Characterization of Single-Isomer, Heptasulfatedâ-Cyclodextrins by Electrospray Ionization MassSpectrometry and Indirect UV Detection CapillaryElectrophoresis

Pavel V. Bondarenko, Barbara Wolf, Hong Cai, J. Bryan Vincent, Ronald D. Macfarlane, andGyula Vigh*

Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255

The molecular weight, the purity, and the degree ofsubstitution for several newly synthesized single-isomer,heptasulfated â-cyclodextrin derivatives, heptakis-6-sulfato-â-cyclodextrin, heptakis(2,3-diacetyl-6-sulfato)-â-cyclo-dextrin, and heptakis(2,3-dimethyl-6-sulfato)-â-cyclodex-trin, were determined by electrospray ionization massspectrometry and indirect UV detection capillary electro-phoresis. The main fragmentation mechanism of thesesingle-isomer, fully sulfated cyclodextrins involves dis-sociation of the sulfate groups. The extent of desulfationincreases with the skimmer-collimator voltage bias andthe number of charges on the electrospray ions. Byreducing the skimmer-collimator voltage bias, the lossof sulfate groups could be reduced, though not completelyeliminated, for the doubly charged molecular ions. Thus,ESI-MS can be used to confirm the degree of substitutionfor the most sulfated isomer, but not to determine theexact concentration distribution for the lower substitutedisomers in a mixture.

Sulfated cyclodextrins (CDs) have been synthesized and usedfor a number of pharmaceutical,1-3 technical,4 and biomedical5

applications. They have also been used as additives in capillaryelectrophoresis (CE) to facilitate the separation of both ionic andnonionic enantiomers.6-8 The commercially available sulfated CDsare complex mixtures of isomers with different degrees and lociof substitution and different chiral selectivities. Therefore, it isof great analytical interest to create single-isomer sulfated CDs.Recently, several heptakis(6-sulfato)-â-CD derivatives were syn-thesized and successfully used for the CE separation of enan-tiomers.9-11 In this paper, we report electrospray ionization (ESI)mass spectrometric studies which augment the indirect UV

detection CE measurements and provide molecular weight (MW)information forsand confirm the structure ofsthe newly synthe-sized compounds.

Mass spectrometry has been used previously to study non-charged derivatized cyclodextrins,12,13 sulfoalkyl ether derivativesof cyclodextrins,14,15 heparin-derived oligosaccharides,16-22 andsulfated oligosaccharides.23-29 The presence of sodium or potas-sium counterions complicates the mass spectrometric investigationof sulfated oligosaccharides. Several matrixes and desaltingprocedures were introduced to increase the yield of molecularions and preserve their integrity during mass analysis. Heparin-derived oligosaccharides were adsorbed onto an immobilizedcationic surfactant film and desalted by a water rinse in a plasma

(1) Jarvinen, T.; Jarvinen, K.; Schwarting, N.; Stella, V. J. J. Pharm. Sci. 1995,84, 295.

(2) Shiotani, K.; Uehata, K.; Irie, T.; Uekama, K.; Thompson, D. O.; Stella, V. J.Pharm. Res. 1995, 12, 78.

(3) Fukunaga, K.; Hijikata, S.; Ishimura, K.; Sonoda, R.; Irie, T.; Uekama, K. J.Pharm. Pharmacol. 1994, 46, 168.

(4) Parmerter, S. M.; Allen, E. E.; Hull, G. A. U.S. Patent 3,426,011.(5) Folkman, J.; Weisz, P. B.; Joullie, M. M.; Li, W. W.; Ewing, W. R. Science

1989, 243, 1490.(6) Fanali, S. J. Chromatogr. A 1996, 735, 77.(7) Vigh, Gy.; Sokolowski, A. D. Electrophoresis 1997, 18, 2305.(8) Chankvetadze, B.; Endresz, G.; Blaschke, G. Electrophoresis 1994, 15, 804.

