Analysis of Oligosaccharides Derived from Heparin by Ion-Pair

Anal. Chem. 2009, 81, 3485–3499

Analysis of Oligosaccharides Derived from Heparin by Ion-Pair Reversed-Phase Chromatography/Mass Spectrometry Catalin E. Doneanu, Weibin Chen,* and John C. Gebler Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757 Current chromatographic and mass spectrometric techniques have limitations for analyzing heparin and heparin oligomers due to their high polarity, structural diversity, and sulfate lability. A rapid method for the analysis of heparin oligosaccharides was developed using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method utilizes an optimized buffer system containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro2-propanol (HFIP), to achieve highly efficient separation together with enhanced mass response of heparin oligosaccharides. Analyses of a heparin oligosaccharide test mixture, dp6 through dp22, reveal that the chromatographic conditions enable baseline resolution of isomeric heparin oligosaccharides (dp6) and produce intact molecular ions with no sulfate losses during mass spectrometric analysis. In addition, the described conditions are amenable to the detection of heparin oligosaccharides in positive ion mode, yield stronger positive ion signals for corresponding oligosaccharides compared to the negative ion mode, and allow identification of structural isomers by an MS/MS approach. Because sensitive detection of oligosaccharides is also achieved with ultraviolet (UV) detection, the method utilizes a dual detection scheme (UV and MS in series) along with IPRP UPLC to simultaneously obtain quantification (UV) and characterization (MS) data for heparin oligosaccharides. The broad potential of this new method is further demonstrated for the analysis of a low-molecular-weight heparin (LMWH) preparation from porcine heparin. This approach will be of particular utility for profiling the molecular entities of heparin materials, as well as for structural variability comparison for samples from various sources. Heparin is a linear polysaccharide with repeating disaccharide units consisting of a hexuronic acid (HexA) and a D-glucosamine (GlcN) residue. The hexuronic acid can either be a D-glucuronic acid or a L-iduronic acid, and the glucosamine may either be N-acetylated or N-sulfonated. The monosaccharide building blocks are joined together by 1 f 4 glycosidic linkages, forming the primary structure of heparin with a range of chain lengths * To whom correspondence should be addressed. Fax: 508-482-3085. E-mail: [email protected]. 10.1021/ac802770r CCC: $40.75  2009 American Chemical Society Published on Web 04/03/2009

Chart 1. Illustration of the Major Structures of Heparin Oligosaccharides Derived from the Enzymatic Depolymerization Processes

(polydispersity). The molecular weight of heparin can vary from 3 to 40 kDa, with a mean molecular weight of 15 kDa (a chain with approximately 45 monosaccharides).1 Additional structural heterogeneity of heparin arises from variable patterns of sulfation. Up to three sulfate substitutions can occur on each disaccharide unit (HexA and GlcN; Chart 1), resulting in a large number of complex sequences. The most highly sulfated and common regions1 on heparin chains contain three sulfate/sulfonate groups per disaccharide unit, 2-O-sulfated HexA, 6-O-sulfated, and 2-Nsulfonated GlcN. Because of the presence of many covalently linked sulfate/sulfonated and carboxylic acid groups, heparin is strongly acidic and has the highest negative charge density of any known biological molecule.2 As a result, heparin is frequently available as a cationic salt resulting from the partial replacement of the acidic protons of the sulfate groups by metal ions. Heparin is known to be involved in a number of important biological processes. Heparin and its derivative, low-molecularweight heparin (LMWH), have been widely used as clinical anticoagulant drugs for decades during surgery and kidney dialysis.3 It has also been demonstrated that heparin possesses several biological activities that extend beyond anticoagulation. For example, heparin oligosaccharide structures have been found to play a role in mediating several major diseases including inflammation,4 Alzheimer’s disease,5 and cancer,6 making them of great interest in new drug discovery.7 Despite its medical and biological importance, heparin is relatively uncharacterized in terms of its chemical structures. The (1) Casu, B. In Heparin: Chemical and Biological Properties, Clinical Applications; Lane, D. A., Lindahl, U., Eds.; Edward Arnold: London, 1989; pp 2549. (2) Zhang, F.; Yoder, P. G.; Linhardt, R. J. In Polysaccharides: Structural Diversity and Functional Versatility, 2nd ed.; Dumitriu, S., Ed.; Marcel Dekker: New York, 2004; p 773. (3) Petitou, M.; Casu, B.; Lindahl, U. Biochimie 2003, 85, 83. (4) Malhotra, S.; Bhasin, D.; Shafiq, N.; Pandhi, P. Expert Opin. Pharmacother. 2004, 5, 329.

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limited success in structural characterization is largely attributed to the chemical heterogeneity and the structural complexity of heparin. In most cases, detailed analysis of heparin structures typically involves the depolymerization of intact unfractionated heparin, either through an enzymatic or chemical method, and the subsequent analysis of heparin-derived oligosaccharides by specialized analytical instruments and techniques. For example, NMR spectroscopy is one of the most effective tools for the structural elucidation of heparin.8-10 It can provide valuable structural details such as monosaccharide composition, glycosidic linkage, and sulfation patterns. Mass spectrometry (MS) is another powerful technique for structural elucidation of biopolymers. It offers accurate molecular weight measurement for intact oligosaccharides and deduces oligosaccharide sequence through fragmentation.11 In comparison with other detection and identification techniques for the analysis of heparin oligosaccharides, mass spectrometric methods have several advantages including low sample consumption (picomoles), short analysis time (few minutes), and analytical versatility. With the development of soft ionization methods such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), MS has been increasingly applied to the characterization of heparin oligosaccharides.12 Nonetheless, both MS and NMR techniques have challenges when they are applied to heparin characterization, especially when no initial sample separation is involved. For instance, NMR analysis requires a relatively large amount (10 nmol to 1 µmol) of pure oligosaccharide material and therefore is not suitable for biologically significant oligosaccharides, which often contain unusual sequences13,14 and are available only in limited quantities. As for MS techniques, the high acidity of sulfategroups and their lability toward fragmentation have made direct MS analyses of heparin oligosaccharides a formidable task. In general, the difficulties increase with increasing number of sulfate groups in the heparin oligosaccharides. Another barrier for the MS analysis of heparin oligosaccharides is their propensity to form adducts with metal cations, which result in greater spectral complexity and signal splitting.15 Careful sample handling is required to avoid contamination by adventitious metal ions (especially sodium) to reduce the chances of adduct formation. Because depolymerization of heparin normally creates heterogeneous mixtures with a distribution of structure-related oligosaccharides, the sample complexity implies that approaches that utilize NMR or MS technique alone will be difficult to extend to more complex mixtures containing both small and large oligosaccharides. A combination of a separation technique with an (5) Scholefield, Z.; Yates, E. A.; Wayne, G.; Amour, A.; McDowell, W.; Turnbull, J. E. J. Cell Biol. 2003, 163, 97. (6) Zacharski, L. R.; Loynes, J. T. Curr. Opin. Pulm. Med. 2002, 8, 379. (7) Linhardt, R. J.; Toida, T. In Carbohydrates as Drugs; Witczak, Z. B., Nieforth, K. A., Eds.; Marcel Dekker, Inc.: New York, 1997; pp 277-341. (8) Pervin, A.; Gallo, C.; Jandik, K. A.; Han, X. J.; Linhardt, R. J. Glycobiology 1995, 5, 83. (9) Yang, H. O.; Gunay, N. S.; Toida, T.; Kuberan, B.; Yu, G. L.; Kim, Y. S.; Linhardt, R. J. Glycobiology 1999, 10, 1033. (10) Mikhailov, D.; Linhardt, R. J.; Mayo, K. H. Biochem. J. 1997, 328, 51. (11) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161. (12) Chi, L.; Amster, J.; Linhardt, R. J. Curr. Anal. Chem. 2005, 1, 223. (13) Rosenberg, R. D.; Lam, L. Proc. Natl. Acad. Sci. U.S.A. 1999, 76, 1218. (14) Kinoshita, A.; Yamada, S.; Haslam, S. M.; Morris, H. R.; Dell, A.; Sugahara, K. J. Biol. Chem. 1997, 272, 19656. (15) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 2060– 2066.

