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Journal of Chromatography A, 1216 (2009) 6838–6843

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

as chromatography–mass spectrometry resolution of sugar acid enantiomersn a permethylated �-cyclodextrin stationary phase

eorge Cooper ∗, Minakshi Sant, Cynthia Asiyopace Science Division, NASA-Ames Research Center, Moffett Field, CA 94035, United States

r t i c l e i n f o

rticle history:eceived 14 March 2009eceived in revised form 15 July 2009ccepted 23 July 2009vailable online 11 August 2009

a b s t r a c t

Analysis of compounds in meteorites revealed a need to simultaneously characterize multiple enan-tiomers of sugar acids (aldonic acids) present in trace amounts. Analyses by gas chromatography–massspectrometry demonstrated that all but two of the three-carbon through six-carbon straight-chainedsugar acid enantiomer pairs could be resolved using a single derivatization procedure and one setof GC parameters. Compounds were analyzed as their ethyl ester/O-triflouroacetyl, isopropyl ester/O-

eywords:as chromatography–mass spectrometrynantiomersugar acidldonic acid

triflouroacetyl and isopropyl ester/O-pentafluoropropionyl derivatives on a capillary column containingpermethylated �-cyclodextrin (Chirasil-Dex CB) as the stationary phase. Characteristic mass fragmentsare related to the ester groups while several ions are also common to derivatized monosaccharides.

Published by Elsevier B.V.

yclodextrinhiralsil-Dex CB

. Introduction

Sugar derivatives, common in the biosphere, are also known toxist in asteroids and possibly comets. A suite of sugar derivatives,redominately sugar acids (polyhydroxy carboxylic acids), was

dentified in carbonaceous meteorites [1]. Extraterrestrial organicompounds have important implications in the study of early solarystem organic and pre-biotic chemistry [2]. Some sugar acids suchs glyceric, erythronic and ribonic are also common in biochemistry.e often observe glyceric acid (predominately thed enantiomer) in

oil samples. Important applications of sugar acid analysis includehe complex problem of molecular and/or enantiomer analysis ofugars by gas chromatography (GC). For example, just one five-arbon aldo sugar can produce at least five peaks (isomers) onchromatogram: commonly, an alpha and beta five-membered

ing (furanose), an alpha and beta six-membered ring (pyranose)nd the open chain. If enantioselective analysis is performed thisotentially gives 10 peaks. However modification of sugar carbonylroups, e.g., in the present case (Section 2.2) oxidation of straight-hain aldo sugars resulting in sugar mono-acids (hereafter referred

o as aldonic acids), allows greatly simplified molecular/enantiomernalysis: there are only one or two chromatographic peaks per com-ound. In the analysis of enantiomers in rare samples a commonhallenge is finding a method that will simultaneously detect and

∗ Corresponding author. Tel.: +1 650 604 5968; fax: +1 650 604 1088.E-mail address: gcooper@mail.arc.nasa.gov (G. Cooper).

021-9673/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.chroma.2009.07.073

resolve as many d/l pairs as possible using small quantities of sam-ple material. Ideally, one could select a single derivatizing agent,column, and set of instrument parameters. Here, we report on ourattempts at GC separation of enantiomers of three to six-carbon (C-3 to C-6) straight-chain aldonic acids by gas chromatography–massspectrometry (GC–MS) in as close to a “one pot” procedure as possi-ble (see Table 1 for the list of compounds). The ultimate goal is theanalyses of d/l ratios of aldonic acids extracted from single sam-ples of extraterrestrial material. In such samples the majority oftarget compounds are present in trace amounts—with the methodsdescribed below we can routinely detect the acids at sub-nanomolelevels.

