Electrospray Mass Spectrometric Detection of Chitobiose in Enzyme

Smithsonian Environmental Research Center, Smithsonian Institution, P.O. Box 28, Edgewater, Maryland 21037. The presence of chitin in algae was determ...
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Anal. Chem. 1996, 68, 1335-1341

Electrospray Mass Spectrometric Detection of Chitobiose in Enzyme Hydrolysates of Marine Phytoplankton Mona Shahgholi,† Mark M. Ross, and John H. Callahan*

Naval Research Laboratory, Chemistry Division, Code 6113, Washington, D.C. 20375 Richard A. Smucker

Smithsonian Environmental Research Center, Smithsonian Institution, P.O. Box 28, Edgewater, Maryland 21037

The presence of chitin in algae was determined by mass spectrometric detection of its enzymatic hydrolysis end product, chitobiose. Electrospray mass spectrometry (ESMS) was used for detection of chitobiose in the enzyme hydrolysate because of its capability to detect underivatized amino sugar oligomers. This strategy affords simplified sample preparation and reduced processing time. Despite the remarkable success in the analysis of purified sugars, ES mass spectrometric detection of sugars in complex matrices remains a challenging task and generally requires sample cleanup. This work demonstrates the use of ES-MS with both off-line and on-line sample cleanup for the detection of chitobiose generated by the enzymatic hydrolysis of chitin. The porous graphitic carbon (PGC) packing material used in the on-line method has been used for chromatographic separation of sugars and is particularly suitable for this assay because EScompatible solvents such as water and acetonitrile can be used for analyte elution. The effectiveness of microscale liquid chromatography ES-MS with PGC was demonstrated for a suite of sugars. Renewed interest in the use of chitinous materials in medicine, agriculture, and industry1-3 has highlighted the need for developing methods for unambiguous identification of chitin. Chitin is a primary, linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine, produced in significant quantities in aquatic environments, where it contributes to the C and N cycles. Chitin is found in the exoskeleton of arthropods, the shells of bivalves, and the walls of many fungi which reside in the aquatic environment as well as various structures in other organisms.4 Most classical methods for chitin identification lack specificity, leading to contradictory interpretations.5 Some physical methods, such as X-ray diffraction6 and infrared spectroscopy,7,8 can † National Research Council postdoctoral associate. (1) Ruprecht, R. M. Biochem. Biophys, Res. Commun. 1991, 174, 489. (2) Pennisi, E. Sci. News 1993, 144 (5), 72. (3) Brine, C. J.; Sandford, P. A. Advances in Chitin and Chitosan; Elsevier Science Publishers, ltd.: New York, 1992; pp 96-105. (4) Muzzarelli, R. A. A. Chitin; Pergamon Press: Oxford, UK, 1977; p 309. (5) Jeuniaux, C.; Voss-Foucart, M. F. Biochem. Syst. Ecol. 1991, 19 (5), 347. (6) Rudall, K. M. Adv. Insect Physiol. 1963, 1, 257. (7) Darmon, S. E.; Rudall, K. M. Discuss. Faraday Soc. 1950, 9, 251.

This article not subject to U.S. Copyright. Published 1996 Am. Chem. Soc.