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identification technique clearly provides an advantage in understanding heparin structures. Recent efforts in the development of analytical methodologies for heparin analysis have coupled electrophoretic and chromatographic techniques with MS to enhance structural characterization.16-26 The liquid-phase separation techniques fractionate oligosaccharides in complex samples that are otherwise not possible for direct ESI-MS analysis. The application of these techniques has generated some success in the past to solve structure-related problems for heparin analysis. For instance, capillary electrophoresis (CE) was directly coupled with MS to analyze heparin oligosaccharide mixtures prepared from porcine mucosa heparin by enzymatic digestion.23 On the basis of analysis of eight known disaccharide standards, the structures of a saturated disaccharide and two tetrasaccharides that were not identified previously were determined. Although CE is a highresolution separation technique and is capable of separating larger oligosaccharides, the choice of buffers is rather limited to couple CE with MS because a number of buffers typically used in CE are nonvolatile. In addition, CE requires daily calibration to maintain day-to-day reproducibility in migration time.23 Many liquid chromatography techniques have also been used to separate and thus simplify oligosaccharide mixtures for heparin analysis. Strong anion exchange chromatography (SAX) seems to be the ideal choice to separate heparin oligosaccharides because the separation mechanism is based upon charge density differences.27,28 However, SAX is difficult to directly couple with MS due to the use of high-concentration nonvolatile salts in the mobile phases. Other chromatographic techniques that are amenable to online ESI-MS analysis include size-exclusion chromatography (SEC),20 hydrophilic interaction chromatography (HILIC),21 and ion-pair reversed-phase (IPRP) chromatography.16-19 However, only limited success was reported in adapting these chromatographic techniques for the LC/MS analysis of heparin oligomers. An online SEC LC/MS method was used to determine the molecular weights for more than 60 oligosaccharide components in an LMWH preparation.20 The SEC method had the benefit of (16) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707. (17) Thanawiroon, C.; Rice, K. G.; Toida, T.; Linhardt, R. J. J. Biol. Chem. 2004, 279 (4), 2608. (18) Henriksen, J.; Roepstorff, P.; Ringborg, L. H. Carbohydr. Res. 2006, 341, 382. (19) Korir, A. K.; Limtiaco, J. F. K.; Gutierrez, S. M.; Larive, C. K. Anal. Chem. 2008, 80, 1297. (20) Henriksen, J.; Ringborg, L. H.; Roepstorff, P. J. Mass Spectrom. 2004, 39, 1305. (21) Naimy, H.; Leymarie, N.; Bowman, M. J.; Zaia, J. Biochemistry 2008, 47, 3155. (22) Venkataraman, G.; Shriver, G.; Raman, R.; Sasisekharan, R. Science 1999, 286, 537. (23) Gunay, N. S.; Linhardt, R. J. J. Chromatogr., A 2003, 1014, 225. (24) Rhomberg, A. J.; Ernst, S.; Sasisekharan, R.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4176. (25) Duteil, S.; Gareil, P.; Girault, S.; Mallet, A.; Feve, C.; Siret, L. Rapid Commun. Mass Spectrom. 1999, 13, 1889. (26) Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, K.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669. (27) Imanari, T.; Toida, T.; Koshiishi, I.; Toyoda, H. J. Chromatogr., A 1996, 720, 275. (28) Rice, K. G.; Kim, Y. S.; Grant, S. C.; Merchant, Z. M.; Linhardt, R. J. Anal. Biochem. 1985, 150, 325.

using simple mobile-phase compositions, but the resolution of the SEC separation was lower compared to other chromatographic techniques. Complete resolution between oligosaccharides with two close chain lengths (e.g., dp6 and dp8) was not demonstrated in the report.20 In addition, the coupling of an online cationexchange column was needed to minimize the massive adduction of various cations with oligosaccharide molecular ions. Recently a HILIC LC/MS platform was developed for screening and quantitative analysis of a library of protein-binding heparin/ heparan sulfate (HS) oligosaccharides.21 The oligosaccharides were separated on a HILIC column on the basis of their overall polarity which is determined by the size, sulfation, and acetylation content. However, the technique was unable to resolve isomeric oligosaccharides efficiently.21 In addition, for SEC and HILIC, the separation times are long, where a typical separation method takes about 60 min per analysis.20,21 Ion-pair reversed-phase chromatography (IPRP-LC) coupled with online MS detection is a promising technique for the analysis of heparin oligosaccharides with minimal sample preparation.16-19 In the IPRP-LC separation of heparin oligosaccharides, ion pairing agents, typically amines with hydrophobic alkyl chains,16,17,29 are added to the mobile phases and the separation is performed on a traditional reversed-phase column (e.g., C18). IPRP-LC separates oligosaccharides on the basis of size, sulfation content, and isomerization of heparin oligosaccharides,16,17,30 while also removing alkali or alkaline earth metal cations from oligosaccharide samples to minimize MS interference from cation adduction.17 Recent progress in the application of IPRP for heparin oligosaccharide analysis includes the use of ultraperformance liquid chromatography (UPLC) to circumvent the long column equilibration time issue associated with the IPRP LC technique.19 UPLC separations are performed at high pressures (up to 15 000 psi) with columns packed with 1.7 µm particles and are known to provide increased speed, efficiency, and resolution compared to traditional HPLC analysis.31 Methods using UPLC for separation achieved complete resolution of 12 common heparin disaccharides in less than 5 min,19 demonstrating the high resolving power of sub-2 µm particles for heparin disaccharide analysis. Although disaccharide analysis of completely depolymerized heparin is valuable toward its complete sequence analysis, as shown previously by Venkataraman et al.,22 the information obtained by this approach is rather limited because it only provides the relative ratios of disaccharide components for the unfractionated intact heparin. Because the functional units of heparin are usually oligosaccharide domains with a chain length of 14-20 monosaccharide residues, analysis of larger portions of the intact chain becomes more useful for mapping the domain structures. On the other hand, because of the presence of a larger number of negatively charged sulfate groups in longer heparin oligosaccharides, their analysis by IPRP-LC ESI/MS is considerably more challenging than the analysis of short ones. Both the propensity of cation-adduct formation and the probability of losing sulfate groups during MS analysis increase drastically with increasing oligosaccharide length. As a result, only a few attempts have been (29) Karamanos, N. K.; Vanky, P.; Tzanakakis, G. N.; Tsegenidis, T.; Hjerpe, A. J. Chromatogr., A 1997, 765, 169. (30) Lawrence, R.; Kuberan, B.; Lech, M.; Beeler, D. L.; Rosenberg, R. D. Glycobiology 2004, 14, 467. (31) Swartz, M. E. J. Liq. Chromatogr. Relat. Technol. 2005, 28, 1253–1263.