There are few literature references to GC enantiomer separa-tion methods for suites of aldonic acids on either chiral or achiralcolumns. In an early investigation, Pollock and Jermany [3] sep-arated d diastereomers of C-3 to C-5 aldonic acids in a test ofthree butyl esters/O-acetyl derivatives on an achiral carbowax cap-illary column. Although this was not dl enantiomer separation theauthors noted that they had formed and separated a dl sugar aciddiastereomer pair using an enantiomerically pure alcohol for ester-ification (they described the results as unsatisfactory). Still, thismay be the only previous report of the GC properties of a suiteof aldonic acids analyzed simultaneously. A subsequent paper by

the same authors [4] reported the results of d diastereomer anal-yses of the C-6 aldonic acids galactonic and gluconic. Kim et al.,[5] separated dl-glyceric acid, and several mono-hydroxy acids, onDB-5 and DB-17 (achiral phases) after formation of diastereomersof the acids. Using a chiral phase (perpentylated �-cyclodextrin),

G. Cooper et al. / J. Chromatogr. A 1216 (2009) 6838–6843 6839

Table 1GC data of aldonic acid enantiomer separations on permethylated �-cylcodextrinwith indicated compound derivative and separation factor, ˛a, where separationswere achieved.

Aldonic Acid Et–TFAb ISP–TFA ISP–PFP d/l order

C-3Glyceric X 1.008 d–lC-4Threonic sep 1.008 d–lErythronic X X 1.007 d–lC-5Ribonica sep sep l–dArabinonic 1.003 X sep l–dLyxonic sep 1.007 d–lXylonic sep 1.004 d–lC-6Allonic 1.002 d–lTalonic 1.003 d–lAltronic 1.003 l–dMannonic 1.003 l–dGulonic 1.003 X X l–dGalactonic 1.006 d–lGluconic 1.006 X d–lIdonic 1.002 l–d

a Separation factor, ˛, is the ratio of adjusted retention times of the enantiomers.All separations listed were achieved with GC condition I. sep indicates at least partialseparation of enantiomers at full peak height – an optimum value of ˛ = 1.002 wasachieved for ribonic acid; X denotes no enantiomer separation; blank fields indicaten

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o attempt at separation.b Et-TFA: ethyl ester/O-trifluoroacetyl; ISP-TFA: isopropyl ester/O-trifluoroacetyl;

SP-PFP: isopropyl ester/O-pentafluoropropionyl.

önig et al. [6] separated enantiomers of dl-glyceric and dl-tartariccid as their methyl ester/O-triflouroacetyl derivatives on a capil-ary column (several other polyols were separated with the samehase).

Our GC trials of aldonic acids included derivatization withnown reagents and relatively straightforward procedures:he ethyl ester/O-triflouroacetyl (Et–TFA), isopropyl ester/TFAISP–TFA,) and isopropyl ester/O-pentafluoropropionyl (ISP–PFP)erivatives were studied. We utilized a capillary column coatedith permethylated �-cylcodextrin (the chiral phase) chemically

onded to polydimethysiloxane: a Chiralsil-Dex CB column. In GC,he most generally used cyclodextrins, �, �, and � (six, seven, andight glucopyranosides/ring, respectively) have been employed forwide range of enantiomer analyses. These include biomarkers [7],lkanes [8,9], and underivatized polar compounds [10]. For a reviewp to 2001 see Schurig [11]. Early use of cyclodextrins as packing inC included the use of acetate and propionate derivatives [12] whileermethylated cyclodextrin was used for the first time in 1979 [13].eparation power was increased by the combination of permethy-ated �-cylcodextrin and capillary GC by Juvancz et al. [14]. Furtherdvances, cyclodextrin dissolved in polysiloxane [15] and cyclodex-rin chemically bonded to the polysiloxane [16,17], are the basisf present cyclodextrin capillary GC columns. Although more lim-

ted than cyclodextrins, linear modified dextrins (“acyclodextrins”),ave also shown the ability to resolve enantiomers including thosef amino acids [18].

. Experimental

.1. Reagents

With the exception of dl-glyceric acid, one or both enantiomers

f individual aldonic acids were synthesized from the correspond-

ng sugar. Sugars were purchased from either Sigma–Aldrich (St.ouis, MO, USA) or Omicron Biochemicals (South Bend, IN, USA).oth enantiomers of glyceric acid were purchased (Sigma–Aldrich).

sopropyl alcohol (IPA)–acetyl chloride kits were obtained from

Fig. 1. General derivatization reactions of aldonic acids.

