. In order to accomplish this we
kept allsystem parameters constant throughout the study other
thancolumn setup and modifier gradient. All of the columns usedwere
uniform in size (4.6 250 mm, 5 m) in order to allow us toverify the
effect of column length and packing material. POE-(4)-NP was used
in all of the diol column studies because of its shortelution
time.
POE-(4)-NP was separated on a combination of one-, two-,
andthree-packed diol columns connected in series to study the
effectof column length (Figure 1). A single diol column poorly
sepa-rated the sample. Baseline separation was not achieved with
a
single column. SFC separation on two diol columns
increasedseparation, but early eluting peaks were not baseline
separated.Using two diol columns, SFC separation was comparable
withnormal-phase HPLC using one diol column. For
comparison,POE-(4)-NP, POE-(5)-tert-OP, and Triton N-101 were
separated bySFC on two diol columns and HPLC on one diol column
(Figures24). The retention time of the chromatographic peaks for
SFCseparation using two diol columns was considerably lower
thannormal-phase HPLC separation using one diol column (Tables Iand
II shows data for the NPEO sample and Table III shows datafor the
OPEO sample). The addition of a third diol column to theSFC system
generated a better separation, but later-eluting peaksbegan to
broaden.
The effect of the stationary phase on separation was
sequen-tially tested using a single diol, amino, and cyano column
(Figure5). The retention of oligomers with longer ethoxylated
unitsvaried with each stationary phase tested. The diol column had
theleast retention, the amino column had intermediate retention,and
the cyano column had the greatest retention. It was not pos-sible
to elute all of the compounds off the cyano column using
thecorresponding gradient. In general, a larger methanol
modifierconcentration was needed to elute longer ethoxylate-chain
com-
Figure 2. Chromatograms of POE-(4)-NP using (A) normal-phase
HPLC-UVwith one Supelcosil LC-Diol column and (B) SFC-UV with two
Supelcosil LC-Diol columns. The peak annotations represent the
number of ethoxylate units.
Figure 1. Packed-column supercritical fluid chromatograms using
stacked diolcolumns: (A) one Supelcosil LC-Diol column, (B) two
Supelcosil LC-Diolcolumns, and (C) three Supelcosil LC-Diol
columns. The sample used in eachchromatogram was POE-(4)-NP (2.0
mg/mL). A linear modifier gradient wasused by the following
program: 10.0% methanol was increased to 26.0% at arate of 0.6%/min
with a 2.0-min hold and then returned to 10.0% in 4.0 minfollowed
by a 2.0-min hold.
-
Journal of Chromatographic Science, Vol. 40, February 2002
64
pounds. Because of this, we can conclude that APEs with a
longerethoxylate chain are more polar than those with shorter
chains.Following this reasoning, the cyano column must be the
mostpolar stationary phase because it retained the more polar
compo-nents longer, and the diol column is the least polar.
Columns with different stationary phases were coupled inseries
to test how the arrangement would affect the retention ofan APE
sample. Two column arrangements were tested. The firstconsisted of
one diol column followed by one cyano column. Thesecond setup
contained three columns, a diol column, a cyanocolumn, and an amino
column in series (Figure 6). A steeper gra-dient was needed than
previously used in order to elute all of thecompounds because of
the presence of the cyano column (as pre-viously mentioned). The
modifier gradient that was used isdescribed in Figure 6.
One of our goals in this study was to achieve separation
thatwould allow us to easily collect individual oligomers for use
asstandards. The combined diolcyano setup rendered shorterretention
times than the combined diolcyanoamino setup;therefore, this
arrangement was used for preparative fraction col-lection. In the
chromatograms of stacked columns using differentstationary phases,
peak splitting was observed for later-eluting
peaks. POE-(4)-NP and POE-(5)-tert-OP were separated, and
fivefractions of each sample were collected. A large volume (75 L)
ofconcentrated sample was injected six to eight times in the
collec-tion process. Isolated fractions were reanalyzed both by SFC
forpurity (Figures 7 and 8) and FIAESIMS for identification.
Theconcentrations used for the semipreparative work caused
thechromatographic peaks to significantly broaden and in somecases
combine. Because of this phenomenon we were not able tocollect
individual fractions of the two initial oligomers of POE-(4)-NP and
fractions of the three initial oligomers of POE-(5)-tert-OP as
evidenced by the SFC-UV of the early fractions.
FIAMS was used to identify the components in each fraction.ESIMS
was chosen because it is amenable to high-molecular-weight analytes
and works well with liquid mobile phases.Samples were dissolved in
methanol (a compatible solvent forESIMS), which made ESIMS a
desirable tool for fraction iden-tification. It was possible to
produce sodium-adducted molecularions rather easily. In order to
create an optimum response, thefractions were first injected and
the cone voltage varied in orderto produce the greatest response
for each individual analyte. AfterMS tuning conditions were
perfected, the fractions were rein-jected into the instrument. A
spectrum was created between
Figure 4. Chromatograms of Triton N-101 using (A) normal-phase
HPLC-UVwith one Supelcosil LC-Diol column and (B) SFC-UV with two
Supelcosil LC-Diol columns. The peak annotations represent the
number of ethoxylate units.
Figure 3. Chromatograms of POE-(5)-tert-OP using (A)
normal-phase HPLC-UVwith one Supelcosil LC-Diol column and (B)
SFC-UV with two Supelcosil LC-Diol columns. The peak annotations
represent the number of ethoxylate units.
-
Journal of Chromatographic Science, Vol. 40, February 2002
65
m/z 200 and 700 by averaging scans of the injected
sample.Figures 9 and 10 show the average mass spectrum of each
fraction.The spectra confirm that each chromatographic peak varied
byone ethoxylated unit (a separation of m/z 44 represents an
ethoxy-late unit). It was possible to identify NP3EO through NP7EO
inbasically pure collected fractions of POE-(4)-NP and OP5EOthrough
OP8EO in fractions collected from POE-(5)-tert-OP.
Major ion peaks consisted of Na+ adduct ions, and minor
peakswere produced by K+ adduct ions under positive electrospray
con-ditions. Trace levels of sodium and potassium must be present
inthe mobile phase that was used for FIAESIMS because elec-trolyte
was not added to the solutions. According to Okadasresearch (17),
APEs have an affinity for alkali metals and have a
flexible structure that allows them to form complexes with
alkalimetals. This explains the ion pairing seen in the mass
spectra.Crescenzi et al. performed an experiment to see if the
detectorresponse would decrease because of the complexation
ofoligomers competing for the limited metal pool available.
Whenequivalent amounts of ethoxylated compounds were analyzed
byESIMS, it was found that the detector response increased
expo-nentially from 1 to 6 EO units and then leveled off at 8 EO
units(the scope of the study) (18). A decrease in signal was most
notice-able for lower ethoxylated oligomers. This can be explained
bynoting that ethoxylated compounds can form increasingly
stablecomplexes with alkali metal ions as the EO unit number
increases(17).