made to adapt IPRP LC/MS for the analysis of longer oligosaccharides. Thanawiroon et al.17 analyzed a preparation of enzymatically digested heparin sample and obtained spectra of heparin oligosaccharides up to tetradecasaccharide (dp14). The experimental setup mandated a second eluent of 5 mM tributylamine in acetonitrile to be simultaneously sprayed into the ion source in order to improve the MS responses of larger oligomers. In a separate report, two different IPRP separation schemes had to be developed17 to meet the needs for the separation of complex mixtures of larger heparin oligosaccharides and for the resolution of isomeric heparin oligosaccharides in fractions with a limited dp range. In addition, each separation scheme required a long gradient time (60 min), and gradient slopes needed to be adjusted for different sample composition to obtain isomer separation. Therefore, the sample throughput was rather limited. This work describes the development of a rapid, robust, and simple method for heparin oligosaccharides analysis using ionpair reversed-phase ultraperformance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (IPRP-UPLC ESI Q-TOF MS). The method was developed for rapid profiling of large heparin fragments prepared from heparin depolymerization processes. A systematic evaluation of a range of ion-pairing reagents and mobile phase compositions was performed. An optimized mobile phase containing a linear pentylamine and a unique additive, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), was developed for heparin oligosaccharides to yield highly efficient separation together with enhanced MS response. In addition, the method took advantage of the speed, efficiency, and resolution offered by UPLC and performs the analysis using reversed-phase C18 columns packed with 1.7 µm particles to significantly shorten the analysis time while maintaining a high separation efficiency. The described method was successfully applied to the analysis of a mixture of heparin oligosaccharides and a LMWH preparation from porcine tissue, where monoisotopic masses of oligosaccharides up to dp22 (with 37 sulfate groups) were measured. EXPERIMENTAL SECTION Materials and Oligosaccharide Samples. All linear and branched aliphatic amines used in this study [n-propylamine (PPA), n-butylamine (BTA), n-pentylamine (PTA), n-hexylamine (HXA), n-octylamine (OTA), tripropyl amine (TPA), and tributyl amine (TBA)] were the highest grade available from SigmaAldrich (St. Louis, MO). Acetic acid (AA), formic acid (FA), 1,1,1,3,3,3-hexafluoro isopropanol (HFIP), Glu-Fibrinopeptide B (GFP), and raffinose were also purchased from Sigma-Aldrich. Ultrapure water (18 MΩ cm) was obtained in-house from a Milli-Q water purifier (Millipore Corporation, Medford, MA). Acetonitrile (ACN) was provided by Honeywell Burdick and Jackson (Muskegon, MI). Sodium salts of heparin oligosaccharide fractions (dp6, 8, 10, 12, 14, 16, 18, 20, and 22) were purchased from V-Laboratories, Inc. (Covington, LA) and were used without further purification. Each oligosaccharide fraction was dissolved in pure water to prepare stock solutions at a concentration of 2 mg/mL. The active ingredient of the LMWH drug Innohep, tinzaparin sodium, was provided by LEO Pharma (Ballerup, Denmark) as a gift. For IPRPUPLC/MS analysis, the tinzaparin sodium solution was prepared Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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at a concentration of 1 mg/mL by dissolving the dry powder in Milli-Q water. LC Separation. LC separation was performed on an ACQUITY UPLC system (Waters Corporation, Milford, MA) equipped with a binary solvent manger, a sample manager, a column heater, and a tunable UV (TUV) detector. All UPLC separations were performed on an ACQUITY UPLC BEH C18 column, 2.1 mm × 150 mm, 1.7 µm (Waters) at a flow rate of 0.4 mL/min. All injections were performed in the full-loop injection mode using a 10 µL sample loop. The column temperature was maintained at 45 °C. UV absorbance was monitored at two wavelengths (220 and 232 nm) with a sampling rate of 2 points/s. As described below in the discussion, different mobile phase compositions with various amines and acids were tested and evaluated during the study. Specific gradient conditions, mobile phase compositions, and other conditions are given in the figure captions. In general, eluent A consisted of a pure aqueous solution and eluent B contained 75% acetonitrile/25% aqueous solution (v/v). Both eluents A and B contained an equal concentration of ion pairing reagent (amines) and were buffered with the same volume of acids to adjust the pH. All eluents were degassed ultrasonically prior to the use. Mass Spectrometry. The ACQUITY UPLC system was connected to a standard ESI source on a hybrid quadrupole timeof-flight mass spectrometer (either a Q-Tof Premier mass spectrometer or a SYNAPT HDMS mass spectrometer, Waters Corp.) via a fused silica capillary (75 µm i.d.). The data acquisition was controlled by Masslynx 4.1 (Waters), and depending on the experimental objectives (see discussion below), spectra were acquired either in negative or positive ion mode. The following instrument settings were common for analyses performed in both positive and negative ion modes: source temperature 120 °C, desolvation temperature 400 °C, collision energy 5 eV. When operated in negative ion mode, the Q-Tof Premier mass spectrometer used the following instrument settings: capillary voltage 2.8 kV, cone voltage 25 V, extraction cone 4 V. The following instrumental parameters were used on the SYNAPT HDMS System for data acquisition in positive ion mode: capillary voltage 2.0 kV, cone voltage 30 V, extraction cone 5 V. The data were acquired on both instruments using V-mode with a fwhm resolution of ∼ 10 000, an m/z range of 400-3000, and a scan time of 0.6 s. The lock-mass solutions for instrument calibration contained 0.1 µM GFP in 50% ACN and 0.1% FA for positive ion mode ([M + 2H]2+, m/z ) 785.8421) and 1 µg/mL raffinose in 50% ACN for negative mode ([M - H]-, m/z ) 503.1617). Monoisotopic masses are reported throughout the text. RESULTS AND DISCUSSION The focus of this work is to develop a rapid and effective IPRPUPLC/MS method for the analysis of heparin-derived oligosaccharides. The method should achieve highly efficient chromatographic separation of the oligosaccharide mixture while greatly improving the quality of mass spectra by enhancing MS responses and minimizing the sulfate group losses. For these purposes, mobile phases consisting of volatile ion-pairing reagents such as tripropylammonium acetate (TPAA) and tributylammonium acetate (TBAA) were evaluated at the beginning of the project. These mobile phases were previously employed in the LC/MS analysis 3488


of highly sulfated carbohydrates.16,17 The experiments using these mobile phases did not yield satisfying results in our laboratory because of low MS sensitivity and inadequate chromatographic resolution for large oligomers. Most of the problems encountered were linked to the mobile phase compositions and the purity of ion-pairing reagents. The observed ESI-MS signals for larger oligosaccharides (>dp8) decreased rapidly with increasing chain lengths due to ion suppression and losses of sulfate groups, making accurate MW measurements difficult. In addition, despite of the use of the highest-grade TPA and TBA reagents that are commercially available, ions corresponding to adducts of the same oligosaccharide with multiple ion-pairing structures (such as dialkyl amines) were readily observed among all the heparin oligomers. Furthermore, mobile phases prepared using the tertiary aliphatic amines tended to produce strong absorbance at wavelength 232 nm, thus limiting the utility of UV detection for heparin oligosaccharides. Because of these limitations, alternative ionpairing reagents and mobile phase compositions were investigated in order to determine how their concentration and chemical properties would affect LC/MS analysis of heparin oligosaccharides. Effect of Mobile Phase Composition on Chromatographic Performance. Linear aliphatic amines have been used as ionpairing reagents for the separation of inorganic anions and carboxylic and sulfonic acids.32,33 They are readily available in high purity and are relatively more soluble in aqueous mobile phases compared to the corresponding tertiary amines with the same alkyl chains. However, the suitability of linear alkyl amines as ion-pairing regents for the separation of heparin oligosaccharides has not been extensively studied.17 In an effort to find an alternative ion-pairing reagent that would enable separation of large heparin oligosaccharides, the influence of the alkyl chain lengths of five linear alkyl amines (PPA, BTA, PTA, HXA, and OTA) on the retention and resolution of heparin-derived oligosaccharides was investigated. To measure the retention times and resolution of oligosaccharides, a test mixture containing three commercially available heparin-derived oligosaccharide fractions (dp6, dp8, dp10) was prepared with each fraction at a concentration of 50 µg/mL. On the basis of separate LC/MS analyses of each individual fraction, as well as the product information from the vendor, it was observed that each dp6/dp8/dp10 fraction contains not only the corresponding fully sulfated oligosaccharides but also many other components that represent under-sulfated oligosaccharide isomers (less than three sulfate groups for every disaccharide repeating unit). This level of complexity created a useful test sample to evaluate the separation efficiency of different ion-pairing reagents and/or different mobile phase compositions. For example, the coexistence of the fully sulfated hexasaccharide (nine sulfate groups) and the hexasaccharide with one less sulfate group (eight sulfate groups) in the mixture provides good reference markers from which the separation efficiency can be measured. For the evaluations of different ion-pairing reagents, the mobile phases were composed of a linear alkyl amine as an ion-pairing reagent (15 mM) and 100 mM HFIP as a buffering acid. Since these linear alkyl amines do not carry a permanent positive charge, (32) Juhasz Marengo, E.; Gennaro, M. C.; Abrigo, C. Anal. Chem. 1992, 64, 1885–1893. (33) Kraak, J. C. J. Chromatogr. 1978, 161, 69–82.

Table 1. Gradient Profiles Used for Each of the Linear Alkyl Amines Investigated As Ion-Pairing Reagents for the Separation of dp6/8/10 Oligosaccharide Fractions ion-pairing reagents

percentage of eluent B at the beginning and end of gradient

name

concentration (mM)

beginning

end

gradient slope (% B/min)

PPA BTA PTA HXA OTA

15 15 15 15 15

0 0 10 25 60

15 30 40 55 90

1.5 3.0 3.0 3.0 3.0

an acid is usually added to the mobile phase to protonate the amines. HFIP is a volatile fluorinated alcohol, and because of the acidity (a 100 mM HFIP aqueous solution has a pH value of 4.5), its addition helps to lower the solution pH to convert all the free alkyl amines into ammonium ions. Additional roles of the HFIP modifier are further discussed in the following sections. For all mobile phases, the pH of the aqueous solutions was in the range of 8.2-8.4. For each ion-pairing reagent, the oligosaccharides in the test mixture were eluted with a linear gradient. The beginning of the gradient profiles was adjusted (see Table 1) in order to obtain similar retention times for the fully sulfated hexasaccharide (dp6, ∼6 min) and the fully sulfated decasaccharide (dp10, ∼9 min), while the flow rate (0.4 mL/min) and the gradient slope (3% B/min) were kept constant across all the mobile phase conditions evaluated (except PPA, which used 1.5% B/min). Separation performance for each ion-pairing reagent was compared based on the calculated peak capacity (P).34 For each hexa-, octa-, and decasaccharide, a pair of peaks that represent the fully sulfated isomer and the corresponding most abundant isomer with one less sulfate group was chosen to calculate the peak capacity. The P value was calculated according to eq 1: P)1+

t2 - t1 ω4σ

(1)

where t1 and t2 are the corresponding retention times for the fully sulfated and most abundant monoundersulfated oligosaccharide isomers, respectively, and ω4σ is the average peak widths at 13.4% of peak height. By definition, the peak capacity reflects the theoretical number of peaks that can be resolved within a specific gradient time. In this particular case, P values predict the separation performance of various mobile phases between heparin oligomers with the same chain length but different sulfate groups. The effect of the chain length of linear alkyl amines on resolution of heparin oligosaccharides is shown in Figure 1A. As a comparison, the peak capacity for the mobile phase containing tripropyl amine (TPA) is also plotted. TPA has been reported in the literature17 as an ion-pairing reagent for the separation of sulfated oligosaccharides. Employing identical experimental conditions, both PTA and HXA provide considerably higher peak capacities than TPA, PPA, BTA, or OTA for the hexa, octa-, and decaoligosaccharides. On average, peak capacities of PTA and (34) Neue, U. D. HPLC Columns: Theory, Technology, and Practice; Wiley-VCH: New York, 1997; p 314.