Grace Davison Discovery Sciences (Waukegan, IL, USA). Triflu-oroacetic anhydride (TFAA) and pentafluoropropionic anhydride(PFPA) were purchased from Sigma–Aldrich and Grace Davi-son Discovery Sciences. Anhydrous ethanol, bromine, and carbontetrachloride were purchased from Sigma–Aldrich. Anhydroustetrahydrofuran (THF) was obtained from Burdick and Jackson andSigma–Aldrich. Ion-exchange resins (AG-50 and AG-1) were pur-chased from Bio-Rad Laboratories (Hercules, CA, USA).

2.2. Synthesis and derivatization procedures

We examined all C-3 to C-6 straight-chained aldonic acids.Where needed, d and/or l aldonic acid enantiomers were preparedby bromine oxidation of the corresponding sugar. Oxidation mix-tures generally consisted of sugar (∼1–3 mg): Br2 in mole ratios of1:2 and NaOH solution was added as needed to bring the initial pHto 7–9. The C-4 and C-5 sugar mixtures (and a C-6 sugar, talose) wereheated for 30 min at 60–70 ◦C; instead of heating, the remaining C-6sugar solutions were allowed to sit at room temperature from 3 to 7days. After reaction, all mixtures were concentrated by rotary evap-oration and applied to a column of excess AG-50 cation-exchangeresin. The product acids were then eluted with ∼5 bed-volumes ofwater, dried (rotary evaporation), and re-dried after the addition ofmethanol.

Enantiomer analyses of all acids were performed on the straight-chain ammonium forms, i.e., lactone rings were opened by heatingsamples to 100 ◦C for 5 min in a slight molar excess (≤2×) of NH4OH(solution) followed by drying. The carboxylic groups were deriva-tized to their ISP or ethyl (Et) esters by heating the acids at 60 ◦Cfor 30 min in excess isopropanol/acetyl chloride or ethanol/acetylchloride mixtures: higher temperatures resulted in the formationof a aldonic acid–chloride substitution product. After drying ofesters, hydroxyl groups were derivatized to their O-TFA derivativesby heating samples in mixtures of TFAA:THF for 5 min at 50–60 ◦Cand allowing them to sit at room temperature from 30 to 60 min(Fig. 1). O-PFP derivatives were prepared in PFPA:ethyl acetate withno heating: samples were sat at room temperature for 45 min.Approximate molar ratios of 1:25, aldonic acid:anhydride (in excesssolvent) were used. After acylation samples were dried and excesscarbon tetrachloride was added to remove trifluoroacetic acid byazeotropic distillation (trifluoroacetic acid is a reaction/hydrolysisby-product of TFAA that does not interfere with enantiomer reso-lution). An appropriate volume of the final solvent (fresh THF, thusfar) was then added for GC injection.

2.3. Instruments and parameters

Two GC–MS units in electron-impact (EI) mode were used foranalyses: a Finnigan (Thermo Fisher Scientific, Waltham, MA, USA)GCQ GC interfaced to a Finnigan GCQ ion trap mass spectrometer

and an Agilent Technologies (Santa Clara CA, USA) 6890N GC inter-faced to an Agilent 5973 inert Mass-Selective Detector (quadrupolemass spectrometer); both are low resolution units. Both GCs wereused in splitless mode. Two Varian (Palo Alto, CA, USA) ChirasilDex-CB columns (25 m length × 0.25 mm diameter × 0.25 �m film

6840 G. Cooper et al. / J. Chromatogr. A 1216 (2009) 6838–6843

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ig. 2. GC enantiomer separations of C-4 and C-5 of (ISP–TFA) aldonic acids using Guickly with column usage. Erythronic acid enantiomers (not shown) are not separatSIM) of characteristic mass spectral ions: threonic acid, 379 amu; 5-C acids, 505 am

hickness each) in series and/or a single 50 m column were usedor GC separations (column film thickness and diameter were con-tant). The GC injector temperature was 200 ◦C and the heliumow rate was constant at 1 mL/min MS conditions: ion trap detec-

or temp., 200 ◦C; quadrupole temp., 150 ◦C; carrier gas, helium;ransfer line, 200 ◦C; electron voltage, 70 eV.