Table I. Chromatographic Peak Retention Times of POE-(4)-NP
(NPEO) Separated by SFC Using TwoSupelcosil LC-Diol Columns and
HPLC Using OneSupelcosil LC-Diol Column
EO unit SFC RT* HPLC RT
2 7.18 9.143 7.86 9.934 8.64 10.665 9.68 11.826 10.61 13.087
11.56 14.468 12.49 15.839 13.43 17.28
10 14.37 18.7411 15.16
* RT, retention time.
Table II. Chromatographic Peak Retention Times of TritonN-101
(NPEOs) Separated by SFC Using Two SupelcosilLC-Diol Columns and
HPLC Using One Supelcosil LC-Diol Column
EO unit SFC RT* HPLC RT
2 7.29 9.883 8.02 10.684 8.83 11.845 9.75 13.086 10.66 14.337
11.57 15.558 12.48 16.809 13.36 18.06
10 14.20 19.3911 15.03 20.6812 15.84 22.2913 16.61 24.1114
17.3715 18.1016 18.8117 19.5818 20.10
* RT, retention time.
Table III. Chromatographic Peak Retention Times of
POE-(5)-tert-OP (OPEOs) Separated by SFC Using TwoSupelcosil
LC-Diol Columns and HPLC Using OneSupelcosil LC-Diol Column
EO unit SFC RT* HPLC RT
2 6.79 9.283 7.48 10.064 8.20 10.925 9.05 12.086 10.00 13.397
10.96 14.758 11.91 16.109 12.88 17.51
10 13.86 18.9611 14.8012 15.76
* RT, retention time.
Figure 5. Packed-column supercritical fluid chromatograms using
singlecolumns of different polar packing material: (A) Supelcosil
LC-Diol column, (B) Spherisorb NH2 column, and (C) Supelcosil
LC-PCN column. The sampleused in each chromatogram was POE-(4)-NP
(2.0 mg/mL). A linear modifiergradient was used by the following
program: 10.0% methanol was increasedto 26.0% at a rate of 0.6%/min
with a 2.0-min hold and then returned to 10.0%in 4.0 min followed
by a 2.0-min hold.
-
Journal of Chromatographic Science, Vol. 40, February 2002
66
It was important to perform chromatographic separations
withabsorbance detection on the fractions as well as MS analysis,
thusallowing us to positively identify sample components because
MScould not detect all of the compounds present. The first
fractionof both POE-(4)-NP and POE-(5)-tert-OP contained more
thanone compound (as seen in their SFC-UV chromatograms). Thesodium
ion affinity of the smaller ethoxylate chain compounds islower than
the larger chain oligomers, and because of this they
were not detectable in the mass spectra.APEs can be categorized
by their average ethoxylate unit value.
According to Wang and Fingas (3), all of the oligomers
havealmost identical molar absorptivity, which allows integrated
chro-matographic peak areas to be used directly to determine the
molefraction of each oligomer. POE-(4)-NP contained NP
predomi-nantly with short ethoxylate chains. NP2EO through
NP11EOwere observed in its SFC-UV separation. An average
ethoxylate
Figure 6. Packed-column supercritical fluid chromatograms using
stackedcolumns of different polar stationary phases: (A) one
Supelcosil LC-Diolcolumn and one Supelcosil LC-PCN column and (B)
one Supelcosil LC-Diolcolumn, one Supelcosil LC-PCN column, and one
Spherisorb NH2 column.The sample used in each chromatogram was
POE-(4)-NP (2.0 mg/mL). Multiplelinear modifier gradients were used
by the following program: 10.0% methanolwas increased to 13.2% by
0.5%/min and then continued to 14.4% at0.7%/min, 16.6% at 0.8%/min,
20.0% at 1.0%/min, 40.0% at 8.0%/min (heldfor 5.0 min), and then
returned to 10.0% at 15.0%/min.
Figure 7. Supercritical fluid chromatograms of collected
POE-(4)-NP fractions.Separation was conducted on one Supercosil
LC-Diol column and oneSupelcosil LC-PCN column in series (the
system settings were the same asFigure 3).
Figure 8. Supercritical fluid chromatograms of collected
POE-(5)-tert-OP frac-tions. Separation was conducted on one
Supelcosil LC-Diol column and oneSupelcosil LC-PCN column in series
(the system settings were the same asFigure 3).
Figure 9. Positive-ion FIAESIMS of POE-(4)-NP fractions operated
in full-scanmode. Ions were in the form of (M+Na)+ and each were
separated by m/z 44(the mass of one ethoxyl unit): (A) fraction 1,
cone voltage of 59 V; (B) fraction2, cone voltage of 53 V; (C)
fraction 3, cone voltage of 62 V; (D) fraction 4, conevoltage of 65
V; and (E) fraction 5, cone voltage of 67 V. Each spectrum
wasaveraged over the sample injection peak.
-
Journal of Chromatographic Science, Vol. 40, February 2002
67
unit value of 4.20 was calculated from peak areas.
POE-(5)-tert-OP had a similar distribution as POE-(4)-NP. Its
average ethoxy-late unit value was calculated as 4.48, and it
contained OP2EOthrough OP12EO in its SFC-UV separation. Triton
N-101 con-tained a greater range of NPEOs. Its calculated average
ethoxylateunit was 9.97. NP2EO through NP18EO were observed in its
SFC-UV chromatogram. Higher EO peaks were detected in SFC
sepa-rations, which were not detected by HPLC analysis. Wang
andFingas produced similar average EO unit values from their
capil-lary SFC data. Their analysis of Igepal CO430 (trade name
forPOE-(4)-NP), Triton X-45 (trade name for POE-(5)-tert-OP),
andTriton N-101 produced average EO values of 4.14, 4.50, and
9.52,respectively (11,12). We used the chromatographic data from
theSFC-UV separations on two diol columns to calculate our
averageEO values.
Conclusion
Normal-phase packed-column SFC produced a similar separa-tion of
APE mixtures compared with normal-phase HPLC.Column length,
stationary phase, and column combinations withdifferent stationary
phases all affected the separation of the APEmixtures tested.
Longer column lengths increased the separationof oligomers.
More-polar stationary phases retained oligomerswith larger
ethoxylate units for a longer time. A combination ofcolumns with
different stationary phases produced separationscombining both the
effects of longer columns and the separationability of each
stationary phase. Retention times for SFC separa-
tions were notably shorter than normal-phase HPLC. One ofSFCs
advantages is its ability to use longer combined columnlengths
without elevated back pressure, which occurs in HPLC.Combining
multiple columns with different stationary phasesseemed to provide
the best separation.