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V. Carbohydr. Res. 1996, 287, 139.(15) Jung, M.; Francotte, E. J. Chromatogr. A 1996, 755, 81.(16) McNeal, C. J.; Macfarlane, R. D.; Jardine, I. Biochem. Biophys. Res. Commun.

1986, 139, 18.(17) Juhasz, P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333.(18) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131.(19) Mallis, L. M.; Wang, H. M.; Loganathan, D.; Linhardt, J. Anal. Chem. 1989,

61, 1453.(20) Lamb, D. J.; Wang, H. M.; Mallis, L. M.; Linhardt, R. J. J. Am. Soc. Mass

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Chromatogr. 1992, 591, 225.(22) Da Col, R.; Silvestro, L.; Naggi, A.; Torri, G.; Baiocchi, C.; Maltrasio, D.;

Cedro, A.; Viano, I. J. Chromatogr. 1993, 647, 289.(23) Dai, Y.; Whittal, R. M.; Hindsgaul, O.; Li, L. Proceedings of the 44th ASMS

Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996; p 230.

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(25) Dai, Y. Q.; Whittal, R. M.; Bridges, C. A.; Isogai, Y.; Hindsgaul, O.; Li, L.Carbohydr. Res. 1997, 304, 1.

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M. J. Biol. Chem. 1995, 270, 13794.(28) Zhu, B. C. R.; Drake, R. R.; Schweingruber, H.; Laine, R. A. Arch. Biochem.

Biophys. 1995, 319, 355.(29) Takagaki, K.; Kojima, K.; Majima, M.; Nakamura, T.; Kato, I.; Endo, M.

Glycoconjugate J. 1992, 9, 174.

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desorption mass spectrometric study.16 Negative ion fast atombombardment (FAB) analysis was performed using triethanol-amine as the matrix.19 Tetrapropylammonium counterions formedadducts with sulfated oligosaccharides which did not fragmentextensively in ESI mass spectrometry.21,22 A flow injection ESIanalysis was performed using a Dionex anion membrane sup-pressor combined with postsuppressor addition of ammoniumhydroxide to eliminate the loss of sulfate groups in the negativeion ESI mass spectra.24 In another study, cation-exchange beadswere used to desalt the sample solution to assist the MALDIprocess.17 Recently, heparin-derived oligosaccharides were mixedwith arginine-rich basic peptides to form gas-phase ionic com-plexes. This technique eliminated sulfate loss and improved thedetection sensitivity in MALDI analysis.17,18 Coumarin 120 (7-amino-4-methylcoumarin) and 6-aza-2-thiothymine were found tobe effective MALDI matrixes for sulfated tri- and tetrasaccha-rides.23,25 The objective of this paper was to study the utility ofelectrospray ionization mass spectrometry for the characterizationof sulfated cyclodextrin derivatives.

EXPERIMENTAL SECTIONChemicals. The single-isomer heptasulfated â-CD derivatives

were synthesized in our laboratory as described in refs 9-11 andare now commercially available from Regis Technologies, Inc.(Morton Grove, IL). The sodium salt of heptakis-6-sulfato-â-CD(C42H63O35(SO3Na)7, MWcalc ) 1849.3) is denoted by the abbrevia-tion HS-â-CD, and the sodium salt of heptakis(2,3-diacetyl-6-sulfato), â-CD (C70H91O49(SO3Na)7, MWcalc ) 2437.8) is denotedby the abbreviation HDAS-â-CD, while the sodium salt of heptakis-(2,3-dimethyl-6-sulfato)-â-CD (C56H91O35(SO3Na)7, MWcalc ) 2045.7)is denoted by the abbreviation HDMS-â-CD. Previous analyticalwork indicates that they are pure compounds which have all sevensulfate groups on their primary face and contain only smallamounts of other isomers as impurities.9-11 HPLC grade waterand methanol were obtained from EM Science (Gibbstown, NJ).Phthalic acid and tris(hydroxymethyl)aminomethane (TRIS) forthe preparation of the CE background electrolytes were fromAldrich Chemical Co. (Milwaukee, WI).