The following set of GC conditions was used for enantiomeresolution (I was the overall best):

I: 45 ◦C – increase at 3 ◦C/min to 70 ◦C, hold for 30 min – increaseat 3 ◦C/min to 200 ◦C.

II: 45 ◦C – increase at 7 ◦C/min to 70 ◦C, hold for 33 min – increaseat 3 ◦C/min to 200 ◦C.

II: 45 ◦C – increase at 5 ◦C/min to 70 ◦C, hold for 40 min – increaseat 3 ◦C/min to 200 ◦C.

. Results and discussion

.1. Derivatizations

All compounds were analyzed in their open-chain ammonium-alt forms. Although some aldonic acid lactones (the cyclic forms)re relatively resistant to ring opening, e.g., mannonic [19], these of low molar ratios of NH4OH with brief heat (Section 2.2)esulted in the open forms with no observable lactones remaining.

hile our efforts at derivatizations and enantiomer separations ofldonic acids continue, esterification of all C-3 to C-5 acids pro-eeded smoothly in both IPA/acetyl chloride or Et/acetyl chlorideixtures. However, C-6 acids were generally much less soluble. For

xample, gluconic acid and its ammonium salt are known to be rela-ively insoluble in common solvents; tables of solubilities list thesewo to be only slightly soluble in ethanol. In our samples C-6 acidolubilities in pure isopropanol (or ethanol) was inadequate for aew mg of the ammonium salts—although the limited solubilities dollow small amounts of ISP ester formation (Table 1). Adequate sol-bility, and therefore esterification, of C-6 acids as a group was onlychieved in the acidic ethanol/acetyl chloride mixtures. In attemptso form O-TFA derivatives, the C-6 esters also displayed limitedolubilities in common aprotic solvents. After trials with several sol-ents, fresh anhydrous tetrahydrofuran (in excess) combined with

he anhydride (TFAA, etc.) was found to be adequate—it routinelyllowed analysis of sub-nanmole quantities of enantiomers. Noigns of racemization were seen during derivatization of sugar acidsr oxidation of sugars. However, oxidations caused relatively smallmounts of individual sugars to decompose to homologous sugar

ition I (Section 2.3). The enantiomer separation of ribonic acid decreases relativelyh isopropyl esters (Table 1). Chromatogram was obtained by selected ion monitoring

acids of lesser carbon number with high specificity for enantiomerretention. For example, d-glucose to d-arabinonic, d-erythronic,and d-glyceric acids, or l-xylose to l-threonic and l-glyceric acids.

3.2. GC separation of enantiomers

Figs. 2 and 3 show selected enantiomers separations achievedto date with GC condition I, Table 1 summarizes all results. Enan-tiomer pairs of unequal amounts (shown in some cases) and pureenantiomers were used to confirm the identities of each enan-tiomer. Although derivatives varied, d/l separation was achievedfor all enantiomers under chromatographic condition I (see Section2.3). Enantiomer resolution at half peak height of the present runsvaried from 3.7 for ISP–TFA lyxonic to 1.3 for Et–TFA idonic (Fig. 3)[resolution = 1.18 (t2 − t1/Wh1 + Wh2), where t is retention time of adesignated enantiomer and W is peak width at half height]. How-ever, the enantiomers of ribonic acid and allonic acid were the mostsensitive to column lifetime. While all other acids display relativelyconsistent enantiomer separation at a given duration of column use,the enantiomer separation of these acids is gradually lost.