Figure 1. Impact of mobile phase composition on the peak capacity of oligosaccharides using IPRP-UPLC. (A) Influence of different ion-pairing reagents on the peak capacity of heparinderived oligosaccharide dp6/8/10. The mobile phases contain 100 mM HFIP and one of the following ion pairing reagents at 15 mM concentration: PPA (n-propylamine), BTA (n-butylamine), PTA (npentylamine), HXA (n-hexylamine), and OTA (n-octylamine). (B) Effect of PTA concentration on the peak capacity of heparin oligosaccharide dp6-dp14 and dp18. The mobile phases contain 100 mM HFIP and one of the following PTA concentrations: 5, 15, 25, and 40 mM. (C) Effect of HFIP concentration on the peak capacity of heparin oligosaccharide dp6-dp14 and dp18. Mobile phases contain 15 mM PTA. The HFIP concentration was varied from 0 to 200 mM. Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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HXA are roughly 2-fold higher compared to TPA, PPA, and BTA. In addition, an increase of retention is observed with increasing alkyl chain length, which is attributed to the increased lipophilic properties of the ion-pairing reagents as a higher degree of stationary phase surface coverage is achieved with increasing chain length. Figure 1A illustrates that the longer alkyl chains such as PTA and HXA enhance the ion-pairing capability of the buffer system and improve the separation performance. Interestingly, Figure 1A also reveals that the peak capacity calculated from the OTA buffer system is less than those from PTA and HXA, indicating poor performance of the buffer system. When examining the data, it was found that the studied oligosaccharides were retained longer on the column with the OTA buffer system (the earliest eluting peaks start at 60% mobile phase B, also see Table 1). However, the peak widths were generally much wider compared to those from the PTA and HXA buffer systems. The influence of the ion-pairing reagent concentration on retention and resolution of heparin oligosaccharides was investigated using PTA as an example. Pentylamine (PTA) was further studied because it yielded superior chromatographic performances as well as the improved mass spectrometric performances (see discussion below). At this point, the test mixture was expanded to include a six-fraction mixture containing dp6, dp8, dp10, dp12, dp14, and dp18 heparin-derived oligosaccharides with each component at a concentration of 100 µg/mL. The effect of ionpairing concentration on peak capacity was studied at 5, 15, 25, and 40 mM PTA, while keeping the HFIP concentration constant at 100 mM. Figure 1B shows that the greatest peak capacity was obtained with 15 mM PTA for all dp6-dp18 oligosaccharides, and the P values calculated at 5 mM concentration are less than half of those at 15 mM PTA. On average, there is about a 7% successive decrease for the calculated peak capacity values for mobile phases containing 25 and 40 mM PTA when compared to 15 mM PTA. Interestingly, the changes in peak capacity for each incremental increase or decrease do not seem to correlate with the size of the oligosaccharides. In addition to the variation in peak capacity, it is also observed that the retention of oligosaccharides increases with the addition of PTA until it reaches 25 mM concentration. Then, a decrease in retention at 40 mM concentration is recorded. Because the pH of the buffer rises to ∼9.0 at 40 mM PTA, the decrease in retention may be attributed to the incomplete protonation of pentylammonium ions as the pH approaches the pKa of PTA (10.6). These results reveal the impact of the intercorrelation of the ion-pairing concentration and mobile phase pH and may be ascribed to the predominance of different interaction mechanisms as a function of ion-pairing concentration, which was already observed in an earlier report.35,36 The effect of HFIP concentration on separation performance was studied in mobile phases containing 15 mM pentylamine (PTA). The concentration of HFIP in this mobile phase was prepared at 0, 10, 25, 50, 100, and 200 mM. Because of the different concentrations of HFIP, the pH of the aqueous mobile phases varied from 9.4 (no HFIP) to 8.1 (200 mM HFIP). Figure 1C shows that the peak capacity improves as the HFIP concentration is increased from 10 to 100 mM, before decreasing at 200 mM. However, the largest increase is observed when the HFIP (35) Hearn, M. T. W.; Grego, B.; Hancock, W. S. J. Chromatogr. 1979, 185, 429–444. (36) Zou, J.; Motomizu, S.; Fukutomi, H. Analyst 1991, 116, 1399–1405.

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concentration is changed from 10 to 25 mM (∼10% increase). In the absence of HFIP, dp14 and dp18 oligosaccharides were not detected. The concentration of the HFIP additive has only a minor impact on the retention of oligosaccharides. In the tested HFIP concentration range, the oligosaccharides eluted 0.1-0.2 min earlier when the HFIP concentration was doubled. On the basis of the these experiments, a mobile phase containing 15 mM pentylamine in 25-100 mM HFIP was determined to be an optimal chromatographic condition for the separation of heparin-derived oligosaccharides. Effect of Mobile Phase Composition on Mass Spectrometric Performance in Negative Ion Mode. It has long been recognized that electrospray ionization heavily depends on a number of solvent properties such as dielectric constant and viscosity.37 This dependence introduces challenges for the development of a high-performance IPRP-LC/ESI-MS method because the optimal mobile phases for IPRP LC often extensively suppress the MS responses in ESI, while the best solvents for electrospray ionization with minimal adduct formation are not suitable for IPRPLC separation.17 Because the objective of this project is to develop an efficient IPRP-LC/MS method for high-resolution chromatographic separation while still obtaining good MS responses for heparin oligosaccharides, all of the mobile phase compositions discussed above were also evaluated for MS performance during the chromatographic optimization process. As was previously mentioned, HFIP is added to the mobile phases as a buffering acid to control the pH and to ensure the protonation of the alkyl amines so they can function properly as ion-pairing reagents. Protonation of amines was traditionally achieved using a volatile weak acid such as acetic acid.16-19 However, acetic acid is generally regarded as the main responsible factor for ion suppression observed with triethylammonium acetate (TEAA) buffers38,39 for the analysis of DNA oligonucleotides with negative electrospray detection. Several reports on the analysis of DNA oligonucleotides have found that the use of HFIP modifier rather than acetic acid enhances the ESI-MS signal of negatively charged oligonucleotides.38,39 Considering the fact that both oligonucleotides and heparin oligosaccharides contain many negatively charged acidic groups (albeit chemically different), it is therefore expected that a similar MS enhancement effect should be observed for highly sulfated oligosaccharides as well. The experimental results (Figure S1 in the Supporting Information) from the same concentration of a fully sulfated hexasaccharide (dp6) with two mobile compositions both containing 15 mM propylamine (PPA) but a different acidic modifier clearly prove this hypothesis. Typically, replacing the acetic acid with HFIP enhanced the MS signal intensity by 10-100-fold depending on the size of the oligosaccharide. Figure 2 is representative ESI-MS spectra obtained for hexasaccharides (dp6) with six, seven, eight, and nine sulfate groups using pentylamine (PTA) as the ion-pairing reagent. Clustered “quasi-molecular” ion peaks corresponding to adducts between the dp6 isomers and PTA dominates the spectrum. The mass (37) Wang, G.; Cole, R. B. In Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; pp 137-174. (38) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320–1325. (39) Gilar, M.; Fountain, K. J.; Budman, Y.; Holyoke, J.; Davoudi, H.; Gebler, J. C. Oligonucleotides 2003, 13, 229–243.

Figure 2. Negative ESI-MS spectra obtained for nona- (A), octa- (B), hepta- (C), and hexa- (D) sulfated hexasaccharides. The monoisotopic peak of the most abundant ion signal is indicated for each oligosaccharide. The presence of acetonitrile adducts is noted by O.

spectra presented in Figure 2 do not show any peaks corresponding to sulfate losses or sodium adducts. The fact that dp6 isomers were detected mostly as PTA adducts rather than naked molecular ions in MS analysis shows that there are strong ion-pairing interactions between the positively charged linear amines and the negatively charged sulfates in the gas phase. As previously noted,16,17,40,41 highly sulfated oligosaccharides can be ionized as either multiply charged deprotonated ions or adduct ions with ion-pairing reagents in the negative ion ESI-MS condition, provided that mild desolvation conditions are used. Formation of adduct clusters in MS is undesirable since the ion current is divided among several ion species. As a result, the sensitivity is reduced and the data analysis can be more complicated. However, there are clear advantages to adduct formation for heparin oligosaccharide analysis. The formation of an ion-pair between the sulfate group and the alkyl amine protects the oligosaccharide from sulfate group losses (Figure S1 in the Supporting Information) and eliminates the formation of other metal ion adducts. This is especially beneficial for the analysis of large heparin oligosaccharides because the prevention of sulfate losses makes it possible to detect the intact molecular ions and obtain accurate molecular weight measurements, from which oligosaccharides with different degrees of sulfation can be unambiguously identified. In order to make the mobile phases containing HFIP friendly to ESI-MS, several factors have to be evaluated that are related to the mobile phase compositions. The effect of different ionpairing reagents on MS signal intensity was investigated in a series of experiments where the test mixture containing three commercially available heparin-derived oligosaccharide fractions (dp6, dp8, dp10) was LC separated and then electrosprayed from mobile phases containing different ion pairing reagents (15 mM, five linear alkyl amines and tripropylamine) along with 100 mM HFIP. Because the ion intensity varies across the chromatographic peaks, the average ion counts (n ) 3) from the monoisotopic peak (40) Zaia, J.; Costello, C. E. Anal. Chem. 2003, 75, 2445–2455. (41) Da Col, R.; Silvestro, L.; Naggi, A.; Torri, G.; Baiocchi, C.; Moltrasio, D.; Cedro, A.; Viano, I. J. Chromatogr. 1993, 647, 289–300.