An advantage of using packed columns over the use of
capillarycolumns is the ability to inject larger amounts of sample
and col-lect eluted fractions. It is possible to isolate and
identify individualAPEs. Additionally, it is possible to identify
the remaining chro-matographic peaks because of each peak differing
by one ethoxy-late unit. Our study demonstrated the importance of
using bothabsorbance detection as well as MS. MS alone did not show
all thecomponents of our initial fractions because of the
decreaseddetector response.
Less solvent waste was produced using SFC compared withHPLC.
Each SFC separation that used cyano packing as part of itscolumn
arrangement used 6.7 mL of methanol. The remainingSFC setups (the
studies of column length and stationary phase)used 11.8 mL of
methanol. All separations performed by normal-phase HPLC used 34.75
mL of hexane and 5.25 mL isopropanolfor a combined volume of 40 mL.
The HPLC system used almost600% more solvent than the SFC system
using a cyano stationaryphase and over 330% more than the other SFC
setups studied(this is not including the volume of solvent needed
to initiallyequilibrate the systems). The reduction of solvent
waste is animportant step of reducing pollution.
Because of the fact that APEs are used as industrial cleaners
andother processing aids, they enter wastewater and end up insewage
treatment plants. Some APE waste is transferred into theenvironment
and metabolized into lower ethoxylated alkylphe-nols, which are
considered endocrine disrupters (2). APEs havebeen found in fish,
river sediment, and other environmental sam-ples through analytical
techniques (1,4,9,10,1822). The resultsof our study could lead to
the further use of the method developedfor applications in the
analysis of environmental samples.Additionally, our method could be
altered for use in a futurelarge-scale separation and collection of
individual ethoxylatedalkylphenols. Access to standards of
individual ethoxylatedalkylphenols is important for their
quantitative analysis.
Acknowledgments
We would like to acknowledge Dr. Clifford P. Rice
(USDAARS/NRI/EQL, Beltsville, MD) for APE information and
AirProducts and Chemicals, Inc. for supplying SFC-grade
carbondioxide.
References
1. B. Thiele, K. Gunther, and M.J. Schwunger. Alkylphenol
ethoxylates:trace analysis and environmental behavior. Chem. Rev.
97: 324772(1997).
2. R. White, S. Jobling, S.A. Hoare, J.P. Sumpter, and M.G.
Parker.Environmentally persistent alkylphenolic compounds are
estrogenic.Endocrinology 135: 17582 (1994).
Figure 10. Positive-ion FIAESIMS of POE-(5)-tert-OP fractions
operated infull-scan mode. Ions were in the form of (M+Na)+ and
each were separated bym/z 44 (the mass of one ethoxyl unit): (A)
fraction 1, cone voltage of 63 V; (B)fraction 2, cone voltage of 68
V; (C) fraction 3, cone voltage of 65 V; (D) frac-tion 4, cone
voltage of 75 V; and (E) fraction 5, cone voltage of 75 V. Each
spec-trum was averaged over the flow injection peak.
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Journal of Chromatographic Science, Vol. 40, February 2002
68
3. Z. Wang and M. Fingas. Rapid separation of nonionic
surfactants ofpolyethoxylated octylphenol and determination of
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5. R.E.A. Escott, S.J. Brinkworth, and T.A. Steedman. The
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6. I. Zeman. Applications of bonded diol phases for separation
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7. A.M. Rothman. High-performance liquid chromatographic
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8. J.L. Gundersen. Separation of isomers of nonylphenol and
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9. P.L. Ferguson, C.R. Iden, and B.J. Brownawell. Analysis
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10. D.Y. Shang, M.G. Ikonomou, and R.W. MacDonald.
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11. Z. Wang and M. Fingas. Quantitative analysis of
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13. A. Giorgettie, N. Pericles, H.M. Widmer, K. Anton, and P.
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Mixed mobile phases and pressure programming in packed column
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pressurepulsed valve. J. Microcolumn Sep. 6: 44957 (1994).
17. T. Okada. Efficient evaluation of poly(oxyethylene) complex
forma-tion with alkali-metal cations. Macromolecules 23: 421619
(1990).
18. C. Crescenzi, A. Di Corcia, R. Sampri, and A.
Marcomini.Determination of nonionic polyethoxylate surfactants in
environ-mental waters by liquid chromatography/electrospray mass
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19. T.L. Keith, S.A. Snyder, C.G. Naylor, C.A. Staples, C.
Summer, K. Kannan, and J.P. Giesy. Identification and
quantification ofnonylphenol ethoxylates and nonylphenol in fish
tissues fromMichigan. Environ. Sci. Technol. 35: 1013 (2001).
20. H.B. Lee, T.E. Peart, D.T. Bennie, and R.J. Maguire.
Determination ofnonylphenol and their carboxylic acid metabolites
in sewage treat-ment plant sludge by supercritical fluid carbon
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21. T.L. Potter, K. Simmons, J. Wu, M. Sanchez-Olvera, P.
Kostecki, andE. Calabrese. Static Die-away of a nonylphenol
ethoxylate surfactantin estuarine water samples. Environ. Sci.
Technol. 33: 11318 (1999).
22. M. Petrovic and D. Barcelo. Determination of anionic and
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Manuscript accepted December 7, 2001.
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69
An isocratic high-performance liquid chromatography (HPLC)method
for the simultaneous determination of -tocopherol,retinol, and five
carotenoids (luteinzeaxanthin, -cryptoxanthin,lycopene, and - and
-carotene) in human serum is described.Serum samples are
deproteinized with ethanol and extracted oncewith n-hexane.
Resulting extracts are injected onto a C18 reversed-phase column
eluted with methanolacetonitriletetrahydrofuran(75:20:5, v/v/v),
and full elution of all the analytes is realizedisocratically
within 20 min. The detection is operated using threechannels of a
diode-array spectrophotometer at 290, 325, and 450 nm for
tocopherol, retinol, and the carotenoids, respectively.An internal
standard is used for each channel, which improvesprecision. The
choice of internal standards is discussed, as well asthe extraction
protocol and the need for adding an antioxidantduring the
extraction and chromatographic steps. The analyticalrecoveries for
liposoluble vitamins and carotenoids are more than85%. Intra-assay
relative standard deviation (RSD) values (n = 20)for measured
concentrations in serum range from 3.3% (retinol) to 9.5%
(lycopene), and interassay RSDs (n = 5) range from
3.8%(-tocopherol) to 13.7% (-cryptoxanthin). The present method
isused to quantitate the cited vitamins in healthy subjects (n =
168)from ages 9 to 55 years old.
Introduction
Retinol (vitamin A) and -tocopherol (vitamin E) are
nonen-zymatic antioxidants (1). Vitamin A acts as a direct
scavengerof reactive oxygen species (ROS) and is also thought to
inhibitfree radical synthesis via increasing the activity of
detoxifyingsystems (2).