unambiguously detect chitin in a biological system. However, these methods are purely qualitative and of low sensitivity. A more specific method based on colorimetric determination of N-acetylglucosamine released by tandem enzymatic hydrolysis (with chitinase and chitobiase) provides qualitative and quantitative information.5 These procedures still suffer from poor mass sensitivity and specificity, which is significant to our assay. The current technique used for detection of chitobiose in enzymatic hydrolysates of whole algal cells uses strong anionexchange (SAE) liquid chromatography at pH > 11, followed by electrochemical detection in oxidative pulsed amperometric mode (PAD).9 SAE-PAD has demonstrated good sensitivity for detection of sugars (limit of detection (LOD) 2-10 nM, 0.4-0.8 pmol/ injection10,11 ), but it lacks specificity, in that compound identification is confirmed only by retention time. By contrast, analytical techniques based on mass spectrometry could potentially be of high specificity and sensitivity. Fast atom bombardment mass spectrometry has been successfully applied to characterization of sugars, derivatized and underivatized.12,13 Derivatized sugars can be analyzed by gas chromatography/mass spectrometry in the electron ionization14-16 or chemical ionization17 modes. Thermospray mass spectrometry has been applied to the analysis of underivatized sugars, sometimes in conjunction with liquid chromatography.18-21 These techniques either are, in general, not very sensitive or require derivatization prior to (8) Pearson, F. G.; Marchessault, R. H.; Lyang, C. Y. J. Polym. Sci. 1960, 43, 251. (9) Smucker, R. A., manuscript in preparation. (10) Mopper, K.; Schultz, C. A.; Chevolot, L.; Germain, C.; Revuelta, R.; Dawson, R. Environ. Sci. Technol. 1992, 26, 133. (11) Hardy, M. R.; Tonwsend, R. R.; Lee, Y. C. Anal. Biochem. 1988, 170, 54. (12) Dell, A.; Egge, H.; Von Nicolai, H.; Strecker, G. Carbohydr. Res. 1983, 15, 41. (13) Kamerling, J. P.; Heerman, W.; Vliegenthart, J. F. G.; Green, B. N.; Lewis, I. A. S.; Strecker, G.; Spik, G. Biomed. Mass Spectrom. 1983, 10, 420. (14) Stellner, K.; Saito, H.; Hakomori, S. Arch. Biochem. Biophys. 1973, 155, 464. (15) Tisza, S.; Sass, P.; Molnar-Perl, I. J. Chromatogr. A 1994, 676, 461. (16) Funakoshi, I.; Yamashina, I. Anal. Biochem. 1980, 107, 265. (17) Games, D. E.; Lewis, E. Biomed. Mass Spectrom. 1980, 7 (10), 433. (18) Simpson, R. C.; Fenseleau, C. C.; Hardy, M. R.; Townsend, R. R.; Lee, Y. C.; Cotter, R. J. Anal. Chem. 1990, 62, 248. (19) Hsu, F. F.; Edmonds, C. G.; McCloskey, J. A. Anal. Lett. 1986, 19 (11, 12), 1259. (20) Marko-Varga, G.; Buttler, T.; Gorton, L.; Olsson, L.; Durand, G.; Barcelo, D. J. Chromatogr. A 1994, 665, 317.