of the most intense adduct at the apex of each chromatographic peak were used to compare the MS performances in negative ion ESI-MS for each of the fully sulfated dp6, dp8, dp10. Figure 3A depicts the influence of different ion-pairing reagents on signal intensity. For each ion-pairing reagent, the MS responses decrease as the size of the oligosaccharide increases. Because of the presence of many other isomeric components (besides the fully sulfated isomers) in each individual fraction, the true molar concentration for each fully sulfated isomer could not be obtained. As a result, it is not feasible to judge how much of the change in MS response was due to the increase of the oligosaccharide size. Nonetheless, it seems reasonable to assume that the MS responses decrease when the oligosaccharide size increases, as reported in the literature.16,17 The results shown in Figure 3A also demonstrate that, for the same heparin oligosaccharide, pentylamine (PTA) produces the best ESI-MS response among all the linear alkyl amines investigated in this study. In addition, the use of PTA results in at least a 2-fold increase in the ion counts for the tested oligosaccharides when compared to the previously reported tripropylamine ([). Lower MS signals for PPA, BTA, OTA, and tripropylamine are expected considering oligosaccharide separation is worse when compared to PTA (Figure 1A). Interestingly, the MS ion counts from mobile phases containing HXA (9) are less than 50% of that from PTA despite the fact that better chromatographic performance was achieved with HXA as the ionpairing reagent (Figure 1A). This may be related to the less satisfying desolvation effect resulting from the higher boiling point of HXA (132 °C for HXA versus 104 °C for PTA). On the basis of overall performance for both LC separation and MS signal intensity, pentylamine was chosen for further MS detection optimization. The influence of ion-pairing reagent concentration on the MS response was investigated using the six-fraction mixture containing dp6, dp8, dp10, dp12, dp14, and dp18 oligosaccharides using four pentylamine concentrations and a fixed HFIP concentration (100 mM). Figure 3B shows that the concentration of pentylamine has no significant effect on MS signal intensity for all of the fully Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Figure 3. Impact of mobile phase composition on the MS responses heparin oligosaccharides. The oligosaccharide response in negative ESI-MS was calculated based on the average ion counts (three injections) recorded at the apex of each chromatographic peak produced by the fully sulfated oligosaccharides during IPRP-UPLC/ MS analysis. (A) Oligosaccharide responses in negative ESI-MS obtained for 50 µg/mL of dp6/8/10 heparin oligosaccharide fractions during IPRP-UPLC/MS analysis using a mobile phase containing 100 mM HFIP and one of the following ion-pairing reagents at 15 mM concentration: PPA (n-propylamine), BTA (n-butylamine), PTA (n-pentylamine), HXA (n-hexylamine), and OTA (n-octylamine). (B) Effect of PTA concentration on oligosaccharide response in negative ESI-MS for 100 µg/mL of dp6-dp14 and dp18. MS responses were acquired using a mobile phase containing 100 mM HFIP and 5-40 mM PTA. (C) Effect of HFIP concentration on oligosaccharide response in negative ESI-MS for 100 µg/mL of dp6-dp14 and dp18 analyzed by UPLC/MS using a mobile phase containing 15 mM PTA. Six HFIP concentrations were investigated in the range of 0-200 mM.

sulfated isomers. This finding seems contradictory with the previous conclusion that higher concentrations of di- or trialkylamines are not suitable for ESI analysis due to the poor MS responses of the eluting analytes.17,19 The discrepancy may be explained by the use of different mobile phase modifiers. In contrast to the current use of a fixed concentration of HFIP as the weak acid to protonate pentylamines, acetic acid was used as 3492


previously reported. As the concentration of trialkyl amines (ionpairing reagents) increases, which is desirable for better chromatographic performance, more acetic acid is needed to protonate the alkylamines. Since the total ion current generated during electrospray ionization is roughly constant, an increase in acetic acid will draw a larger portion of the total ion current and a further decrease in the intensity of analyte ions. When mobile phases contain HFIP as a modifier, HFIP (boiling point ) 58 °C) is more volatile than pentylamine (boiling point )104 °C) and thus evaporates quickly at the droplet surface during the electrospray/ desolvation process. Regardless of the initial pH values of the four mobile phases investigated (from 8.0 to 9.0), the pH at the electrospray droplet surface rises (∼10) due to the evaporation of HFIP. This dynamic adjustment of the pH in the electrospray droplets due to the preferential removal of HFIP was first proposed by Apffel et al.38 and is attributed to the observations herein on the MS responses using the four different ion-pairing concentrations. Up to this point, the mobile phase containing 15 mM pentylamine were observed to yield the best overall performance for both LC separation and MS detection. For optimization of the HFIP concentration, the pentylamine concentration was maintained at 15 mM and the dp6-dp18 test mixture (100 µg/mL) was analyzed using mobile phase containing 0, 10, 25, 50, 100, and 200 mM HFIP. Figure 3C shows the MS responses of all fully sulfated isomers in the mixture at the different HFIP concentrations. It is observed that 25 mM HFIP produces the best overall MS responses for all oligosaccharides except the two shortest ones, dp6 and dp8. The lower MS responses from mobile phases with 0 or 10 mM HFIP are expected based on their chromatographic performances (lower peak capacity). It is noted that there is about a 20-50% reduction in MS responses for mobile phases containing 50 or 100 mM HFIP, despite the fact that their P values are higher (See Figure 1C). This is likely caused by the more pronounced ion suppression at higher HFIP concentrations. HFIP appears to generate a background ion at m/z 336.08 ([M - H]-). As the HFIP concentration increases, the ion current that corresponds to this ion increases, resulting in a loss of MS responses for heparin oligosaccharides. In summary, after the systematic evaluation, a mobile phase containing 15 mM PTA and 25/50 mM HFIP gives the best compromised performances with respect to IPRP-LC separation and negative ion MS response. This mobile phase composition is selected as optimal for IPRP-LC/ESI-MS analysis of large heparinderived oligosaccharides. IPRP-UPLC Coupled to ESI-MS for Analysis of Oligosaccharides in Negative Ion Mode. Figure 4 shows the MS and UV chromatograms obtained for the separation of the dp6-dp18 oligosaccharide test mixture using a 10 min gradient condition. Because these oligosaccharides were generated via an enzymatic depolymerization process, a double bond exists between the C4 and C5 position of the terminating hexuronic acid residue at the nonreducing end of each oligosaccharide, producing a characteristic absorption maximum at 232 nm for UV detection.42 IPRPUPLC provides a high-resolution separation, where individual oligosaccharide size classes (differing by the two repeating disaccharide units) are well-resolved with a 10 min linear gradient. (42) Lohse, D. L.; Linhardt, R. J. J. Biol. Chem. 1992, 267, 24347–24355.

Figure 4. Total ion chromatogram (A) and UV trace at 232 nm (B) obtained for the IPRP-UPLC separation of a text mixture containing 100 µg/mL each of the dp6-dp14 and dp18 oligosaccharide fractions. Peaks labeled with an asterisk in the UV trace correspond to the fully sulfated oligosaccharides. Separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 µm) at a flow rate of 0.4 mL/min and a column temperature of 45 °C. Gradient conditions: 10-40% B in 10 min; eluent A ) 15 mM PTA, 50 mM HFIP, pH ) 8.8. Eluent B ) 75% ACN, 15 mM PTA, 50 mM HFIP.

