Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass

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Anal. Chem. 2004, 76, 6628-6634

Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass Spectrometry in Human Serum Maryline Gasthuys,†,‡ Sandra Alves,† Franc¸ ois Fenaille,§ and Jean-Claude Tabet*,†

LCSOB, University Pierre et Marie Curie, Boite 45, 4 place Jussieu 75252 Paris Cedex 05, France, Laboratoire National de Depistage du Dopage, 143 avenue Roger Salengro 94290 Chatenay-Malabry, France, and Nestle´ Research Center, Nestec Ltd., Vers-Chez-les-Blanc, 1000 Lausanne 26, Switzerland

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMSn experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ionssm/z 299 and 399sthat were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g‚L-1. Blood substitutes have long been sought after as alternatives to blood for use in the medical field, particularly in case of lack of red blood cells following surgery or accident. Moreover, since the 1980s, there has been an unfortunate decrease in the availability of donated blood units, partly due to public misapprehension of risks from donations. The development of new drugs to rectify the lack of available whole blood was required. Blood substitutes can be either synthetic perfluorocarbon solutions or hemoglobin-based oxygen carriers (HBOCs).1 First studies on “artificial blood” have led to administration of purified hemoglobin * Corresponding author: (e-mail) [email protected]. † University Pierre et Marie Curie. ‡ Laboratoire National de Depistage du Dopage. § Nestec Ltd. (1) Re´my, B.; Deby-Dupont, G.; D’Ans, V.; Ernest, P.; Lamy, M. Ann. Fr. Anesth. Reanim. 1999, 18, 211-224.

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solutions.2,3 Although showing promising results, cell-free hemoglobin tetrameric structure can dissociate in plasma into R,βdimers, causing nephrotoxicity.4 To maintain molecular stability and improve oxygen off-loading, intra- and intermolecular crosslinking has been performed on ultrapurified hemoglobin. For example, the two R-chains of diaspirin cross-linked hemoglobin (DCLHb; HemAssist, Baxter) are linked by bis(3,5-dibromosalicyl) fumarate bridges.5,6 Several preparations (PolyHeme, Northfield; Hemolink, Hemosol) using either bovine or human hemoglobin intermolecularly cross-linked by different agents have been developed. Hemopure (HBOC-201), which has been approved for human application in South Africa, and Oxyglobin (Biopure Corp.) were produced from ultrapurified bovine hemoglobin polymerized by glutaraldehyde, thus leading to molecular species with molecular weights up to 500 000.7 Besides the indicated medical use of HBOCs, the use of these compounds can be misapplied for doping purposes. Reticulated hemoglobins are more efficient than normal hemoglobin for oxygen transport8 and could therefore drastically improve an athlete’s performance. As a consequence, artificial oxygen carriers have been added to the list of prohibited substances by the World Anti-Doping Agency (WADA).9 Owing to this fact, the need to find a way to detect and identify this type of molecule has become a priority for doping control. For the present, the detection is commonly achieved using size exclusion chromatography.10 Recently, a general electrophoresis method for screening several HBOCs in human serum has been developed.11 Mass spectrometry has proved to be an efficient method to analyze high molecular mass compounds such as biomolecules (2) Rabiner, S. F.; Helbert, J. R.; Lopas, H.; Friedman, L. H. J. Exp. Med. 1967, 126, 1127-1142. (3) Rabiner, S. F.; O’Brien, K.; Peskin, G. W.; Friedman, L. H. Ann. Surg. 1970, 171, 615-622. (4) Debaene, B.; Barbot, A. Reanimation 2003, 12, 580-591. (5) Sloan, E. P.; Koenigsberg, M.; Gens, D.; Cipolle, M.; Runge, J.; Mallory, M. N.; Rodman, G. J. Am. Med. Assoc. 1999, 282, 1857-1964. (6) Yu, Z.; Friso, G.; Miranda J. J.; Patel, M. J.; Lo-Tseng, T.; Moore, E. G.; Burlingame, A. L. Protein Sci. 1997, 6, 2568-2577. (7) Rausch, C. W.; Gawryl, M. S.; Houtchens, R. A.; Laccetti, A. J.; Light, W. R. U.S. patent 5,840,852, 1998. (8) Pearce, L. B.; Gawryl, M. S. Adv. Exp. Med. Biol. 2003, 530, 261-270. (9) The 2004 prohibited list, Internal Standard, The World Anti-Doping Code, update 25 November 2003, http://www.wada-ama.org. (10) Trout, G. J.; Kazlaukas, R. Chem. Soc. Rev. 2004, 33, 1-13. (11) Lasne, F.; Crepin, N.; Ashenden, M.; Audran, M.; De Ceaurriz J. Clin. Chem. 2004, 50, 410-415. 10.1021/ac049275d CCC: $27.50