Vitamin E protects unsaturated fatty acids located in both
celland organelle membranes against endo- and exogenous free
rad-icals and ROS, which are involved in the initiation and extent
ofmembrane damages caused by nonenzymatic lipid peroxidation(3,4).
Carotenoids act as ROS and free radical scavengers (5),stimulants
of immune response (6), and anticarcinogenic agents(7). Because of
their wide variety of functions and biologicalroles, clinical
interest in the evaluation of retinol, -tocopherol,and carotenoids
has increased in recent years owing to their roleas antioxidants,
which may be important in reducing the risk ofnumerous diseases
including cancer (811), coronary heart dis-ease (12,13), and
diabetes mellitus (1418).
Thus, rapid, simple, sensitive, and selective methods for
thesimultaneous determination of these antioxidants in
biologicalfluids are needed. As a matter of fact, the measurement
of anindividual class of antioxidants such as thiols (19),
hydrophilic,or liposoluble vitamins provides more information for
the mech-anistic evaluation of a clinical disease linked to
oxidative stressthan a total antioxidant status assay (20).
Numerous spectroscopic and separative methods have alreadybeen
reported for the assay of retinol, -tocopherol, andcarotenoids in
plasma or serum, and among them high-perfor-mance liquid
chromatography (HPLC) is one of the most pow-erful analytical tools
for this purpose (2125).
Both normal-phase (2628) and reversed-phase (2935)
HPLCconditions have been widely used. However, many of thesemethods
include gradient elution (3639), flow rate (34,36),wavelength
time-programmation (36,40), a switching devicebetween coupled
columns (41,42), and the use of two differentdetectors in series
(43,44). All of these approaches are time-con-suming because of
their long-equilibration period between eachrun and troublesome
because of the hyphenated systems needed.
Indeed, the main difficulty for the simultaneous determinationof
liposoluble vitamins and carotenoids results from their dif-ferent
spectral characteristics (absorption maxima vary in the
Abstract
An Isocratic Liquid Chromatographic Method withDiode-Array
Detection for the SimultaneousDetermination of -Tocopherol,
Retinol, and Five Carotenoids in Human Serum
Sonia Gueguen1, Bernard Herbeth1, Grard Siest1, and Pierre
Leroy21Inserm U525, Centre de Mdecine Prventive, 2 rue du Doyen
Jacques Parisot, 54500 Vandoeuvre-ls-Nancy, France and 2Thiols
andCellular Functions, Facult de Pharmacie, Universit Henri Poincar
Nancy 1, 30 rue Lionnois, 54000 Nancy, France
Reproduction (photocopying) of editorial content of this journal
is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
* Author to whom correspondence should be addressed: email
[email protected].
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Journal of Chromatographic Science, Vol. 40, February 2002
70
range of 292 to 450 nm). This problem has been solved by
usingmultichannel UVvis spectrophotometric detectors
(31,37,40,4547). More recently, a technique combining both
isocratic elu-tion in reversed-phase mode and diode-array detection
wasreported, providing selectivity between the three classes
ofliposoluble vitamins and thus a convenient way for their
simul-taneous measurements (32).
For all these methods, the preanalytical treatments,
especiallythe extraction procedure relying upon either
liquidliquid(2628,3035,39,43,47,48) or solidliquid (38,49,50)
partition,are critical steps to obtain reliable data.
This study deals with some improvements of a previouslyreported
method (32); the full validation of the optimized assay;and its use
to quantitate retinol, -tocopherol, luteinzeaxan-thin,
-cryptoxanthin, lycopene, and - and -carotene inhealthy
subjects.
Experimental
Chemicals, reagents, and standardsAll solvents and reagents used
were of analytical- or HPLC-
grade. Ultrapure water was prepared using a Milli-Q system
(Millipore Milford, MA). Tert-butylated hydroxytoluene (BHT)was
purchased from Sigma-Aldrich (St. Quentin Fallavier,France).
All-trans retinol (henceforth simply referred to as
retinol),retinol acetate, -tocopherol, -tocopherol acetate, and
-carotene standards were obtained from Fluka (Buchs,Switzerland).
Zeaxanthin and -cryptoxanthin were a generousgift from
Hoffman-Laroche (Basle, Switzerland). Lycopene andechinenone were
purchased from CaroteNature (Lupsingen,Switzerland). Stock
solutions of retinol, -tocopherol, and theircorresponding internal
standards (acetate form) were preparedin ethanol (EtOH) added with
0.01% (w/v) BHT. Carotenoidswere prepared in tetrahydrofuran (THF)
added with 0.01% BHT.Stock solutions were protected from light in
ambered glass bot-tles, titrated by spectrophotometry using their
specificabsorbance (Table I), and stored under nitrogen at 80C for
upto 2 mo. The concentrations of stock solutions were 0.250.5mg/mL
for retinol and retinol acetate, 34 mg/mL for -toco-pherol and
-tocopherol acetate, and 0.10.2 mg/mL forcarotenoids.
Daily working solutions for calibration curves were preparedby
diluting stock solutions in EtOH containing 0.01% BHT. Theranges of
tested concentrations are indicated in Table II. Aninternal
standard mixture containing retinol acetate, -toco-
pherol acetate, and echinenone was also prepareddaily following
a similar procedure (combining100 L of each stock solution of
internal standardand diluting the volume to 20 mL withEtOH0.01%
BHT). All the operations were per-formed by handling solutions in
darkness and ice.
The standards of -carotene and zeaxanthinwere used to quantitate
-carotene and bothlutein and zeaxanthin, respectively.
Blood collection and storage conditions Blood was collected at
the antecubital vein of
168 healthy control subjects from ages 9 to 55years old
(informed consent was obtained, andthe research protocol was in
agreement with theHelsinki Declaration) in a reclined position in
drytubes (Vacutainer Tube, Becton Dickinson,Grenoble, France).
Blood samples were cen-
Table I. Characteristics of Standards Used
MaximumMolecular weight wavelength
Compounds (g/mol) (nm) A1%1 cm* (mol1/L/cm1)
Retinol 286.5 325 1835 (32,61) 52573Retinol acetate 328.5 326
1550 (32,61) 50912-Tocopherol 430.7 292 75.8 (45) 3265-Tocopherol
acetate 472.8 290 40 (32) 1891Echinenone 550.9 458 2244 123622
(Hoffmann-Laroche data source)
Luteinzeaxanthin 568.9 452 2765/2416 (45)
157301/137446-Cryptoxanthin 552.9 452 2486 (45) 137451Lycopene
536.9 472 3450 (32,61) 185231-Carotene 536.9 450 2620 (35) 140667*
In EtOH as the solvent. Data references appear in the
parentheses.