Typical baseline peak widths for this chromatogram are 6-8 s, and the peaks become narrower as the size of the oligosaccharide increases. The last eluting peak in each size class, marked with an asterisk in the UV trace, is the fully sulfated oligosaccharide and corresponds to the structure shown in Chart 1. Within each size class, a number of other peaks that represent under-sulfated oligosaccharides are also observed. These species share the same chain length but differ in the number of sulfate and acetyl groups at glucosamine residues. The most intense peak eluting right before each fully sulfated peak is the corresponding monoundersulfated oligosaccharide. These are also the peaks used in the previous section to calculate the peak capacity under different experimental conditions. As shown in Figure 4, the increase in the number of sulfate groups results in greater retention on the column, indicating that there are more ion pairs generated between the positively charged amines and the negatively charged sulfate/carboxylate groups. In the mass spectrometric analysis of highly sulfated oligosaccharides, the partial ion-pairing of sulfate groups with various metal cations (e.g., sodium, potassium) should be eliminated if possible, as these ions can result in highly complex spectra and decrease sensitivity. The removal of cations is successfully achieved in the ESI-MS using the developed method. The predominant peaks in all of the negative ion ESI mass spectra are always associated with the pentylamine adducts. No sodium adducts were observed even for the oligosaccharide with a size up to dp18. The fully sulfated dp18 oligosaccharide contains 27 sulfate groups and 9 carboxylate groups, and these 36 sites could all potentially form ion pairs with sodium ions. The spectrum displayed in Figure 5 does not show any sodium adduction or any significant sulfate group losses. The lack of salt adducts in this spectrum is attributed to the strong ion-pairing interaction between the highly charged polyanion and the positively charged pentylammonium ions. Eluents containing higher concentrations of the pentylammonium ion are obviously more efficient in replacing cations at the sulfate/ carboxylate groups during IPRP-LC separation of heparin oligosaccharides.

Figure 5. Negative ESI-MS spectrum of fully sulfated dp18 (27S dp18). The inset shows the partially resolved isotopic distribution of this oligosaccharide.

Compared to di- or trialkyl amines, the linear alkyl amines are more prone to form adducts with heparin oligosaccharides in the gas phase due to less steric hindrances imposed by the side chains. Although generally believed harmful to the sensitivity of MS analysis, the formation of adducts is important for detecting large heparin oligosaccharides as intact species without the loss of labile sulfate groups. This accomplishment has implication for elucidating the structure-function relationship for large size heparin oligosaccharides because it enables the alignment of critical functional groups in a long linear chain. The adducts take the form [M + mPTA - nH]n- with n being the number of protons and m the number of pentylammonium counterions. The number of ion-pairing molecules that are included in the adducts depends on the number of sulfate groups in the oligosaccharide molecule, as well as the instrument settings. For example, the mass spectrum corresponding to the fully sulfated dp6 oligosaccharide (Figure 2A) has five intense peaks at m/z 951.49, 995.04, 1038.58, 1082.14, and 1125.70, which can Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Table 2. Measured and Calculated MW for Major Quasi-Molecular Ions Observed in the ESI Mass Spectra for Fully Sulfated Heparin-Derived Oligosaccharides (dp6-dp18) in Negative Ion ESI-MS with PTA as the Ion-Pairing Reagenta monoisotopic peak of the most intense adduct ions

fully sulfated oligosaccharide

retention time (min)

measured

calculated

charge state

no. of PTA molecules included in the quasi-molecular ionsa

dp6 dp8 dp10 dp12 dp14 dp16 dp18

6.23 7.79 8.99 9.92 10.67 11.27 11.75

1038.63 884.40 1192.85 1501.30 1780.71 2089.17 1623.75

1038.66 884.43 1192.89 1501.36 1780.78 2089.25 1623.82

2333344-

2, 3, 4, 5, 6 2, 3, 4, 5, 6 5, 6, 7, 8, 9, 10, 11 8, 9, 10, 11, 12, 13,14 13, 14, 15, 16, 17 16, 17, 18, 19, 20, 21, 22 12, 13, 14, 15, 16, 17,18,19,20

a

The number of PTA adducts producing the most abundant ESI-MS response is underlined.

be assigned to [M + 2PTA - 2H]2-, [M + 3PTA - 2H]2-, [M + 4PTA - 2H]2-, [M + 5PTA - 2H]2-, and [M + 6PTA 2H]2-, respectively. The negative ion ESI spectra for larger oligosaccharides are similar, but the charge states are also higher. The largest oligosaccharide in the test mixture that shows a discernible isotopic distribution from its adducts is an oligosaccharide of dp18 (charge state of four). The fractions containing dp20 and dp22 oligosaccharides were detected in negative ion ESI-MS after injecting more concentrated solutions (500 µg/mL), but their isotopic envelopes could not be resolved due to the lower signal-to-noise ratio of MS signal recorded. The losses of sulfate groups are also influenced by the mass spectrometer settings. Among all the instrumental parameters, it was found that the cone voltage was critical to minimize the sulfate loss during the analysis. Optimization of the MS instrument settings was performed with the fully sulfated hexasaccharide from the dp6 fractions. Negligible sulfate group losses were observed by setting the cone voltage at or below 25 V. When the cone voltage was set above 25 V, sulfate losses became more prominent. At higher cone voltage values, (e.g., >40 V), a series of peaks with a 167 Da mass difference was observed, which corresponds to the removal of the sulfate-PTA pairs altogether. As a result, the cone voltage was set to 25 V for the duration of this study. Because of the high resolving power of the time-of-flight analyzer, accurate molecular weights for all oligosaccharides (dp6-dp18) can be assigned based on the charge states and monoisotopic peaks in the negative ion ESI spectra. From these measurements, the number of saccharide units, acetyl groups, and sulfate groups in each molecule can be determined for any given oligosaccharide up to dp14.22 The relative molecular weights calculated based on monoisotopic mass and the observed m/z values for the major peaks corresponding to the fully sulfated oligosaccharides in the test mixture are summarized in Table 2. Analysis of Structural Isomers of Heparin Oligosaccharides by IPRP-UPLC/MS. Because of the variability of sulfation on heparin chains, many structural isomers are produced during the depolymerization process. These isomeric oligosaccharides have identical composition with respect to the number of saccharide units, sulfate groups, and acetyl groups, but the spatial arrangement of the sulfate and/or acetyl groups are dissimilar. They are isobaric and indistinguishable by mass spectrometry. Resolving such oligosaccharide mixtures would increase confidence relating to the homogeneity of any prepared sample for mass spectrometric analysis. The high-resolution IPRP-UPLC 3494


method developed in this work is demonstrated to be capable of separating positional isomers of isobaric oligosaccharides having identical charge and mass. Figure 6 shows the separation of multiple isobaric hexasaccharides within 10 min. These extracted ion chromatograms are generated by extracting the monoisotopic mass (±50 ppm) of the most abundant PTA adducts (See Figure 2) of the four hexasaccharides shown. The ion chromatograms of m/z 1038.66, 911.58, 871.60, and 788.09 correspond to hexasaccharides with nine, eight, seven, and six sulfate groups, respectively. None of the four hexasaccharides contain an acetyl group in the molecular structure. As expected, the fully sulfated hexasaccharide (dp6, 9S) produced a single peak (Figure 6A) because there are no other structural isomers. The other three hexasaccharides all display multiple peaks with different retention times in their respective extracted ion chromatograms. For example, for the hexasaccharide with eight sulfate groups (dp6, 8S), Figure 6B shows four major peaks eluting between 5.2 and 5.8 min, all of which correspond to m/z 911.58, suggesting there are four major isomers for this hexasaccharide. The separation of structural isomers has been previously shown using IPRP-HPLC.17,18 The earlier work was achieved using a very shallow gradient and a long separation time. High-resolution separation of the positional isomers achieved in this work was obtained on a much shorter time scale (10 min, 3% B/min) using the same chromatographic conditions developed for the analysis of complex oligosaccharide mixtures. This increase in performance can be attributed to the use of UPLC technology in conjunction with optimized mobile phases. To further investigate the ability of IPRP-UPLC for the separation of isomeric oligosaccharides, the gradient slope was decreased to 1% B/min in order to maximize the resolution of the structural isomers from the hexasaccharides with eight sulfate groups (dp6, 8S). In panels A and B of Figure 7, the four chromatographic peaks observed in Figure 6B have been resolved into seven peaks in both the UV chromatogram and extracted ion chromatogram. The experiment was repeated with hexylamine (HXA) as the ion-pairing agent (Figure 7C). Seven peaks were also obtained with this reagent, suggesting that the seven peaks are truly caused by the positional isomers. Since there are no commercial standards available for the positional isomers (dp6, 8S), the structural identity for each of these peaks could not be verified chromatographically. In addition, it is not clear whether all of the structural isomers present in the dp6 fraction are completely resolved because the hexasaccharide fraction may not contain all nine isomers that are

Figure 6. Chromatographic separation of four hexa-saccharides with different degrees of sulfation (6, 7, 8, and 9S dp6). The monoisotopic peaks displayed in Figure 2 were used to generate the four extracted ion mass chromatograms ((50 ppm) shown in panels A-D. All chromatographic conditions are identical to those specified for Figure 4.