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analysis. In recent years, electrospray mass spectrometry22-25 has demonstrated promising results in the analysis of sugars.26-28 Electrospray is capable of detecting underivatized sugars with good sensitivity, thereby eliminating sample derivatization steps.27 However, ES sensitivity significantly deteriorates when the analyte is present in a complex matrix or with high salt concentrations.29 The above problems have been alleviated for peptides and proteins in complex matrices, by using microscale liquid chromatography (micro-LC) in conjunction with ES, for on-line sample cleanup and introduction into the mass spectrometer.30,31 Micro-LC ES mass spectrometry may also be a suitable method for routine analysis of sugars in complex matrices. The objective of this study was to demonstrate the utility of ES-MS for detection of chitobiose in enzymatic hydrolysates of whole algal cells. These hydrolysates are complex matrices with a notably high salt content. The mass spectrometric method would supplement the standard method for detection of chitobiose, which identifies the latter by its retention time in a chromatographic analysis with pulsed amperometric detection. To be of use, the mass spectrometric method should be able to detect low analyte concentrations, similar to the levels analyzed by SAEPAD.10 In the studies described here, initial evaluation of electrospray mass spectrometric results demonstrated that matrix complexity was interfering with the detection of chitobiose in the enzymatic hydrolysates of whole algal cells; therefore, preliminary efforts were focused on sample cleanup. Sample cleanup was carried out off-line using activated charcoal, and the mass spectrometry was performed on a lab-built electrospray interface.32 Comparison of the initial results with those obtained by SAE-PAD suggested the necessity to improve the ES-MS limits of detection for chitobiose. This was accomplished by performing on-line microLC for sample cleanup and introduction using porous graphitic carbon (PGC) as the LC packing material. Chitobiose elutes from PGC with solvents that are compatible for direct interface to an ES mass spectrometer, e.g., water and acetonitrile. This is in contrast to anion-exchange LC-MS, where the high ionic strength solutions often necessary for liquid chromatography of sugars are not directly suitable for ES mass spectrometry.33,34 The utility of PGC for micro-LC of sugars was further explored in two examples. Preliminary results suggest a need for further examination of PGC for chromatographic separation of other types of sugars. (21) Niessen, W. M. A.; van der Hoeven, R. A. M.; van der Greef, J.; Schols, H. A.; Lucas-Lokhorst, G.; Voragen, A. G. J.; Bruggink, C. Rapid Commun. Mass Spectrom. 1992, 6, 474. (22) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451. (23) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64. (24) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642. (25) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 927A. (26) Reinhold, B. B.; Recny, M. A.; Knoppers, M. H.; Reinherz, E. L.; Reinhold, V. N. In Techniques in Protein Chemistry; Angeletti, R. H., Ed.; Academic Press, Inc.: New York, 1991; Vol. III, p 287. (27) Duffin, K. L.; Welply, J. K.; Huang, E.; Henion, J. D. Anal. Chem. 1992, 64, 1440. (28) Karlsson, K. E. J. Chromatogr. 1993, 647, 31. (29) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63, 2709. (30) Roboz. J.; Yy, Q.; Meng, A.; van Soest, R. Rapid Commun. Mass Spectrom. 1994, 8, 621. (31) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605. (32) Shahgholi, M.; Callahan, J. H., manuscript in preparation. (33) Black, G. E.; Fox, A.; Fox, K.; Snyder, A. P.; Smith, P. B. Anal. Chem. 1994, 66, 4171. (34) Conboy, J. J.; Henion, J. Biol. Mass Spectrom. 1992, 21, 397.

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EXPERIMENTAL SECTION Apparatus. The experiments were performed on two different electrospray sources, fitted to a TSQ-70 (Finnigan-MAT Corp., San Jose, CA) mass spectrometer. The first source was built in this laboratory, following the design of Chait et al.,35 which uses a heated capillary for desolvation of the electrosprayed droplets. It consists of a removable inlet probe, similar in design to that of Sproch and Kruger,36 and a modified TSQ-70 thermospray source. The heated capillary and skimmer, individually biased to appropriate voltages, are axially arranged in this source design. Ions at the exit of the skimmer are directed into the mass analyzer region using the original lenses of the thermospray source. The nebulizer-assisted electrospray needle assembly was patterned after that reported by Henion et al.24 The needle assembly was mounted in an x-y-z manipulator, which was used to optimize the position of the needle in front of the heated capillary inlet. A small flow of nitrogen was used to stabilize the ES plume. The analyte solution was delivered with a Model 341A syringe pump (Sage Instruments, Orion Research Inc., Cambridge, MA) at ∼3 µL/min. Details of the laboratory-built source will be provided in a separate communication.32 The second source is the standard Finnigan electrospray source that was procured later during these studies. The Finnigan electrospray source also employs heat for desolvation of electrosprayed ions, a flow of sheath gas to assist in droplet formation, and an auxiliary gas to assist in the evaporation of less volatile solvents. Special features of this source are the presence of a tube lens in the capillary (nozzle)-skimmer region and an rf-only octapole beyond the skimmer. The tube lens is used to focus ions and pass them through the skimmer, which is held at ground potential. The rf-only octapole focuses ions at the exit of the skimmer and passes them to two lenses, from which the ions enter the first quadrupole analyzer of the triple quadrupole spectrometer. Further details on this source can be obtained from Finnigan-MAT Corp. Reagents. Glucose, N-acetylglucosamine, glucosamine, trehalose, chitobiose, chitin, chitinase (Streptomyces griseus), and angiotensin I were obtained from Sigma Chemical Co. (St. Louis, Mo). Raffinose pentahydrate was from Pfanstiehl Laboratories, Inc. (Waukegan, IL). Ultrex-grade hydrochloric acid was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). HPLC-grade methanol was purchased from EM Science (Gibbstown, NJ) and HPLC-grade acetonitrile from Fisher Scientific (Pittsburgh, PA). The methanol used for the ES experiments was distilled over a bed of activated charcoal in order to lower the background in the mass spectra. Water used for preparation of the standards was distilled and deionized. Other chemicals were obtained from commercial suppliers. Phytoplankton Cultures and Growth Media. Single species algal cultures were obtained from national culture facilities and maintained in Guillard’s37 filtered and nutrient-supplemented seawater medium. This medium is hereafter noted as F/2 medium. Cyclotella cryptica (CCMP 333), Thalassiosira weissflogii (CCMP 1336), and Tetraselmis verrucosa (CCMP 918) were obtained from the Provasoli-Guillard Center of Culture of Marine (35) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81. (36) Sproch, N.; Kruger, T. J. Am. Soc. Mass Spectrom. 1993, 4, 964. (37) Guillard, R. R. L. In Culture of Marine Invertebrate Animals; Smith, W. L., Chanley, M. H., Eds; Plenum Publishing Corp.: New York, 1975; pp 2960.