© 2004 American Chemical Society Published on Web 10/01/2004

thanks to soft ionization techniques, such as MALDI12,13 and electrospray.14 Primary structure as well as posttranslational modifications of proteins can be determined using tandem mass spectrometry (MS/MS) experiments.15,16 Electrospray ionization enables coupling with liquid chromatography and has been widely used to characterize hemoglobin variants17,18 as well as modified hemoglobins.19-21 The identification of specific fragments of bovine hemoglobin in human plasma after enzymatic digestion22 can constitute a way to establish the presence of Oxyglobin (bovine and human hemoglobins differ in amino acid sequence23). However, such methodology could be of limited interest since any type of hemoglobin can be used to manufacture glutaraldehydereticulated hemoglobin, either bovine, human, or recombinant.24 Therefore, we have developed an approach for the identification of exogenous polymerized hemoglobin involving the detection of modified residues, methodology that could be used whatever the origin of the hemoglobin. In the present study, we report a method based on mass spectrometry analyses for the identification of glutaraldehydepolymerized bovine hemoglobin in comparison with normal bovine hemoglobin. Bovine hemoglobin and Oxyglobin were first totally hydrolyzed with 6 N hydrochloric acid. Since they have been reduced by NaBH4 during the cross-linking process, the crosslinks are thought to be resistant to acid hydrolysis.25 Acid hydrolysis of Oxyglobin is expected to lead to some modified amino acid residues that could generate specific ions on the corresponding electrospray ionization (ESI) mass spectrum. The amino acid mixtures were first analyzed by direct infusion on a triple quadrupole mass spectrometer and that allowed us to highlight two potential Oxyglobin-specific ions, both able to lose a 159 u neutral under collision-induced dissociation (CID) conditions. LC-ESIMS experiments performed on natural and crosslinked bovine hemoglobin provides confirmation of the specificity of these species for Oxyglobin hydrolysate. Further constant neutral loss experiments brought results on hydrolysates of human serum samples spiked with 4 and 10 g‚L-1 of Oxyglobin. LC-ESIMS experiments provided an alternative method to detect those Oxyglobin-specific ions in serum samples spiked with Oxyglobin at a concentration of 4 g‚L-1. In a last set of experiments, the analysis of a glutaraldehyde cross-linked human hemoglobin (PolyHeme) showed the applicability of the current methodology to detect fraudulent administration of glutaraldehyde cross-linked hemoglobins from different origins. (12) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (13) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (14) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse C. M. Science 1989, 246, 64-71. (15) Roepstorff, P. Biomed. Mass Spectrom. 1984, 11, 601. (16) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (17) Rai, D. K.; Landin, B.; Alvelius, G.; Griffiths, W. J. Anal. Chem. 2002, 74, 2097-2102. (18) Wada, Y. J. Chromatogr., B 2002, 781, 291-301. (19) Nakanishi, T.; Miyazaki, A.; Kishikawa, M.; Yasuda, M.; Tokuchi, Y.; Kanada, Y.; Shimizu, A. J. Mass Spectrom. 1997, 32, 773-778. (20) Roberts, N. B.; Green, B. N.; Morris, M. Clin. Chem. 1997, 43, 771-778. (21) Rai, D. K.; Landin, B.; Griffiths, W. J.; Alvelius, G. Rapid Commun. Mass Spectrom. 2002, 16, 1793-1796. (22) Thevis, M.; Orgozalek Loo, R. R.; Loo, J. A.; Scha¨nzer, W. Anal. Chem. 2003, 75, 3287-3293. (23) http://www.expasy.org, P01966, P02072, P01922, P02023. (24) Hoffman, S. J.; Nagai, K. U.S. patent 5,661,124, 1995. (25) Monsan, P.; Puzo, G.; Mazarguil, H. Biochimie 1975, 57, 1281-1292.