Table II. Equations of Calibration Curves and Values of LODs and
LOQs*
Equations of calibration curves
Concentration Slope Intercept Correlation LOD LOQ range (mol/L)
(SD, n = 5) (SD, n = 5) coefficient (mol/L) (mol/L)
Retinol 0.457.50 0.16 (0.015) 0.021 (0.016) 0.998 0.45
0.66-Tocopherol 4.8080.0 0.01 (0.000) 0.027 (0.008) 0.996 2.64
5.36Luteinzeaxanthin 0.101.90 0.35 (0.034) 0.024 (0.006) 0.997 0.06
0.11-Cryptoxanthin 0.091.50 0.34 (0.031) 0.022 (0.019) 0.996 0.03
0.09Lycopene 0.121.90 0.24 (0.018) 0.018 (0.020) 0.997 0.03
0.08-Carotene 0.132.00 0.35 (0.019) 0.014 (0.006) 0.997 0.03 0.06*
Each calibration curve included six points, and each point was
assayed in five replicates. Calculated by internal standardization:
(standard peak area/internal standard peak area)/standard
concentration. SD, standard deviation.
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Journal of Chromatographic Science, Vol. 40, February 2002
71
trifuged (1500 g for 15 min at 4C) within 2 h after
collection,and resulting serum samples were frozen in liquid
nitrogen untilHPLC analysis.
Serum sample treatmentAll the handling operations were carried
out in darkness. The
serum samples were rapidly thawed at room
temperature,homogenized, and 200 L was transferred into a
borosilicateglass tube kept on ice and 300 L of the internal
standard mix-ture added. After mixing with a vortex for 20 s,
proteins were pre-cipitated by adding 200 L of EtOH0.01% BHT, and
the volumewas diluted to 1 mL with ultrapure water. After mixing
with anorbital shaker at 2500 rpm for 1 min, 2 mL of
n-hexane0.01%BHT was added. The samples were shaken for 1 min and
cen-trifuged at 2700 g for 20 min at 4C.
The organic layer was carefully transferred into a glass tubeand
evaporated to dryness under a stream of nitrogen at
roomtemperature. The dried residue was redissolved in 25 L
ofTHF0.01% BHT and vortexed for 30 s. A 75-L amount ofmobile phase
was added, and the resulting mixture was vortexedfor another 30 s.
Samples were then transferred to 200-L insertvials and placed into
the HPLC autosampler.
HPLC system and operating conditionsThe HPLC system consisted of
an isocratic solvent delivery
pump (Model Kontron Instruments 422), an autosamplerequipped
with a 20-L injection loop, a cooling sample tray anda column oven
(Model AS-300, ThermoQuest, Les Ulis, France),a UVvis diode-array
detector (Model Gold LC-168, BeckmanCoulter, Fullerton, CA), and
data-processing software (Gold New,Beckman).
A guard column (8- 3-mm i.d.) packed with Nucleosil C18 (5 m)
(Macherey Nagel, Duren, Germany) and an analyticalcolumn (250- 3-mm
i.d.) packed with Nucleosil 100 C18 (5 m)(Macherey Nagel) were
eluted with a mobile phase consisting ofa mixture of
methanolacetonitriletetrahydrofuran (75:20:5,v/v/v) containing
0.01% (w/v) BHT. The mobile phase was filteredthrough a 0.45-m
Nylon membrane and was used at a columntemperature of 35C and a
flow rate of 0.6 mL/min. Three chan-nels corresponding with
different wavelength values were usedto acquire data for the
selective monitoring of -tocopherol (290nm), retinol (325 nm), and
carotenoids (450 nm) and theirrespective internal standard. During
analysis, the tray compart-ment containing sample vials was cooled
at 5C. After eachworking period (approximately 50 samples), it was
necessary torinse the column with methanol at a flow rate of 0.6
mL/min for20 min to eliminate highly hydrophobic compounds and
preventthe loss of column efficiency.
CalculationThe vitamin concentrations were determined from a
standard
curve of the peak-area ratio of the analyteinternal
standardplotted against the concentration of analyte (expressed in
micro-moles per liter). A linear least-square regression analysis
wasperformed for each analyte, and the standard curve was
repeatedif the correlation coefficient was below 0.990.
The detection limit (LOD) and the quantitation limit (LOQ)were
expressed, respectively, as:
LOD = (a0 + 3sa0) / a1 Eq. 1
and
LOQ = (a0 +10sa0) / a1 Eq. 2
where a1 is the slope, a0 the intercept, and sa0 the standard
devi-ation of the intercept (51).
Quality controlA human serum pool made with 1 mL of fresh serum
from 100
healthy subjects and stored at 80C was used for the
routinequality control. Aliquots were extracted and analyzed
accordingto the same procedure that was described previously.
Evaluationof the method performance was assessed by comparing
theresults of the quality control with the means and relative
stan-dard deviations (RSDs) calculated using results from several
pre-liminary runs (n = 20 per day for five days).
Results and Discussion
Optimization of sample treatment and HPLC techniqueThe basic
method used in this study has been described by
Talwar et al. (32). Some modifications relating to the
internalstandards, the sample preparation procedure, and the use of
anantioxidant during both the extraction and chromatography
pro-cesses have been made. We chose this method because it allowsin
a fast and easy way the simultaneous separation of the twoclasses
of lipophilic vitamins (namely retinol, -tocopherol,
andcarotenoids). Our main objective was to measure
simultaneouslylipophilic vitamins and carotenoids, which are the
most abun-dant in human serum. Thus, the separation of the isomers
ofretinol, -tocopherol, and carotenoids did not appear relevantfor
our present epidemiological studies.
In most methods, the use of an antioxidant during
sampletreatment was demonstrated to be necessary to prevent a
signif-icant loss in carotenoid contents, especially lycopene and
-carotene (32,37,39,40,47). Thus, we initially added 0.01%ascorbic
acid to the organic solvents used for the standardspreparation
(EtOH and THF) and to the mobile phase, as indi-cated by Talwar et
al. (32). After analyzing the same sample several times, we
observed a decrease of the carotenoid concen-trations, indicating
degradation as a function of time. We testedanother antioxidant
(BHT) that is widely used in other methods(37,39,47) and added it
to the mobile phase and all the solvents(EtOH, THF, and hexane)
used for the standard and samplepreparation. Indeed, hexane
containing BHT efficiently pro-tected the carotenoids from
degradation during the evaporationof the extractive organic layers,
and the addition of BHT to themobile phase also prevented any loss
of these analytes and prob-ably helped increase the longevity of
the column by neutralizingperoxides present in THF. Moreover, we
observed that decreasingthe evaporation temperature from 40C to
room temperaturesignificantly increased carotenoid recoveries, as
already noted bydifferent authors (39,43).