Phytoplankton (West Boothbay Harbor, ME). Chlorella capsulata (LB 2074) and Synnechococcus sp. (LB 2380) were obtained from the phytoplankton collection at the University of Texas at Austin (Austin, TX). Individual isolated algal cultures were maintained by transfer of algal isolates to silicate-supplemented F/2 medium. Individual cultures were scaled up to 300 mL cultures (in 1 L flasks), starting with 25 mL of medium in 50 mL flasks. Principles of aseptic transfer were employed at all stages of culture transfer. Once daily, flasks were gently swirled to achieve gas exchange. Temperature was maintained at 18 °C, and a diurnal cycle of 14 h light and 10 h dark was provided by fluorescent light. Replicate flasks of respective single algal cultures were pooled. After several minutes of mixing with a magnetic stir bar, aliquots of intact culture were collected over a 25 mm Whatman GF/F glass fiber filter, using low vacuum (125 mmHg) in order to minimize disruption of cellular integrity. Typically 5-15 mL of culture fluid was processed, depending upon cell density and filtration rates. One-half milliliter of 0.5 N HCl was applied to the filter paper and allowed to react for 10 min in order to remove inorganic carbon. The acid was then removed by vacuum, and 1 mL of deionized water was used to rinse residual HCl acid. Each filter was stored independently in a conventional freezer until analyzed. The freezing step disrupts the algal cell envelopes, thereby exposing the interior of the cells. Organic Carbon. Algal cell mass was expressed by organic carbon elemental analysis according to EPA Method 440.4.38 Organic carbon loads on the filters ranged from 70 to 350 µg of C per filter. Enzymatic (Chitinase) Hydrolysis of Chitin in Phytoplankton. (i) Enzyme Preparation. Chitinase produced by the filamentous bacterium Streptomyces griseus was selected because of its previously demonstrated production of a singular endproduct, the disaccharide chitobiose. Commercial chitinase from S. griseus (Sigma PN, C-1525) was first purified because it carries strongly bound residual chitooligosaccharides with degree of polymerization 4 or higher. These oligosaccharides need to be processed prior to analytical chitinase hydrolysis of algal cells in order to remove endogenous background and aid in standardizing starting conditions for the enzymatic hydrolysis. Toward this end, prewashed 10 mm dialysis tubing (SpectraPor, MWCO 10 000) was equilibrated in 10 mM sodium acetate buffer (pH 4.5-5.0) containing 0.02% sodium azide and 5 mM CaCl2 (NACA buffer). Ten units of chitinase dissolved in NACA buffer was sealed in the prewashed dialysis tubes, which were maintained at 37 °C throughout the digestion-dialysis process. The NACA dialysis buffer was replaced three times during the 6-8 h digestion-dialysis period. Processed chitinase was pooled and either used immediately or stored at 5 °C for up to 12 h. (ii) Sample Pretreatment and Chitinase Hydrolysate Management. Algal samples, standards, and blanks were heated at 100 °C for 60 min in order to eliminate endogenous cellular enzyme activity. The algal samples collected on filter papers were transferred (along with the filter paper) to individual 2 or 3 mL thick-wall reaction vials and stirred in 1 mL of NACA buffer. One milliliter of pure chitinase in NACA buffer was added to each (38) Method 440.0. Determination of carbon and nitrogen in sediments and particulates of estuarine and coastal waters using elemental analysis.EPA Methods for Determination of Chemical Substances in Marine and Estuarine Environmental Samples; U.S. Government Printing Office: Washington, DC, 1992.