Indirect UV Detection Capillary Electrophoretic Separa-tions. Indirect UV detection CE separations26 were carried outon a Beckman P/ACE 2200 unit (Beckman Instruments Inc.,Fullerton, CA) which was equipped with a UV detector operatedat 214 nm and connected to a 80486DX IBM PC computer. TheSystem Gold version 8.01 software package (Beckman) was usedto control the instrument and generate the electropherograms.An untreated, 25-µm-i.d., 144-µm-o.d., 17/25-cm fused silica capil-lary (Polymicro Technologies, Phoenix, AZ) was used for theseparations. The electric field strength was 650 V/cm. Thecapillary coolant was thermostated at 20 °C. The backgroundelectrolyte (BE) was made by adjusting the pH of a 20 mMphthalic acid solution to 8.5 with TRIS. All samples were pressureinjected for 1 s.

ESI-MS Analysis. Electrospray ionization mass spectra wereobtained with a Vestec model 201-A single-quadrupole massspectrometer, which was equipped with the Vestec electrosprayion source30 (PerSeptive Biosystems, Vestec Products, Framing-ham, MA). The mass range was limited to m/z 1200. Each

spectrum shown in this paper is an average of five scans. Thesingle-isomer sulfated cyclodextrin solutions were introduced bya model 341B SAGE syringe pump (Orion Research, Boston, MA)via a Valco injector with a 1-µL internal loop (Valco, Houston, TX).The single-isomer sulfated cyclodextrins were dissolved in 75:25(v/v) mixture of methanol/water at a concentration of 4 mg/mL.The apparent pH of the sample solution, measured with acombination glass electrode calibrated with aqueous referencebuffers, was 6.6. These sulfated cyclodextrin solutions wereloaded into the loop of the sampling valve and introduced intothe ion source at a flow rate of 1.6 µL/min. The ion source blockand the spray chamber were maintained at 250 and 50 °C,respectively. Voltages on the needle, nozzle, and collimator ofthe electrospray ion source were set at -2.4 kV, -0.2 kV, and-10 V, respectively.30 Two different skimmer potentials, -10 and-20 V, were tested, generating skimmer-collimator voltage biasvalues of 0 and 10 V, respectively. The skimmer-collimatorvoltage bias in this source serves the same purpose as the nozzle-skimmer voltage bias in other ion source designs.31,32 Maximumion transmission typically occurs at a voltage bias of 10 V. A lowervoltage bias (0 V) was also used to create softer ionizationconditions (see Results and Discussion).

RESULTS AND DISCUSSIONSHeptakis-6-sulfato-â-cyclodextrin (HS-â-CD). Figure 1a

shows the indirect UV detection electropherogram of the single-isomer heptakis-6-sulfato-â-CD (HS-â-CD), indicating the presenceof the target compound and a small amount of impurities (hexa-and octasulfated cyclodextrin isomers, respectively) on both sidesof the main peak. In this paper, the time scale of all electrophero-grams is converted into effective mobility (µeff) scale to allow directcomparison of the electropherograms, independently of thevelocity of the electroosmotic flow.

Figure 1b,c shows the negative ion ESI mass spectra of HS-â-CD recorded under the soft and normal ionization conditions,respectively. The inserted pictograms33 represent the two differentionization modes (soft, Figure 1b; normal, Figure 1c). Thenumbers immediately above the peaks denote the number ofsulfate groups on the particular electrospray ion. The boldnegative italic numbers above the brackets indicate the numberof charges on the electrospray ions. The negative gas-phase ionsare formed by dissociation of Na+ cation(s) from the sulfatedcyclodextrin anion.