Other parameters have to be optimized in order to provide
thebest conditions for the extraction of liposoluble vitamins
and
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Journal of Chromatographic Science, Vol. 40, February 2002
72
carotenoids. The addition of ultrapure water to the
deproteinizedserum with EtOH has been noted to improve the
recoveries ofcarotenoids and liposoluble vitamins (37,52). We
tested severalEtOHwater proportions in the 1:4 to 1:1 range (v/v)
in order toobtain the highest recoveries, and we selected the 1:1
(v/v) pro-portion. Single and double extraction steps with n-hexane
(anincrease of the shaking period) were tested, but no
significantimprovement of recoveries was observed.
The method previously described (32) used two internal
stan-dards: retinol acetate as an internal standard for retinol,
and -tocopherol acetate as an internal standard for both
-toco-pherol and carotenoid. We used a third internal standard
(echi-
nenone) for the quantitation of the carotenoids. Echinenone is
asynthetic carotenoid and has a structure and chemical
propertiesvery similar to the naturally occurring carotenoids in
serum.Thus, the use of echinenone is preferable to the use of
retinolacetate and -tocopherol acetate or tocol currently used in
othermethods (34,43,47), because it is detected at the same
wave-length as the other carotenoids and is light- and
temperature-sensitive as other carotenoids. Thus, the use of three
internalstandards allows for a better quality control and helps to
correctanalytical variations occurring for each liposoluble vitamin
andcarotenoid during the extraction and chromatography
pro-cesses.
Because no loss of analytes was observed in serum extractskept
in darkness for at least 24 h at 5C, as already reported (39),the
automation of the technique was possible with a highthroughput of
samples (approximately 30 per day).
Several methods have been developed to measure the main
Figure 1. Typical chromatograms corresponding with a mixture of
retinol, -tocopherol, and carotenoid standards: (A) channel 1,
diode-array detectionat 290 nm for -tocopherol and -tocopherol
acetate; (B) channel 2, diode-array detection at 325 nm for retinol
and retinol acetate; and (C) channel 3,diode-array detection at 450
nm for carotenoids and echinenone. The peaknumbers are as follows:
(1) 26 mol/L -tocopherol, (2) -tocopherol acetate(the internal
standard), (3) 2.43 mol/L retinol, (4) retinol acetate (internal
stan-dard), (5) 0.62 mol/L luteinzeaxanthin, (6) 0.49 mol/L
-cryptoxanthin, (7)echinenone (internal standard), (8) 0.62 mol/L
lycopene, (9) -carotene, and(10) 0.65 mol/L -carotene.
A
B
C
Figure 2. Typical chromatograms corresponding with an extract of
a humanserum sample: (A) channel 1, diode-array detection at 290 nm
for -tocopheroland -tocopherol acetate; (B) channel 2, diode-array
detection at 325 nm forretinol and retinol acetate; and (C) channel
3, diode-array detection at 450 nmfor carotenoids and echinenone.
Peak numbers are the same as Figure 1.
A
B
C
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Journal of Chromatographic Science, Vol. 40, February 2002
73
carotenoids present in serum in one run simultaneously with
-tocopherol and retinol (3034,37,47). Most carotenoids aredetected
at 450 or 473 nm, but -tocopherol and retinol can onlybe detected
at 290 and 325 nm, respectively. Most of the previ-ously mentioned
methods therefore require the use of severaldetectors in series
(43,44) and a multiwavelength detector eitherwith simultaneous
monitoring at different wavelengths(31,36,37,40,53) or a change in
the detection wavelength duringthe run (30,32,44,47). The need for
simultaneous detection atdifferent wavelengths is illustrated by
the retinol andluteinzeaxanthin that elute within a 0.3-min
interval and haveto be detected at 325 and 450 nm, respectively.
Typical chro-matograms of a standard mixture and an extracted
humanserum are shown in Figures 1 and 2. The chromatogramsrevealed
elution and baseline resolution between all the analytesof interest
except for lutein and zeaxanthin, which are not sepa-rated by this
method. The internal standard echinenone waseluted between
-cryptoxanthin and lycopene and thus did notinterfere with the
other carotenoids analyzed. Several additionalcarotenoids not
identified as of yet appeared between the peak ofluteinzeaxanthin
at 4 min and -cryptoxanthin at 8 min. Beforevalidation of the HPLC
method, we have realizeda selectivity study, and BHT has been
analyzedwith other analytes to see any potential chro-matographic
interference. BHT elutes with ashort retention time (within 3 min)
and is onlydetectable at 290 nm, thus no interference withvitamins
was observed.
Assay validation and quality control of theHPLC method
The quantitation was achieved using theinternal standardization
mode. Data concerninglinearity (the linearity range for each
liposolublevitamin was selected according to its physiolog-ical
values), LOD, and LOQ are indicated in TableII (and precision in
Table III).
The LOD and LOQ values agree with previous data in the
liter-ature (32). In order to calculate recoveries, a pooled serum
wasspiked with 20 L of combined standards to provide the
addedconcentrations of 0.7 mol/L retinol, 8.7 mol/L -tocopherol,and
0.15 to 0.2 mol/L carotenoids. The spiked serum samples (n = 5)
were then extracted using a single extraction step with n-hexane.
Recoveries found were 99.6% 11.1% for retinol,91.2% 2.0% for
retinol acetate, 109.4% 13.4% for -toco-pherol, 101.2% 3.0% for
-tocopherol acetate, 112.6% 22.2% for luteinzeaxanthin, 104.3% 9.1%
for -crypto-xanthin, 109.4% 31.0% for lycopene, 85.1% 8.5% for
-carotene, and 95.6% 9.5% for echinenone. The differentbehaviors of
carotenoids with regard to extraction using n-hexane has already
been reported by Barua et al. (48). The cal-culated recoveries in
this study are satisfactory and comparablewith previously reported
values (30,32,33).
In order to check the precision of the method, a human serumpool
was analyzed 20 times during the same day to assess
therepeatability. This operation was repeated 5 times over a
periodof one month to evaluate the interassay precision. The intra-
andinterassay variations calculated for each vitamin are shown
in
Table III. Precision of the HPLC Assay of Liposoluble Vitamins
andCarotenoids in Serum
Within run Between run
Analyte Concentration* (mol/L) %RSD Concentration (mol/L)
%RSD
Retinol 1.90 (0.06) 3.3 2.1 (0.09) 4.4-Tocopherol 34.9 (1.31)
3.8 29.3 (1.1) 3.8Luteinzeaxanthin 0.65 (0.02) 3.8 0.51 (0.02)
4.5-Cryptoxanthin 0.13 (0.01) 7.8 0.10 (0.01) 13.7Lycopene 0.53
(0.05) 9.5 0.28 (0.04) 12.5-Carotene 0.18 (0.02) 8.8 0.14 (0.02)
12.1-Carotene 0.57 (0.04) 6.7 0.52 (0.05) 9.1* Mean (standard
deviation), n = 20. Mean (standard deviation), n = 5.