reaction vial. After 30 s of mixing, the first sample (200 µL) was taken as the background. After 10 h of incubation, the final sample was taken. The optimum hydrolysis period was determined to be 10-12 h.9 At each sampling point, a 200 µL aliquot from each sample was injected into 400 µL of deionized water which had been placed over a conditioned 100 mg Supelco C-18 solid phase extraction (SPE) cartridge (Bellefonte, PA). After being mixed for 5 s, the diluted sample was slowly displaced through the cartridge (1 drop/s) to allow optimal retention of proteins and other polymers on the C-18 matrix. Disaccharides in the void volume were preserved by freezing. Samples were thawed just prior to analysis by SAE-PAD and ES-MS. (iii) SAE HPLC and PAD Detection (SAE-PAD) of Monomeric and Oligomeric Saccharides. Samples were directly analyzed by strong anion-exchange (SAE) chromatography using 16 mM NaOH, followed by pulsed amperometric detection (PAD) with a Dionex system (Dionex Corp., Sunnyvale, CA).11 Identity of sugars was assigned using reproducibility of retention time and cochromatography of internal standards. Recovery of Chitan Standard in a Chlorella capsulata Matrix. To estimate the recovery of chitin from the whole algal cell matrix, a chitin standard, chitan,39 was added to a culture of C. capsulata, a phytoplankton which does not contain chitin. Enzyme hydrolysis was performed as described earlier, and the levels of chitobiose in the hydrolysates were measured by ES. Chitan is a preparation of natural chitin fibrils which we isolated from T. weissflogii.39,40 These fibrils, when purified, are fully N-acetylated, crystalline, and free from bound amino acids. For this experiment, we ran enzyme, chitan, and F/2 medium blanks in order to check for their respective contribution to background chitobiose levels. Recovery calculations were based on the premise that chitobiose values observed for the positive chitan controls (no algal cells present) should be identical to the chitobiose values when chitan was spiked into cultures of C. capsulata. Acid Hydrolysis of Chitin. Chitin oligosaccharides were prepared from chitin by partial acid hydrolysis (HCl) of commercially purified crab shell chitin. A 0.1 g sample of chitin was dissolved in 1.9 mL of cold 11 M HCl and maintained at 0 °C for 2 h to allow the polymer to swell. Hydrolysis was accomplished by maintaining the above suspension at 40 °C for an additional 2 h, conditions under which the yield is high yet chitin oligomers remain N-acetylated.41 The hydrolysate was then cooled to 0 °C and its pH brought to 1 with 50% NaOH. The precipitated, unhydrolyzed chitin (0.015 g) was removed by vacuum filtration. The filtrate was brought to 10 mL with deionized water. An aliquot was diluted 1000-fold with water for the chromatographic analysis. Off-Line Sample Cleanup. Off-line desalting of the samples was tested with Bio-Rad P-2 desalting gel (Hercules, CA) and activated charcoal. In desalting with the P-2 gel, the molecule of interest is eluted in the void volume, while molecules smaller than MW < 200 (nominal exclusion limit of the P-2 gel) are retained in the gel pores. The activated charcoal, on the other hand, is a nonporous support which adsorbs organics. In this case, desalting is achieved by adsorption of the sugars on the charcoal, while inorganic salts are removed by rinsing with deionized water. (39) McLachlan, J.; McInnes, A. G.; Falk, M. Can. J. Bot. 1965, 43, 707. (40) Smucker, R. A. Biochem. Syst. Ecol. 1991, 19 (5), 357. (41) Rupley, J. A. Biochem. Biophys. Acta 1964, 83, 245.