Using Et–TFA derivatives and one set of GC parameters (GC con-dition I), all but 2 of the 15 enantiomer pairs can be resolved ina single run. The exceptions, dl-glyceric acid and dl-erythronicacid, can be resolved as their ISP–TFA and ISP–PFP derivatives,respectively (below). In some cases overlap in retention time (e.g.,some of the Et/O-TFA C-6 acids) can be alleviated by changesin GC chromatography parameters. With ISP–TFA derivatization,five of the seven C-3 through C-5 acids can be resolved intodistinct GC peaks in a single run. The exceptions, erythronicacid and arabinonic acid, were separated as their ISP–PFP andEt–TFA derivatives respectively; arabinonic acid enantiomers werealso separated as ISP–PFP derivatives. As a group, the C-6 acidsshowed that only the ethyl esters allow enantiomer resolutionof all pairs in a single run. A further increase in resolution ofd/l glyceric acid is achieved with condition III while condition IIincreases resolution of d/l threonic acid. As mentioned, d/l sep-arations of arabinonic and erythronic acids can be achieved intheir O-PFP forms: condition II is optimum for erythronic acidand condition I for arabinonic acid. However, no further trialswere done with PFP derivatives as they apparently cause irre-

versible column damage: all enantioselectivity is lost after severalruns.

The molecular and enantiomer (of each compound) elutionorder of all compounds are the same regardless of derivative(Table 1). C-3 and C-4 enantiomer elution orders were d fol-

G. Cooper et al. / J. Chromatogr. A 1216 (2009) 6838–6843 6841

donic

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Fig. 3. GC enantiomer separations of C-6 (Et–TFA) al

owed by l; C-5 acids were evenly split in enantiomer order.hile resolution of C-6 acids is ongoing, they are also evenly split

n enantiomer order. The total amount (d+ l) of individual com-

ounds injected was approximately 0.1–1.0 nmoles. Preliminaryd/leparations of four deoxy aldonic acids, threo- and erythro- 2,3-ihydroxy butyric; 2,4-dihydroxy butyric; 3,4-dihydroxy butyrics their ISP–TFA derivatives have also been achieved under GCondition I.

ig. 4. Mass spectra of derivatized C-3 to C-6 aldonic acids. Glyceric acid spectrum is fromf fragmentations.

acids under GC condition I. SIM detection: 631 amu.

3.3. Mass spectra

3.3.1. Fragmentation unique to aldonic acids

Mass spectra representative of each carbon number are shown

in Fig. 4: glyceric acid (ISP–TFA) is from ion trap MS while the othersare quadrupole MS. Noticeable in the ion trap mass spectra of sev-eral ISP–TFA aldonic acids is the protonated molecular ion (MH+),e.g., m/z 341 for glyceric acid (Fig. 4). Significant fragments in the

an ion trap MS—others are from quadrupole MS. See Section 3.3 for an explanation

6842 G. Cooper et al. / J. Chromatogr. A 1216 (2009) 6838–6843

in ald

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Fig. 5. Proposed gas phase formation of TFA-lactones

pectra of C-3 and C-4 acids are attributed to the loss of O-alkylrom the molecular ion (M): m/z 281 and 407, respectively (O-alkyls from the ester group). As a group, the most characteristic frag-

ent of the acids is the loss of the carboxyl-alkyl (CO2-R) group:olecular ion-87 amu (M-87) in isopropyl esters and M-73 in ethyl

sters—these are m/z 253, 379, 505, and 631, in C-3 through C-6,espectively. Et–TFA derivatives were not run with the ion trap MS.n all acids a possible displacement product, [lactone(TFA)XH]+, isormed, e.g., erythronic acid, Fig. 5. These ions are represented by

/z 185, 311, 437, and 563, for C-3 through C-6 acids, respectivelym/z 563 is more prominent in ion trap MS, not shown). In any case,hese ions are consistent with lactones as they retain the num-er of native carbons (i.e., skeleton carbons of the acids—beforeerivatization) of each acid as shown by 13C labeled aldonic acidssynthesized in related work). That is, ions 185, 311, 437, and 563hift to 188, 315, 442, and 569, respectively, in the spectra of 13Cabeled acids. Related to this, m/z 299 in the ion trap spectrum oflyceric acid likely results from a 2H “McLafferty + 1” rearrange-ent [20] by the ester group of M. Such losses, in this case 41 amu

C3H5), are characteristic of esters and the resulting ions (e.g., M-1+) are usually formed in low abundances [20]. Loss of HO-TFAsee below) from m/z 299 forms the four-membered ring lactone,/z 185. Loss of HF, 20 amu, from all lactones is seen: it is more

rominent in C-5 and C-6 acids, e.g., m/z 543 in the spectrum ofdonic acid and m/z 417 for lyxonic acid (Fig. 4).