theoretically possible. Nonetheless, the results clearly demonstrate the separation power of UPLC technology and fully optimized ionpairing mobile phases. IPRP-UPLC/ESI-MS for the Analysis of Oligosaccharides in Positive Ion Mode. The analysis in positive ion mode was performed using the dp6-dp18 oligosaccharide test mixture. The experiment was performed using the same optimized mobile phases that were used for the analysis in negative ion mode. To our surprise, the analysis of heparin oligosaccharides in positive ion mode yielded greater MS responses for all the oligosaccharides in the test mixture. Again, the ion species observed in positive ion mode represent adducts formed between the oligosaccharides and counterions, with the exception that they all contain a greater number of PTA molecules compared to those detected in negative ESI-MS for the same oligosaccharide. Figure 8A-D shows the positive ESI-MS spectra obtained for the same hexasaccharides (dp6, 6S-9S) presented in Figure 2. Multiple peaks are observed in the mass spectrum for each oligomer, all of which are separated by 87.5 Da, corresponding to the increased number of pentylammmonium counterions in the adducts. For each oligosaccharide, the overall MS ion counts in positive ion mode are about twice as high as the signals in negative ion mode. It appears that the number of PTA molecules present in the most abundant ion species correlates with the number of sulfate groups in the biomolecule. For example, for hexasaccharides with nine, eight, seven, and six sulfate groups, the most abundant ion species contains nine, eight, seven, and six pentylammonium ions, respectively (Figure 8A-D). Not surprisingly, sodiated peaks (labeled by ×) that correspond to ion species containing multiple pentylammonium ions and one extra Na+ are also observed (Figure 8). Generally, ESI-MS studies have shown that sodium adducts are more likely to be observed when acidic compounds are analyzed by positive ion ESI-MS. Because of the superior MS spectra obtained in positive ion mode, it was possible to acquire an ESI-MS spectrum of the fully sulfated dp22 (containing 33 sulfate groups), which was previously not possible using negative ion mode with the same amount of

injected material. Figure 8E displays the cluster of ion species and the isotope envelope of the most abundant adduct ion (4+) in the cluster observed using positive ESI-MS. The dp22 oligosaccharide molecule has 33 sulfate groups, and the most abundant adduct contains 32 PTA counterions. In addition, despite the presence of a large number of sulfate groups in the molecule, no significant sulfate group loss is observed in the spectrum. The presence of Na+ adduction is clearly more visible in this spectrum (∼30%) with this size of oligosaccharides compared to the smaller ones. However, the more prominent peaks still come from PTA adducts. The spectrum shown in Figure 8E demonstrates that it is possible to obtain accurate molecular weight measurement for highly sulfated oligosaccharides with molecular weights up to 6 300 Da (the relative MW of fully sodiated/sulfated dp22 is 7 315 Da) using the current conditions. The detection of highly sulfated oligosaccharides in both negative and positive mode implies that many different types of adduct ions are generated during the electrospray ionization process. The detection in either positive or negative ion mode would only monitor a subgroup of the total ion species generated in the electrospray process. Because the MS conditions were optimized based on the MS response using negative ion mode, the observed MS responses in positive ion mode may not represent the best ion signal one can obtain with the optimized mobile phases. Further evaluations may be needed in order to optimize the MS conditions for the analysis of heparin oligosaccharide using positive ion mode. MS analysis in positive ion mode for polysulfated oligosaccharides was reported previously using alternative analytical strategies.43,44 The pioneering work from Biemann and Termeij-Groen utilized basic peptides to form complexes (adducts) with heparinderived oligosaccharides in order to measure their molecular weights by positive ion MALDI MS.43 In a separate report, Gunay (43) Termeij-Groen, C. P.; Biemann, K. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333–4337. (44) Gunay, N. S.; Tadano-Aritomi, K.; Toida, T.; Ishizuka, I.; Linhart, R. J. Anal. Chem. 2003, 75, 3226–3231.


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Figure 7. IPRP-UPLC separation of isomeric hexasaccharides containing eight sulfate groups (dp6, 8S) with two ion-pairing reagents. (A) LC/UV chromatogram (232 nm) recorded simultaneously with the MS chromatogram displayed in panel B. Separations were performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) at a flow rate of 0.4 mL/min and a column temperature of 45 °C. (B) Extracted ion chromatogram for m/z 1174.90 in positive ion mode using a 5 Da mass window. The ions originate from the adduct between the dp6 and PTA with a formula of [M + 8PTA + 2H]2+. Gradient conditions: 13-23% B in 20 min. Eluent A ) 15 mM PTA, 50 mM HFIP, pH 8.8. Eluent B ) 75% ACN, 15 mM PTA, 50 mM HFIP. (C) Extracted ion chromatogram for [M + 2HXA - 2H]2- (m/z 925.59) in negative ion mode using a 5 Da mass window. Gradient conditions: 30-40% B in 20 min. Eluent A ) 15 mM HXA, 50 mM HFIP, pH 8.8. Eluent B ) 75% ACN, 15 mM HXA, 50 mM HFIP.

et al.44 were able to acquire positive ion ESI spectra of sucrose octasulfate by infusing solutions containing quaternary ammonium. Although both approaches were useful at their own settings, the ion-pairing reagents used in these studies could hardly be adopted for the development of an effective online LC/ MS method due to the inherent limitation in their chemical properties. Peptides do not work as ion-pairing agents in chromatographic separations; quaternary ammoniums are not volatile and greatly suppresses MS signals.16 The data presented here is the first example of positive ion ESI-MS spectra acquired after highly efficient LC separation for heparin oligosaccharides. It is expected that the approach has a broader applicability for profiling heparin-derived oligosaccharides and for the analysis of other compounds with similar chemical properties. Tandem MS Coupled with IPRP-UPLC for Analysis of Oligosaccharide Isomers Using Positive Ion Mode. On the basis of the results of the previous LC/MS experiments, the fragmentation behavior of the oligosaccharide adduct ions was investigated in an attempt to acquire informative MS/MS spectra 3496


from which oligosaccharide sequence and/or sulfation pattern can be deduced. The MS/MS data from the direct fragmentation of adduct ions quickly proved to be futile because the adduct ions, upon high-energy fragmentation, produced a series of ions corresponding to the consecutive losses of sulfate groups and pentylammonium (167 Da). The data had little value for mapping sulfation pattern, as these ion series did not contain much characteristic structural information. However, because of the preferential loss of the sulfate groups, the fragmentation data also indicated that all of the sulfate/sulfonate groups in an oligosaccharide can be completely removed prior to any glycosidic bond breakage, provided that the applied collision energy is high enough. As a result, an ion corresponding to the oligosaccharide backbone (without any sulfate/sulfonate group attached) is generated. For oligosaccharides that do not contain any acetylated glucosamine, these ion species essentially represent a linear carbohydrate structure with alternating HexA and GlcN. There are no added values for structural sequencing upon further fragmentation of these ions. For oligosaccharides containing N-acetylated glucosamine, the subsequent fragmentation of such ions provides valuable information to reveal the positioning of the N-acetylated glucosamine in the linear chain of the parent oligosaccharides. Such information cannot be directly obtained by a simple molecular weight measurement. A similar approach was previously undertaken by Tissot et al.45 to locate acetyl residues in a large heparin fragment by MALDI mass spectrometry. The feasibility to obtain more structural information regarding the positioning of the acetylated glucosamine (GlcN) residue in the oligosaccharide backbone was explored by fragmenting the doubly charged precursor ions from two hexasaccharides in positive ion mode. The corresponding MS/MS spectra are displayed in Figure 9A,B. Figure 9A shows the MS/MS spectrum obtained from the fragmentation of the doubly charged ion (m/z 1174.90), corresponding to the most abundant adduct from the hexasaccharide with eight sulfate groups and eight pentylammonium ions (see the ESI-MS spectrum displayed in Figure 8B). Most all of the sulfate groups, along with the PTA adducts, are cleaved after applying a collision energy ramp from 60 to 80 eV on the MS instrument. One of the prominent ion signals generated after the sulfate/sulfo groups are stripped corresponds to the singly charged intact hexasaccharide backbone (m/z 1012.30). The ion species undergo further fragmentation in the applied collision energy regime generating the spectrum shown in Figure 9A. A series of B/Y and C/Z ions observed in Figure 9A indicates that these ions come from glycosidic bond cleavage, and the spectrum matches the typical CID spectrum from a linear oligosaccharide. The spectrum further confirms the composition of the hexasaccharide by revealing the backbone sequence of the oligosaccharide. Figure 9B shows the MS/MS spectrum obtained from the fragmentation of the adduct ion from the hexasaccharide with one N-acetyl group and seven sulfate groups (m/z 1155.93). Similarly, a prominent peak corresponding to the intact oligosaccharide backbone (m/z 1054.31) is obtained upon fragmentation. The mass difference between the peak at m/z 1054.31 in Figure 9B and the (45) Tissot, B.; Gasiunas, N.; Powel, A. K.; Ahmed, Y.; Zhi, Z.; Haslam, S. M.; Morris, H. R.; Turnbull, J. E.; Gallagher, J. T.; Dell, A. Glycobiology 2007, 17, 972–982.