EXPERIMENTAL SECTION Material and Chemicals. Bovine hemoglobin and 10 mM phosphate-buffered saline were obtained from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (12 N) was purchased from Prolabo (Paris, France), and acetonitrile and formic acid were purchased from SDS (Marseille, France). All solutions and buffers were prepared using deionized water (MilliQ grade). Oxyglobin solution was a generous gift from Biopure Corp. (Cambridge, MA) by the intermediate of the French Laboratoire National de Depistage du Dopage (LNDD). The human serum samples were obtained by simple centrifugation of blood samples from healthy volunteers. Acid Hydrolysis Step. According to the literature,26 acid hydrolysis was performed using 6 N HCl. In parallel, 2 mg of bovine hemoglobin and 20 µL of Oxyglobin solution (equivalent to ∼2 mg of protein) were diluted in 6 N HCl (final volume 2 mL), and the tubes were further sealed before being placed at 110 °C for 3, 6, 16, or 24 h. The reaction products were then dried under a nitrogen stream. Finally, 500 µL of deionized water was added to the dried products (stock solutions). Aliquots of 10 µL of stock solutions were mixed with 990 µL of CH3CN/H2O + 0.1% HCOOH (60/40, v/v) solution and were introduced into the mass spectrometer without any preliminary purification. Doping Human Serum Samples. For therapeutic purposes, a hemodilution of 20% is needed for an adult. Supposing this efficient dose is the same for increasing an athlete’s performance and considering a total dilution of the product in the blood volume, the concentration of Oxyglobin in the circulatory system would be ∼16 g‚L-1.27 To reproduce experimentally these physiological conditions, 50 µL of human serum has to be spiked with 800 µg of Oxyglobin. To stand below the theoritical detection limit, we deliberately chose to spike 50 µL of human serum samples aliquots with either 3.9 (500 µg, 10 g‚L-1) or 1.5 µL (200 µg, 4 g‚L-1) of Oxyglobin solution (13 g‚dL-1). Three milliliters of 6 N HCl was added to these spiked samples. The tubes were sealed and placed at 110 °C for 6 h. A blank sample was also prepared by direct acid hydrolysis of 50 µL of human serum. After being dried under nitrogen stream, the samples were diluted into 1 mL of deionized water (stock solutions). Aliquots of 10 µL were mixed with 990 µL of CH3CN/H2O + 0.1% HCOOH (60/40, v/v) solution and were introduced into the ESI source of the mass spectrometer. Mass Spectrometry. Direct infusion mass spectrometry experiments were performed in the positive electrospray ionization mode on a triple quadrupole Quattro I (Micromass Inc., Manchester, England) mass spectrometer. Analyses were realized by infusing the solution at a flow rate of 6.7 µL‚min-1 using a KD Scientific Inc. syringe pump. The spray voltage was set at 3.0 kV. Nitrogen was used as both nebulizing gas (20 L‚h-1) and disolvation gas (400 L‚h-1). The cone voltage was set at 20 or 30 V, and full-scan spectra were recorded from m/z 60 to 2000. CID experiments were performed using argon at a pressure of 1.5 × 10-4 mbar, and laboratory collision energy was optimized for each ionic species based on its spectral intensity and on the production of useful fragment ions. Product ion scan experiments were performed by scanning of the first quadrupole while constant neutral loss experiments were obtained by linked scan of the first (26) Moore, S.; Stein, W. H. Methods Enzymol. 1963, VI, 819-831. (27) Toussaint-Hacquard, M.; Devaux, Y.; Longrois, D.; Faivre-Fiorina, B.; Muller, S.; Stoltz, J. F.; Vigneron, C.; Menu, P. Life Sci. 2003, 72, 1143-1157.