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To examine the performance of these materials, chitobiose standards (Sigma) were prepared in the enzyme hydrolysis medium, the NACA buffer, in order to duplicate the makeup of the samples that were to be eventually analyzed by ES-MS. A 30 µM chitobiose solution, prepared in NACA buffer, was processed through the P-2 gel as described by Chen et al.42 The desalted standard was diluted to ∼10 µM with 2 volumes of methanol and electrosprayed. The ES response for chitobiose was better for the gel-desalted sample compared to that for one similarly prepared that had not been desalted; however, the signal intensity was still very low. To achieve more complete desalting, samples were processed with charcoal as described below. Graphite SPE cartridges with 250 mg of 120/400 mesh graphitized, nonporous carbon (Supelco, Inc., Bellefonte, PA) were repacked in glass Pasteur pipets in order to avoid extraction of contaminants from the SPE cartridges with methanol or acetonitrile. Silane-treated glass wool plugs were placed at the inlet and outlet of each pipet in order to form a uniformly packed bed. The packed glass pipets were washed sequentially with 1 mL of acetonitrile, 1 mL of methanol, and 1 mL of water prior to use. Next 100 µL of a 30 µM chitobiose standard prepared in the NACA buffer was transferred to the clean charcoal beds, rinsed with two 1 mL volumes of deionized water, and eluted with three sequential 1 mL volumes of methanol. Each fraction was analyzed by direct infusion electrospray at ∼3 µL/ min. Most of the chitobiose was eluted in the second fraction, with small amounts being detected in the first and third fractions. Comparison of the ES intensities of a treated and untreated standard, as before, demonstrated that the activated charcoal was a more suitable support for desalting of the hydrolysates in this work. Activated charcoal was therefore chosen for the off-line desalting of the enzymatic hydrolysates of whole algal cells. For these samples, 100 µL of the C-18 SPE-processed hydrolysate was transferred to the clean charcoal beds and treated as described above. When no chitobiose was detected within individual fractions, the three fractions were pooled, concentrated under nitrogen, and reanalyzed. Recovery of chitobiose from charcoal in this procedure was estimated with the aid of an internal standard, raffinose. This effort is described in the Results and Discussion section. Microscale Liquid Chromatography. Microcolumns were made according to the method of Kennedy and Jorgenson,43 using a 50 cm segment of 75 µM i.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ). The columns were packed to a length of 7 cm with 7 µm Shandon Hypercarb (Shandon Scientific Inc., UK) PGC. The fritted end of the column was inserted into the ES needle. A 20 µM NaI solution prepared in 70:30 (v/v) methanol/water or 100% methanol was used as sheath liquid at a flow of 2 µL/min. Samples were loaded on the column using a high-pressure injector, as described by Hunt et al.44 After the desired volume, 0.05-2 µL, had been loaded on the column, the column was placed on-line with a Model 140B Applied Biosystems (Foster City, CA) microgradient dual-syringe pump. The eluent flow to the column was adjusted to 0.5 µL/min with the aid of a splitting tee and a length of restriction tubing (50 µm i.d. fused (42) Chen, T.; Yu, H.; Barofsky, D. F. Anal. Chem. 1992, 64, 2015. (43) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 56, 1128-1135. (44) Hunt, D. F.; Alexander, J. E.; McCormack, A.; Martino, P. A.; Michel, H.; Shabanowitz, J.; Sherman, N.; Moseley, M. A.; Jorgenson, J. W.; Tomer, K. B. In Techniques in Protein Chemistry II; Villafranca, J. J., Ed.; Academic Press: New York, 1991; pp 441-454.