Table IV. Concentrations of Retinol, -Tocopherol, and
Carotenoids in Millimoles per Liter Measured in the Serum of
168Healthy Subjects from Ages 9 to 55 Years Old and a Comparison
with Other Studies
Present study*Men Women Talwar Steghens Olmedilla Sowell
Compound 920 years old 2155 years old 920 years old 2155 years
old et al.*, (32) et al.*, (37) et al.*, (54) et al.**, (31)
Retinol 1.37 (0.36) 2.18 (0.43) 1.36 (0.31) 1.86 (0.53) 2.00
(0.60) 1.84 (0.80) 1.71 (0.39) 1.91 (1.052.97)-Tocopherol 20.6
(4.08) 29.7 (8.16) 23.6 (10.9) 26.6 (6.38) 29.6 (7.60) 33.0 (6.67)
32.7 (7.40) 25.7 (13.947.0)Lutein 0.42 (0.12) 0.43 (0.24) 0.49
(0.23) 0.52 (0.25) 0.26 (0.11) 0.71 (0.30) 0.24 (0.21) 0.36
(0.140.74)
Zeaxanthin 0.09 (0.05) 0.07 (0.04)
-Cryptoxanthin 0.13 (0.08) 0.13 (0.11) 0.19 (0.14) 0.17 (0.12)
0.55 (0.11) 0.35 (0.27) 0.60 (0.47) 0.22 (0.050.52)Lycopene 0.33
(0.16) 0.28 (0.16) 0.31 (0.16) 0.32 (0.22) 0.37 (0.18) 0.56 (0.43)
0.42 (0.24) 0.40 (0.110.80)-Carotene 0.08 (0.06) 0.10 (0.13) 0.13
(0.11) 0.14 (0.14) 0.07 (0.04) 0.36 (0.26) 0.07 (0.05) 0.08
(0.020.22)-Carotene 0.49 (0.43) 0.42 (0.29) 0.60 (0.37) 0.64 (0.72)
0.38 (0.20) 0.81 (0.45) 0.37 (0.23) 0.34 (0.070.88)* Means
(standard deviation). Concentrations in serum for men and women
ranging from ages 19 to 62 years old, n = 111. Concentrations in
serum for women ranging from ages 35 to 50 years old, n = 96.
Concentrations in serum for women ranging from ages 25 to 59 years
old, n = 54.
** Concentrations in serum for men and women ranging from ages 4
to 93 years old, n = 3480. Means (concentration range). Sum of
lutein and zeaxanthin (peaks not separated).
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Journal of Chromatographic Science, Vol. 40, February 2002
74
Table III. The RSDs ranged from 3.3% (retinol) to 9.5%(lycopene)
for intra-assay precision and 3.8% (-tocopherol) to13.7%
(-cryptoxanthin) for interassay precision. The RSDvalues obtained
for some carotenoids were comparable withthose reported for most of
the other assays (16,31,32). However,the RSDs obtained for retinol
and -tocopherol were lower thanthose reported in these methods.
This serum pool was then usedfor routine quality control.
Assay of liposoluble vitamins and carotenoids in a healthy
population
The validated method was applied to the measurement ofretinol,
-tocopherol, and five carotenoids in the serum of 168healthy
Caucasian subjects (Table IV). In comparison with previ-ously
published studies, including more than 25 subjects(31,32,37,54),
the value ranges were comparable for most of theliposoluble
vitamins and carotenoids measured except forluteinzeaxanthin, which
was higher than the value found byTalwar et al. (32) but similar to
other studies (31,54). Similarresults were demonstrated in a
previous study by De Leehneer etal. (55). This fact can probably be
explained by differencesbetween the populations involved in the
different studies. We canalso notice that luteinzeaxanthin and
lycopene were in theserum in significant quantities, thus -carotene
could not bemeasured alone as a representative marker of the
serumcarotenoids. As a matter of fact, the carotenoids exhibited
dif-ferent distributions between subjects, tissues (56,57), and
food(58). Moreover, their antioxidant capacities and functions
maydiffer at the cellular level (59). More recently, an HPLC
systemcoupling two different C18 columns has been reported for
theseparation of 13 carotenoids in plasma (60), but the overall
runtime for one sample reached 50 min, which limits thethroughput,
and thus no important additional epidemiologicalinformation was
given.
Conclusion
The reported HPLC method devoted to the assay of
liposolublevitamins and carotenoids in serum permits the separation
of themain carotenoids (luteinzeaxanthin, -cryptoxanthin,
lyco-pene, - and -carotene, retinol, and -tocopherol) within 20min,
which allows a high throughput of samples. The methodwas run for
several months in the routine laboratory and hasclearly proven its
reliability. Because of its specificity and sensi-tivity for a
great number of liposoluble vitamins correspondingwith important
serum antioxidant biomarkers, this method hasan evident interest
for nutritional and epidemiological studiesand is now applied to
various pathological groups such as alco-holic and Type I diabetic
patients.
Acknowledgments
This project was supported in part by a grant from
theAssociation de la Recherche sur le Cholesterol (ARCOL). The
authors gratefully acknowledge the technical staff of the
Centerof Preventive Medicine for their kind participation. The
authorsthank the Socit Francophone des Biofacteurs et Vitamines
forgiving them the opportunity to participate in an
interlaboratoryquality control.
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77
A procedure for the sampling and analysis of energetics and
relatedcompounds in the atmosphere is described. The basic
procedureconsists of the collection of air samples using sampling
cartridgescontaining XAD-2 resin, extraction of the resin with
isoamyl acetate,and an analysis of the extract using gas
chromatography withelectron capture detection. Modifications and
additions to thisprocedure are discussed, such as the use of a
prefilter before theresin sampler to collect particulates and the
use of a mass selectivedetector to analyze for some propellant
compounds of interest orfor quantitative confirmation purposes. Two
differing sizes ofsamplers are evaluated according to the air
volumes required forcollection. The procedure is tested through the
analysis of spikedresin samples, which had air pulled through them
for periods oftime corresponding with the required sampling
volumes. Thisprocedure has application toward the measurement of
energeticresidues in atmospheres resulting from weapons testing
andoperations during training exercises involving munitions.
Introduction
The quantitative measurement of the residual amounts of
ener-getics and related compounds in the environment has been
rou-tinely performed for over three decades. There are
numerousmethods used to analyze soils and waters for
nitroaromatics,nitramines, and other compounds related to U.S.
munitions(16). The U.S. Army has used many of these methods in
thecourse of environmental monitoring to protect the health
andsafety of soldiers and the general population. It has also
relied onthese methods to measure soil and water contamination
fromexplosives during environmental cleanup operations. The
proce-dures generally involve gas chromatographic (GC) and
high-per-formance liquid chromatographic (HPLC) analyses, but there
arealso thin-layer chromatography and immunoassay methods thatare
useful as field screening tests (78).