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and the third quadrupoles. Data acquisition and processing were carried out using the Micromass MassLynx software. LC-MS and LC-MSn Analyses. Chromatographic separation was carried out using a HP1100 HPLC system equipped with a Waters Nova-Pak C18 column (3.9 × 150 mm, 4 µm, 60 Å). The volume of stock solutions injected was 20 µL. The mobile phase consisted of phase A, H2O + 0.1% HCOOH, and phase B, acetonitrile containing 0.1% HCOOH. The percentage of B increased according to a linear gradient of 100% A for 2 min, 0-15% B in 8 min, 15-21% B in 5 min, 21% B for 5 min, 21-26% B in 5 min, 26% B for 5 min, 26-30% B in 5 min, 30-60% B in 5 min, and finally 5 min at 60% before re-equilibrating the column. The flow rate was set at 0.7 mL‚min-1 and split to 70 µL‚min-1 before entering into the ESI source. The analyses were performed in the positive ESI mode with an ion trap instrument (ESQUIRE 3000, Bruker Daltonics, Bremen, Germany) operating in the datadependent mode. The analytical conditions were as follows: capillary voltage of 3.5 kV and source temperature of 350 °C. Heated nitrogen was used as both nebulizing gas (35 psi; 1 psi ) 6894.76 Pa) and drying gas (12 L‚min-1). Solution flew at atmospheric pressure through a grounded stainless steel capillary. The m/z ratio range (noted in Thomson unit28) used for the analytical scan was from 50 up to 1000 Th. Resonant ion ejection occurs at βz ) 2/3 (qz ) 0.78) within a ∆(m/z) value of 0.4 Th. All CID mass spectra were recorded with a low-mass cutoff set to m/z ∼60. RESULTS AND DISCUSSION Analysis of Reticulated Hemoglobin. Synthesis of Oxyglobin consists of the reaction of a dialdehyde compound, the glutaraldehyde (CHO(CH2)3CHO), with ultrapure bovine hemoglobin to produce cross-links between noncontiguous residues. The reaction occurs via Schiff base formation between the aldehyde groups and the amino groups of the protein, either NH2-terminal or -NH2 groups of the lysine residues. However, glutaraldehyde is assumed to also react with other amino acids such as tyrosine, cysteine, arginine, and histidine.29 In the case of bovine hemoglobin, NH2terminal amino acids are valine (R chain) or methionine (β chain). The nature of the cross-links is thought to be cyclic, but other different structures have been proposed30-32 (Chart 1). Up to now, mass spectrometry-based studies dedicated to the detection of Oxyglobin in human plasma have been performed after enzymatic digestion of the protein. Enzymatic digests of the reticulated protein were then compared to normal human22 or bovine hemoglobin10 digestion products. As we have also observed in our laboratory (data not shown), LC-MS analysis of trypsin digestion products of Oxyglobin displayed a missing fragment RT11 (from RV93 to RK99) in comparison with natural bovine hemoglobin digestion product. This suggests that RK99 residue of bovine hemoglobin is involved in a cross-link, and therefore, the trypsin (28) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 93. (29) Haney, C. R.; Buehler, P. W.; Gulati, A. Adv. Drug Delivery Rev. 2000, 40, 153-169. (30) Meade, S. J.; Miller, A. G.; Gerrard, J. A. Bioorg. Med. Chem. 2003, 11, 853-862. (31) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, 1996. (32) Henderson, A. P.; Bleadsdale, C.; Clegg, W.; Golding, B. T. Chem. Res. Toxicol. 2004, 17, 378-382.

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Chart 1a

a Propositions for the structure of the cross-link by glutaraldehyde by (a) Meade et al.,30 (b) Hermanson31, (c) personal communication, (d) Monsan et al.,25 and (e) Henderson et al.32 These structures do not take into account the reduction step after modification by glutaraldehyde and prior to acid hydrolysis.

cleavage does not occur after this residue.33 But Oxyglobin-specific tryptic fragments were not detected by this way, probably due to their poor relative abundance in the peptide mixture and the heterogeneity of the adducts. However, even if the RK99 could be preferentially modified, the reaction should also occur randomly on other sites of the sequence.27,34 For instance, previous experiments performed on trypsin digests showed that the relative intensities of the chromatographic peaks of all the tryptic fragments were lower for Oxyglobin when balanced by the heme moiety chromatographic peak (data not shown). Consequently, chances could be increased to detect modified species by working on amino acids rather than on digest fragments and sensitivity could also be drastically improved. We have oriented our work toward the comparison of acid hydrolysates of Oxyglobin and bovine hemoglobin to detect modified residues. Direct Infusion MS Analyses of Amino Acid Mixtures Using the Triple Quadrupole Mass Spectrometer. To confirm the presence of the modified amino acids in the produced complex amino acid mixture, ESI experiments were performed on the triple quadrupole mass spectrometer by direct infusion. Attention was first focused on the amino acids’ mass-to-charge ratio below 210 Th in the different mass spectra. Both bovine hemoglobin and Oxyglobin hydrolysates allow the detection of most of the amino acids of the hemoglobin sequence in the m/z ratio range from 50 to 210 Th (Figure 1). As is well known, glutamine and asparagine (33) Alma, C.; Trout, G.; Woodland, N.; Kazlankas, R. Proceedings of the ManfredDonike Workshop, Cologne 2002, Sport & Buch Strauss, 2002; pp 169-178. (34) Riess, J. G. Chem. Rev. 2001, 101, 2797-2919.