.3.2. Common fragments of aldonic acids and sugarsThe aldonic acids appear to have multiple ions in common with

FA-sugars (here, “sugars” refers to straight-chained aldo sugarsnly). Sequential losses of 113 (O-TFA) and 114 (HO-TFA) amu frag-ents from M-87 as well as sequential losses of 114 amu from

he above-postulated lactones (e.g., m/z 449, 335, and 221 in thepectra of C-6 acids) are likely (Fig. 4). In the mass spectra ofFA pentoses and hexoses, losses of O-TFA and HO-TFA (initiallyrom M) are known [21]. In the spectrum of (ISP–TFA or Et–TFA)lyceric acid, the odd-electron ion m/z 140 likely results from-87–113. m/z 140 retains two native carbons as does m/z 112

ormed by loss of CO from m/z 140. We also observe an ion of m/z40 in TFA–glyceraldehyde (monomer)—the corresponding sugarf glyceric acid. m/z 265 (TFAO–CH CH–CH O+TFA), known in TFAentose and hexose sugar spectra [21], is seen in all of the present-4 to C-6 aldonic acids (Fig. 4) and C-4 to C-6 sugars (related work).

The fragment of m/z 248 in the spectra of C-4 acids, threonicFig. 4) and erythronic retains three native carbons and is far lessbundant in higher aldonic acids and (monomer) sugars. It couldave formed by rearrangement and loss of OH (17 amu) from ion

65. We observe a prominent ion of m/z 248 in the spectra of glyc-raldehyde dimers (≤25% of base peak), C-4 sugar monomers (7%n threose, 13% in erythrose), and C-4 sugar polymers (≤15%). m/z37 in C-4 spectra could also result from m/z 265 (by loss of CO): ifo, this CO must be from a TFA group as m/z 237 also contains three

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onic acid mass spectra. Erythronic acid is an example.

native carbons. Another odd-electron ion, m/z 278 (M-87–113–114),in C-5 acids (Fig. 4) is also seen in C-5 sugars; it retains four nativecarbons. m/z 221 (above) is seen in the spectra of C-6 aldonic acids(Fig. 4) and C-6 sugars. Loss of CO from m/z 221 gives m/z 193 thatis seen in C-6 acids and C-5 and C-6 sugars. m/z 97, common in TFAderivatives, results from the loss of OH from trifluoroacetic acid in EIspectra (or from the loss of H2O from protonated trifluoroacetic acidin CI spectra) [22]. In the present study m/z 97 generally increasesin relative abundance with carbon number (Fig. 4), consistent withincreasing losses of HO-TFA.

4. Conclusions

The goal of this project was to analyze the enantiomer ratiosof a homologous series of C-3 to C-6 aldonic acids with a singlederivatization procedure and one GC analysis. These compoundsare found as a group in target samples and the majority is presentin trace amounts. The described procedures allow sub-nmole enan-tiomer analyses of 13 of 15 acids with one procedure using a chiralGC stationary phase of permethylated �-cylcodextrin. Fragmentsin the mass spectra of the alkyl-TFA derivatives are interpretablein terms of carboxyl-group losses while other ions are commonto TFA-aldoses. While analyses with standards are ongoing, thecurrent method is suitable for the analysis of rare and/or limitedsamples.

Acknowledgements

The authors are grateful to Dr. S. Pizzarello, Dr. J. Blank, andanonymous reviewers for their helpful suggestions and com-ments on the manuscript. We thank N. Ahuja, D. Nguyen, C.Reed, and M. Carter for assistance with preparation of C-6 aldonicacids. This work was supported by NASA’s Astrobiology Pro-gram.

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[

[[

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