Additionally, there are methods used to monitor selected
com-pounds such as trinitrotoluene (TNT) and dinitrotoluenes
inworkplace atmospheres (910). This monitoring is used to
ensurethat munitions workers are not exposed to harmful levels of
these
compounds. However, there has been little done toward
environ-mental air monitoring for energetics other than the
specific caseof stack emissions produced during weapons destruction
byincineration. The primary impetus for stack monitoring has beento
determine destruction efficiencies associated with the pro-cesses
used to burn the munitions feedstocks. The measurementof energetic
and related compounds in the general atmospherefrom a health-risk
standpoint has become an issue only in the lastfew years.
The U.S. Army has recognized the need to perform air moni-toring
for energetics, partially because of public concern
aboutair-quality issues in areas near U.S. military reservations.
Thereare operations during weapons testing and training that
arepotentially capable of putting measurable quantities of
energeticsand related compounds into the atmosphere. As a result,
theArmy Center for Health Promotion and Preventive
Medicine(USACHPPM) has determined the need to modify current
air-sampling methodologies and analytical techniques to
providemonitoring efforts for a suite of explosives compounds,
includingthose commonly analyzed for by soil and water methods. The
listof compounds of concern includes the nitroaromatics (such
asTNT, tetryl, and their precursors and breakdown products)
andnitramines (such as hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX)
and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine(HMX)). There
are also other propellant compounds of occasionalconcern, including
nitroglycerin, dibutyl- and dioctyl-phthalates,diphenylamine, and
pentaerythritol tetranitrate (PETN).
There are U.S. Environmental Protection Agency (EPA)
air-sampling procedures that employ sampling devices
containingXAD-2 resin to trap polynuclear aromatic hydrocarbons
fromambient air and semivolatile organic hazardous compounds
instack emissions (11,12). USACHPPM has successfully used
modi-fications of several types pertaining to the XAD-2 sampling
trainsfor the collection of stack emissions for energetic residues.
Wedecided therefore to investigate the use of glass cartridges
packedwith XAD-2 resin for general atmospheric sampling for the
ener-getics and propellant compounds. Preliminary tests were
con-ducted using PS-1 cartridges manufactured for EPA Air
ToxicsMethod TO-13 for polynuclear aromatic hydrocarbons, and a
fieldstudy was successfully performed using these
cartridges.Recently, newly designed cartridges have been employed.
These
Abstract
A Procedure for Sampling and Analysis of Air forEnergetics and
Related Compounds
Michael A. Hable, Joseph B. Sutphin, Curtis G. Oliver, Robert M.
McKenzie, Eleonor F. Gordon, and Richard W. BishopThe U.S. Army
Center for Health Promotion and Preventive Medicine, 5158 Blackhawk
Road, Aberdeen Proving Ground, MD 21010-5403
Reproduction (photocopying) of editorial content of this journal
is prohibited without publishers permission.
Journal of Chromatographic Science, Vol. 40, February 2002
-
Journal of Chromatographic Science, Vol. 40, February 2002
78
cartridges are somewhat more robust during shipping and
han-dling than the original types and are compatible with the
sam-pling requirements of the U.S. Army during weapons
testing.These cartridges are of two types: the first being a
modification ofthe original PS-1 design used for high-volume
sampling (Figure1A) and the second a smaller two-section cartridge
designed forshorter test intervals (Figure 1B).
The analytical approach has generally been to use a
modifica-tion of existing USACHPPM GC procedures for the
nitro-con-taining compounds. These procedures use
electron-capturedetection and have been used for many years in our
laboratoriesto reliably quantitate these compounds from a variety
of matrices(3,4,10,13). GC using a mass selective detector (MSD)
was chosenas the most expedient means of analyzing for the
phthalate estersand diphenylamine, because all can be done in a
single GC run.The XAD-2 resin was solvent desorbed with isoamyl
acetate inorder to place the analytes into solution prior to
analysis. Isoamylacetate has proven to be an excellent solvent for
the compoundsof interest. It also provides superior response and
reproducibilitywith the electron-capture detector compared with
other solventstried (such as acetonitrile). Finally, it helps to
minimize chro-matographic problems that can arise with
moisture-laden sam-ples because it is not water miscible (and thus
does not retain thewater).
The sampling cartridges, the chromatographic and
analyticalprocedures used to analyze for the compounds of concern,
andthe test results from the spike studies conducted with the
sam-plers will be described.
Experimental
Air-sampling cartridgesThe initial testing was done using PS-1
sampling cartridges
packed with 55 g of XAD-2 resin. Subsequent tests with these
samplers used 50 g rather than 55 g of the XAD-2, primarily as
amatter of convenience. These cartridges are designed such thatthe
glass cartridge contains a metal screen at the bottom to retainthe
resin during sampling, and the resin is sandwiched above thescreen
between two sections of glass wool. Design specificationsfor these
cartridges vary, but the basic size of the inside samplingbed is 4
2.25 inches. The actual sampler and its calibration anduse has been
described elsewhere (11).
The modified sampling cartridges were manufactured by AceGlass
Inc. (Vineland, NJ). The larger size being tested stillemployed 50
g of XAD-2 resin, but the resin was retained by ametal screen/metal
mesh combination at both ends of the car-tridge, with some glass
wool at the outlet end only. The glass car-tridge body and contents
were held together using Teflon endfittings. The smaller size
cartridge used two 10-g sections of XAD-2 resin separated by glass
wool. It also contained metal screensand mesh at both ends and
glass wool between the screen/meshand resin at the outlet end of
the cartridge. The inner diameter ofthe glass body was smaller, but
the cartridge was similar to thelarge one in its use of metal
screens and Teflon end fittings (asshown in Figure 1B). The second
section of the smaller cartridgewas used as a back-up to measure
breakthrough. If more than20% of the total of an analyte was found
in this section, then thecartridge was considered to have been
oversampled. Both types ofcartridges were compatible with the
sampling devices used withthe original PS-1 cartridges and were
used in the same fashion.
The XAD-2 resin used for packing the cartridges was
astyrenedivinylbenzene porous polymer. It was purchased fromRestek
Corporation (Bellefonte, PA) under the name Ultra CleanXAD-2 Resin.
It was found to be sufficiently clean because it didnot require
further purification for application toward energeticsampling. It
was noted, however, that its appearance variedbetween different
lots of the material. This did not seem to affectthe resins
adsorbent properties, but it had other effects (as will
bedescribed).
Recovery testsThe ability of the XAD-2 cartridges to retain the
compounds of
concern while large volumes of air were passed through them
wastested. Solutions containing known amounts of the analytes
inacetonitrile were spiked into the front part of the resin within
acartridge (in the case of a two-section cartridge the front
sectionwas spiked). The cartridges were placed in a PS-1 sampling
appa-ratus, and clean ambient air was pulled through in the same
wayas it is generally done with actual sampling in the field. The
car-tridges were then returned to the laboratory for the analysis
andevaluation