It was reported previously,31 and observed in our spectra, thatsulfated carbohydrate molecules with more than one sulfate groupcan generate multiply charged electrospray ions: the maximumnumber of charges on the ions is equal to the number of sulfategroups. The loss of one or more sulfate groups is the prominentfragmentation pattern for the 3-, 4-, 5-, and 6- charged ions(Figure 1c), and the extent of fragmentation increases with thenumber of charges for the higher charge-state electrospray ions.One reason for the increased fragmentation could be Coulombicenergy, the value of which increases with the number ofcharges.34-36 The other reason could be collision-induced dis-sociation (CID) in the skimmer-collimator region. In Figure 1b,c,

(30) Allen, M. H.; Vestal, M. L. J. Am. Soc. Mass Spectrom. 1992, 3, 18.

(31) Bruins, A. P.; Covey, T. R.; Henion, J. Anal. Chem. 1987, 59, 2642.(32) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988,

2, 207.(33) Lehmann, W. D. J. Am. Soc. Mass Spectrom. 1997, 8, 756.

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the (4-, 6)/(4-, 7), (4-, 5)/(4-, 7), and (4-, 4)/(4-, 7)ion intensity ratios increase dramatically when the skimmer-collimator voltage bias is increased from 0 to 10 V. This can beexplained by postulating that ions (4-, 6), (4-, 5), and (4-, 4)originate from the heptasulfated cyclodextrins (7) in the solutionand lose one, two, or three sulfate groups, respectively, duringthe ion formation process. The skimmer-collimator bias voltageaccelerates the multiply charged molecular ions with respect tothe neutral molecules of the residual gas and causes energeticion-molecule collisions.30-32 The collisional energy is propor-tional to the number of charges and to the skimmer-collimatorvoltage bias. By reducing this voltage, we were able to reduce,though not completely eliminate, most of the fragmentation forthe 2- ions.

The fragmentation of (7-, 7) ions cannot be directly demon-strated in this experiment because this fragmentation yields the(6-, 6) daughter ion, which is superimposed on the (6-, 6)fragmentation products of (6-, 7) ions.

It was first reported in a negative ion FAB study that the lossof a NaSO3 (103 u) group is accompanied by the addition of a H(1 u) atom which, most likely, comes from the matrix.19 Our data(Figure 1c) also show that the mass difference between thefragments is 102 u, which suggests an analogous fragmentationpattern.

This fragmentation mechanism prevents the ESI-MS determi-nation of the true concentration distribution of the differentlysulfated cyclodextrin isomers in their mixtures, because thefragment ions from a more highly sulfated species and themolecular ions from a lesser sulfated CD have identical MWvalues. One can, though, by minimizing the fragmentation of themolecular ion, correctly determine the degree of sulfation for themost highly substituted isomer of the mixture. This feature isindicated by the measured molecular weight of HS-â-CD (MWmeas

) 1849.1 ( 0.6), which is in good agreement with the calculatedone. (The standard deviation was calculated from all multiplycharged ions.)

Heptakis(2,3-diacetyl-6-sulfato)-â-cyclodextrin (HDAS-â-CD). The indirect UV detection electropherograms and ESI massspectra of HDAS-â-CD recorded under soft ionization conditionsare shown in Figure 2a,b, respectively. The material once againcontains only a small amount of isomeric contamination. Theappearance of the entire mass spectrum and the fragmentationpattern is similar to what was observed for HS-â-CD, except that,due to the m/z limitation of our instrument, only the ions withthe 3- charge state or higher can be observed. A low-intensity(M - SO3 + 3H - 3Na)3- fragment accompanies the molecularion, similarly to the other triply charged single-isomer sulfated

(34) Rockwood, A. L.; Busman, M.; Smith, R. D. Int. J. Mass Spectrom. Ion Phys.1991, 111, 103.

(35) Schnier, P. D.; Gross, D. S.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1995,6, 1086.

(36) Ishikawa, K.; Nishimura, T.; Koga, Y.; Niwa, Y. Rapid Commun. MassSpectrom. 1994, 8, 933.

