Metanol analysis

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

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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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Donald E. Willis

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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


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


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.


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.


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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


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


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


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


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.


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.


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.


68

3. Z. Wang and M. Fingas. Rapid separation of nonionic surfactants ofpolyethoxylated octylphenol and determination of ethylene oxideoligomer distribution by C1 column reversed-phase liquid chro-matography. J. Chromatogr. 673: 14556 (1993).

4. C. Whalberg, L. Renberg, and U. Wideqvist. Determination ofnonylphenol and nonylphenol ethoxylates as their pentafluoroben-zoates in water, sewage sludge, and biota. Chemosphere 20: 17995(1990).

5. R.E.A. Escott, S.J. Brinkworth, and T.A. Steedman. The determinationof ethoxylate oligomer distribution of nonionic and anionic surfac-tants by high-performance liquid chromatography. J. Chromatogr.282: 65561 (1983).

6. I. Zeman. Applications of bonded diol phases for separation ofethoxylated surfactants by high-performance liquid chromatography.J. Chromatogr. 363: 23330 (1986).

7. A.M. Rothman. High-performance liquid chromatographic methodfor determining ethoxymer distribution of alkylphenoxy poly-oxyethylene surfactants. J. Chromatogr. 253: 28388 (1982).

8. J.L. Gundersen. Separation of isomers of nonylphenol and selectnonylphenol polyethoxylates by high-performance liquid chro-matography on a graphitic column. J. Chromatogr. A 914: 16166(2001).

9. P.L. Ferguson, C.R. Iden, and B.J. Brownawell. Analysis ofalkylphenol ethoxylate metabolites in the aquatic environment usingliquid chromatographyelectrospray mass spectrometry. Anal.Chem. 72: 432230 (2000).

10. D.Y. Shang, M.G. Ikonomou, and R.W. MacDonald. Quantitativedetermination of nonylphenol polyethoxylate surfactants in marinesediment using normal-phase liquid chromatographyelectrospraymass spectrometry. J. Chromatogr. A 849: 46782 (1999).

11. Z. Wang and M. Fingas. Quantitative analysis of polyethoxylatedoctylphenol by capillary supercritical fluid chromatography. J. Chromatogr. 641: 12536 (1993).

12. Z. Wang and M. Fingas. Analysis of polyethoxylated nonylphenolsby supercritical fluid chromatography and high-performance liquidchromatography. J. Chromatogr. Sci. 31: 50918 (1993).

13. A. Giorgettie, N. Pericles, H.M. Widmer, K. Anton, and P. Datwyler.

Mixed mobile phases and pressure programming in packed column supercritical fluid chromatography: a unified approach. J. Chromatogr. Sci. 27: 31824 (1989).

14. M. Takeuchi and T. Saito. Combination of semi micro and micropacked column supercritical fluid chromatography with some otherinstruments for qualitative analysis. J. High Resolut. Chromatogr. 14:34751 (1991).

15. T.A. Dean and C.F. Poole. Solventless injection for packed columnsupercritical fluid chromatography. J. High Resolut. Chromatogr. 12:77378 (1989).

16. E.S. Francis, M.L. Lee, and B.E. Richter. Modifier addition in micro-column supercritical fluid chromatography with a high 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 spec-trometry. Anal. Chem. 67: 17971804 (1995).

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 dioxide extraction. J. Chromatogr. A 785: 38594 (1997).

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 nonionicsurfactants, their degradation products, and endocrine-disruptingcompounds in sewage by liquid chromatography/mass spectrometry.Anal. Chem. 72: 456067 (2000).

Manuscript accepted December 7, 2001.

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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



* Author to whom correspondence should be addressed: email [email protected].


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.


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


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


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).


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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Manuscript accepted December 7, 2001.

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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




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

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