Figure 8. Positive ESI-MS spectra obtained for nona- (A), octa- (B), hepta- (C), and hexa- (D) sulfated hexasaccharides. The monoisotopic peak of the most abundant ion signal is indicated for each oligosaccharide. The presence of sodium adducts is noted by the ×. (E) Positive ESI-MS spectrum of fully sulfated dp22 (33S dp22). The inset shows the partially resolved isotopic distribution of this oligosaccharide with a relative MW of 7 315 Da.

peak at m/z 1012.30 in the spectrum Figure 9A suggests that this hexasaccharide contains one N-acetyl group in the oligosaccharide molecule. For a heparin hexasaccharide containing an N-acetyl group, there are three possible isomers with regards to the position of the acetyl group: the N-acetyl group may be localized either on the second, fourth, or sixth saccharide residue, starting from the nonreducing end. The MS/MS spectrum in Figure 9B indicates that the spectrum results from a mixture of two of three positional (isobaric) hexasaccharides. The signature ion *B4 at m/z 699.20 indicates one of the hexasaccharides has the N-acetyl group located on the fourth saccharide residue from the nonreducing end. The other isomer, which is identified by two unique ions, *Z1 ion at m/z 204.08 and *Z2 ion at m/z 380.11, has an N-acetyl group attached to the saccharide residue at the very reducing end (6th from the nonreducing end). Considering the

complex sequences that heparin oligosaccharides have, the occurrence of the two positional (isobaric) hexasaccharides in this dp6 fraction is not unusual. It is very unlikely that these positional isomers can be resolved chromatographically even using a highresolution technique like IPRP-UPLC because the separation is based on the number of sulfate groups in the molecules. In addition, N-acetyl groups are not involved in the ion-pairing interaction. The spectra displayed in Figure 9A,B clearly demonstrate the utility of the MS/MS approach in differentiating the structural isomers of heparin oligosaccharides. UV and MS Quantitation of Heparin-Derived Oligosaccharides. The analysis of oligosaccharide composition with respect to quantification is helpful to understand the structural variability of heparin. It is also a useful tool to compare samples from different sources.26 Because heparin oligosaccharides deAnalytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Figure 9. Positive ion ESI-MS/MS spectra from hexasaccharides. (A) The isotopic distribution of the most abundant ion signal observed in the ESI-MS spectrum of octa-sulfated dp6 (monoisotopic peak at m/z ) 1174.90, see Figure 2) was isolated (5 Da mass window) and fragmented using a collision energy ramp from 60 to 80 eV. (B) The isotopic distribution of the most abundant ion signal observed in the ESI-MS spectrum of N-acetyl heptasulfated dp6 (monoisotopic peak at m/z ) 1155.93) was isolated/fragmented under the same conditions. All fragment ions highlighted in bold and noted with an asterisk are N-acetylated. This spectrum indicates the presence of two coeluting positional isomers of N-acetyl 7S dp6 having the N-acetyl group located either on the fourth or on the sixth oligosaccharide unit starting from the nonreducing end.

rived from an enzymatic depolymerization process exhibit characteristic UV absorption maximum at 232 nm, this UV absorption, when coupled with mass spectrometry, provides a simple method for the quantitative analysis of heparin oligosaccharides. The calibration plots of UV peak area vs sample concentration for fully sulfated dp6/dp8/dp10 yield good linearity (R2 greater than 0.9995) over a concentration range of 0.5-500 µg/mL, providing a linear response over 3 orders of magnitude. The lower limit of detection, at a signal-to-noise ratio of 3:1, is obtained for 10 ng of hexasaccharides loaded on the UPLC column. The precision of the method, tested by four repeat injections, gives relative standard deviations of 2-4%. One common problem with UV detection for IPRP chromatography is the purity of the ion-pairing reagents. Contaminants in the ion-paring reagents often have strong UV absorption, which can produce high background absorbance or add extraneous peaks to the UV chromatogram. Among all the ion-paring reagents evaluated in this work, pentylamine provides the lowest background absorbance at 232 nm, allowing the most sensitive UV detection for oligosaccharides. On the other hand, di- or trialkyl amines tend to yield stronger background absorbance and are 3498


more likely to contain other impurities. For most of branched amines tested in our work, their purities are seldom found to be acceptable for UV quantification purposes. The quantitative analysis by MS was investigated using the fully sulfated dp6 oligosaccharide as an example. The integrated peak area from the extracted ion chromatograms or the average number of ion counts measured at the apex of the chromatographic peak was used to construct the calibration plots. For the hexasaccharide, all of the adduct ions, along with the corresponding isotopes, were combined to give a single measure in the final calculation for either peak areas or peak intensities. The MS responses are linear only at the lower concentration range (0.5 to 10 µg/mL). A correlation coefficient of 0.98 was obtained for the fully sulfated hexasaccharide in this range. Application of IPRP-UPLC/ESI-MS for the Analysis of a LMWH Drug. Tinzaparin sodium is the sodium salt of the LMWH drug, Innohep, which is derived from controlled enzymatic digestion of heparin from porcine intestinal mucosa. The sample contains a range of oligosaccharides with different molecular weights and is an appropriate sample to test the potential use of the developed method for characterization of pharmaceutical drugs. For this purpose, the IPRP-UPLC/ESI-MS method was used for the analysis of tinzaparin sodium. Figure 10 shows the total ion chromatogram of the drug, on which peaks corresponding to oligosaccharide size classes in the range of dp4-dp24 are labeled. As expected, the chromatogram demonstrates that there are many oligosaccharide components in the sample. Shorter oligosaccharides were separated to their respective homogeneities, whereas several components coeluted under one chromatographic peak for larger oligosaccharides. Components with longer chains tend to be retained longer on the column because of the association with more sulfate groups, while an increase in chain length without changing the number of sulfate groups leads to a decrease in retention time. These components can be readily identified by combining the information from the retention time in chromatography and the MS spectra. There are multiple peaks observed between each size class, suggesting the presence of many components with a wide range of sulfate substitution. However, all of the prominent peaks in each size class (Figure 10) belong to the fully sulfated isomers, and the corresponding mass spectra indicate that sulfation does not exceed the expected fully sulfated substitution of three sulfate groups per disaccharide unit. This result is expected because the oligosaccharide that is responsible for the binding activity of the heparin drug is a specific pentasaccharide sequence containing a nonsulfated HexA and a GlcN residue with additional 3-O-sulfation. The largest oligosaccharide for which accurate molecular weight information could be obtained at the specified injection amount (100 µg on-column) is an oligosaccharide with a dp22, which has a molecular weight of ∼6 300 Da. No useful information can be obtained from the MS spectra of oligosaccharides longer than dp22, although chromatographic peaks from the presumed larger oligosaccharides are recognizable. According to the product information, tizaparin sodium contains about 22-36% oligosaccharides with molecular weight higher than 8 000 Da. The failure to observe heparin oligosaccharides larger than dp22 in tinzaparin sodium may be attributed to the low MS sensitivity for large

Figure 10. Total ion chromatogram for the active ingredient of the LMWH drug Innohep (tinzaparin sodium) analyzed in positive ion mode. Separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 µm) at a flow rate of 0.4 mL/min and a column temperature of 45 °C. Gradient conditions: 15-55% B in 20 min. Eluent A ) 15 mM PTA, 50 mM HFIP, pH 8.8. Eluent B ) 75% ACN, 15 mM PTA, 50 mM HFIP.

oligosaccharides. This suggests that the method developed in this work is particularly useful and robust for heparin oligosaccharides up to dp22 in length, for which single sulfate resolution can be achieved chromatographically and accurate molecular weight of the intact species can be obtained. CONCLUSIONS In this work, a method combining IPRP-UPLC, UV, and ESIMS was developed for the analysis of heparin-derived oligosaccharides. The method enables the effective online coupling of IPRP-UPLC to ESI-MS such that the performance characteristics of the UPLC separation and the quality of the MS data are not compromised. The online combination of UPLC, UV, and ESIMS with a time-of-flight analyzer offers a fast and simple method for characterizing heparin-derived oligosaccharides with a size up to 22 residues. In addition to high-resolution separations achieved by UPLC technology, the mobile phases used in this work improve MS responses through effective reduction of ion suppression that is traditionally observed with acetic acid in negative ion mode. Furthermore, the ion-pairing reagents form adducts with the oligosaccharides to prevent the loss of sulfate groups during MS analysis, thus allowing accurate mass measurement of large intact oligosaccharides. The formation of adducts also makes it feasible

to analyze the highly charged polysulfated oligosaccharides in positive ion mode and allows identification of structural isomers with the same composition but varying sequence through tandem mass spectrometry. An application of the method was demonstrated for an active pharmaceutical ingredient, tinzaparin sodium, which is derived from the enzymatic treatment of unfractionated heparin. This approach will be of particular utility for profiling the molecule entities of heparin materials and for structural variability comparison for samples from various sources. ACKNOWLEDGMENT The authors acknowledge LEO Pharma, Ballerup, Denmark, for providing the tinzaparin sodium used in this study and Drs. Sean McCarthy and Martin Gilar for helpful discussions on ionpair reversed-phase chromatography. We thank Ken Fountain and Hui Wei for critical review of the manuscript. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 31, 2008. Accepted March 6, 2009. AC802770R


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Analysis of Oligosaccharides Derived from Heparin by Ion-Pair

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