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silica capillary). Water and pure acetonitrile were used for desalting and gradient elution of chitobiose from the column. The chromatographic run consisted of a 2 min isocratic run with water, followed by a linear gradient from 0% to 80% acetonitrile over 13 min, back to 0% acetonitrile in 1 min, and reequilibration with water for another 5 min. As a result, the analyte was focused (concentrated) on the PGC, desalted during the first 2 min of the program, and eluted with the acetonitrile gradient around 8 min into the run. Mass Spectrometry. The initial tuning of the TSQ-70 triple quadrupole mass spectrometer was performed with perfluorotributylamine (PFTBA) and the standard electron ionization source. The electrospray ion source was then installed and tuned with the angiotensin standard, 10 µM in methanol, infused at ∼3 µL/min. The parameters optimized were the needle-capillary position, capillary temperature, nebulizing gas flow rate, voltages applied to the capillary and skimmer, and, finally, the optical lens settings. The source was then finely tuned (unit mass resolution) for analysis of sugars by infusing a freshly prepared chitobiose solution containing 10 µM NaI. For the off-line experiments, spectra were acquired over a 600 amu range at 1.5 s/scan. Each spectrum was an average of 15 such scans. For the on-line experiments where chitobiose was monitored, spectra were acquired at 0.5 s/scan over a 300 amu range while the peaks eluted. A wider scan range (m/z 400-1500) was used for the on-line micro-LC ES-MS of the sugar mixtures. RESULTS AND DISCUSSION The objective of this work was to develop a mass spectrometric method for the detection of chitobiose in whole cell algal hydrolysates with limits of detection comparable to those achievable by SAE with pulsed amperometric detection (2-10 nM10). Interest in the development of a mass spectrometric method stems from its inherent specificity for establishing the presence of chitobiose; however, its sensitivity is severely compromised when the analyte (chitobiose) is present in a complex matrix and with a high salt content. To determine the utility of the mass spectrometric analysis, several taxa of phytoplankton were hydrolyzed enzymatically and tested by SAE-PAD and ES for the presence of chitobiose. The enzymatic hydrolysates of whole algal cells consist of mixtures of proteins, polysaccharides, and oligo- and monosaccharides in a buffer medium of high salt content. The proteins and polysaccharides were removed with SPE cartridges, leaving behind a sample with a simpler matrix consisting of short-chain sugars and inorganic salts. Following this treatment, the samples were directly analyzed by SAE-PAD; however, they required further treatment (desalting) for the mass spectrometric analysis in order to achieve reasonable detection limits for chitobiose. Initial efforts for desalting the hydrolysates were performed offline, and the samples prepared were analyzed on the lab-built ES interface that was briefly described in the Experimental Section. Desalting was subsequently performed in an on-line fashion because it resulted in lower limits of detection for chitobiose. Direct Infusion ES-MS. A simple calibration plot was first generated in order to assess the ES response for chitobiose with the lab-built ES interface. Chitobiose standards (0.22-8.0 µM) were prepared in charcoal-purified methanol containing 3 µM raffinose and 10 µM NaI. Raffinose was added as internal standard in the absence of radiolabeled chitobiose; NaI was added in order to improve the signal intensities for the sugars.27 The calibration

Table 1. ES-MS Results of the Enzymatic Chitin Assay of Marine Phytoplankton sample standarda

Chitan T. weissflogiib C. crypticac C. capsulatad D. brightwelii Synnechoccus. sp. T. verrucosa F/2 culture medium

SAE-PAD analysis

direct infusion ES

micro-LC ES

+ + + -f -

+ + -e -

+ + + -g -

a Chitan isolated from T. weissflogii.38,39 b 41 µg of C from chitin; 270 µg of total carbon. c 3.3 µg of C from chitin; 75 µg of total carbon. d 322 µg of total carbon. e In this column, (-) signifies