Figure 1. Indirect UV detection CE separation (a) and negative ionESI mass spectra (b and c) for the single-isomer heptakis-6-sulfato-â-CD (HS-â-CD). Numbers at the peaks denote the number of sulfategroups on the cyclodextrin electrospray ions. The skimmer-collimatorvoltage bias is 0 V (soft ionization conditions) in (b), and 10 V (normalionization conditions) in (c). Number with a minus sign indicate thecharges on the electrospray ions.

Figure 2. Indirect UV detection CE separation (a) and negative ionESI mass spectrum (b) for the single-isomer heptakis(2,3-diacetyl-6-sulfato)-â-CD (HDAS-â-CD). Numbers at the peaks denote thenumber of sulfate groups on the cyclodextrin electrospray ions. Theskimmer-collimator voltage bias is 0 V (soft ionization conditions) in(b). Numbers with a minus sign indicate the charges on the electro-spray ions.

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CD derivative ions. The measured molecular weight (MWmeas )2438.0 ( 0.7) is in agreement with the calculated one.

Heptakis(2,3-dimethyl-6-sulfato)-â-cyclodextrin (HDMS-â-CD). The indirect UV detection electropherograms and ESImass spectra of HDMS-â-CD recorded under soft ionizationconditions are shown in Figure 3a,b, respectively. This materialis also quite pure and contains only a small amount of isomericcontamination. It is interesting to note that, unlike the othersingle-isomer sulfated CD derivatives, HDMS-â-CD produces 5-,6-, and 7- HDMS-â-CD ions of relatively low intensities (Figure3b). Consequently, this m/z region is less “congested” with peaksthat originate from the single-isomer sulfated CD. This featureof HDMS-â-CD can be important in the CE-MS analysis of low-

mass analytes which can be electrophoretically separated usingthis single-isomer sulfated CD. The measured molecular weightof HDMS-â-CD (MWmeas ) 2046.0 ( 0.6) is in agreement withthe calculated value.

CONCLUSIONSThe combination of negative ion ESI mass spectrometry and

indirect UV detection capillary electrophoresis made possible thereliable determination of the molecular weight, purity, andsubstitution degree for several newly synthesized single-isomer,heptasulfated â-CD derivatives. Dissociation of the sulfate groupsseems to be the dominant fragmentation pathway: the extent ofdissociation increases with the skimmer-collimator voltage biasand the number of negative charges on the ion. By reducing theskimmer-collimator voltage, the extent of sulfate group dissocia-tion could be reduced, but not completely eliminated, for the 2-(and, presumably, the 1-) ions, indicating that the true degreeof sulfate substitution can always be established correctly for themost highly substituted cyclodextrin isomer. In the negative ionESI technique, the loss of a NaSO3 group is accompanied by theaddition of a hydrogen atom which can originate from the matrixwith the hydrogen transfer taking place during the ionization/desorption event. Further fragmentation studies of the single-isomer sulfated CDs would benefit from an ESI instrument witha broader m/z scale and with MS/MS capabilities, permitting theobservation of the singly charged ions.

ACKNOWLEDGMENTPartial financial support by the Texas Coordinating Board of

Higher Education ARP program (Project No. 010366-016), theWelch Foundation (A-0258), and Beckman Instruments (Fullerton,CA) is gratefully acknowledged. The Vestec V-201A mass spec-trometer was purchased with funds provided by the NationalScience Foundation (CHE-8705697). We are indebted to Cerestar(Hammond, IN) for the â-cyclodextrin sample used in thesynthesis of the single-isomer sulfated cyclodextrin derivativesand Beckman Instruments for the loan of the P/ACE unit.

Received for review October 6, 1997. Accepted March 31,1998.

AC971103B

Figure 3. Indirect UV detection CE separation (a) and negative ionESI mass spectrum (b) for the single-isomer heptakis(2,3-dimethyl-6-sulfato)-â-CD (HDMS-â-CD). Numbers at the peaks denote thenumber of sulfate groups on the cyclodextrin electrospray ions. Theskimmer-collimator voltage bias is 0 V (soft ionization conditions) in(b). Numbers with a minus sign indicate the charges on the electro-spray ions.

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