Figure 1. ESI mass spectra of acid hydrolysis products of (a) bovine hemoglobin and (b) Oxyglobin. The ESI experiments are performed in positive ion mode on the triple quadrupole at a cone voltage of 20 V. The amino acid ion peaks are noted according to the one-letter code, and the “*” symbol designates the impurities frequently observed on ESI mass spectra. Peak resolution has voluntarily been decreased to optimize sensitivity in the interesting ions m/z domain. Consequently, lysine and glutamate amino acids cannot be differentiated on the ESI mass spectra.

residues (Q and N) are hydrolyzed into glutamic and aspartic acids (E and D) residues, respectively.35 Tryptophan residues are also destroyed by this drastic hydrolysis step. Moreover, methionine and cysteine residues are not detected because of their poor occurrence in the protein sequence. The relative abundances of each protonated amino acid compared with the total ionic intensities of all amino acids (Iaai/∑inIaai) are similar in both mass spectra and show a difference of less than 3%, except for the leucine residue ion relative intensity that is lower in Oxyglobin acid hydrolysate (-11%). This trend was expected because the primary structures of Oxyglobin and that of natural bovine hemoglobin are perfectly identical except for the cross-linked amino acids, which are statistically dispersed along the sequence on the basic amino acids. These observations also confirm that using a proteolysis time of 6 h is suitable for obtaining reliable and reproducible values. However, these results cannot be used for differentiation of the different hemoglobin species. On the contrary, several species with m/z ratio higher than 280 Th enable a good differentiation of the native and cross-linked proteins (Figure 1). It must be specified that all results shown in this article were obtained with a hydrolysis time of 6 h. Similar results can be obtained with an overnight acid hydrolysis. The presence of dipeptide and tripeptide ions persisted, and between the two protein hydrolysates, some of them showed different intensities. However, we did not focus on this observation to differentiate the two proteins because the comparison of the two hydrolysate mass spectra allowed observation of the presence of two specific ions in the ESI mass spectrum of Oxyglobin hydrolysate. Two ions at (35) Fountolakis, M.; Lahm, H.-W. J. Chromatogr., A 1998, 826, 109-134.

m/z 299 and 399 could be observed and were strictly not detected in the ESI mass spectrum of native bovine hemoglobin hydrolysis solution, and their relative intensities are highly reproducible. Those ions could potentially correspond to modified amino acids as they are single-charge ions (shown by the natural isotopic cluster) and their respective mass to charge values do not match with intact amino acids or with dipeptides. These additional peaks were detectable with enough sensitivity and to be selected for the CID experiments in order to establish their fragmentation patterns. Tandem Experiments Performed on the Triple Quadrupole Instrument. CID spectra of the m/z 299 and 399 ions show that they are not related by the loss of 100 u (Figure 2). It has already been observed that glutaraldehyde is able to react on itself to form carbohydrate oligomers25 that are able to lose 100 u under CID conditions. Thus, the m/z 299 and 399 ions do not correspond to oligomers of glutaraldehyde. However, both ions are able to lose a neutral 159 u, which suggests that they are probably close in structure. Although the energy at the center of mass is higher for the m/z 399 ion than for the m/z 299 ion, the abundance of m/z 399 is higher than the abundance of m/z 299, which suggests that m/z 399 is more stable than m/z 299. Moreover, the m/z 399 ion produces other consecutive fragmentations such as neutral loss of water from m/z 240. Liquid Chromatography Combined with Sequential MS3 Experiments: Study of the Discriminant Species. To increase sensitivity of these two ions in the mass spectra, we chose to use an ion trap mass spectrometer coupled to a chromatographic column allowing a rough separation of the different species of the mixture and also facilitating the sequential MSn experiments, Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Figure 2. CID spectra of (a) m/z 299 and (b) 399 ions of Oxyglobin acid hydrolysate solution. The experiments were performed on a triple quadrupole mass spectrometer under pressure of collision gas (argon) of 1.5 × 10-4 mbar, with energies at the center of mass of (a) 3.5 and (b) 4.5 eV. The cone voltage was set at 30 V for both analyses.

for structural characterization purposes. Chromatographic separation of amino acids is most of the time performed using ion exchange liquid chromatography36,37 or reversed-phase liquid chromatography after chemical derivatization of the amino acids.38,39 In our case, an effort was made to detect specific species of Oxyglobin and not to separate the several common amino acids of the mixture. Therefore, we have used a reversed-phase HPLC method without any derivatization. The total ionic current chromatograms provided from both bovine hemoglobin and Oxyglobin hydrolysates are shown in Figure 3. Amino acids elute from the column relatively early in the analysis, followed by dipeptides and tripeptides. The presence of small peptides persisted because of the short duration (6 h) of the acid treatment in comparison with classical acid hydrolysis for quantification experiments.40 Numerous coelutions were observed, but the separation was not optimized for the common species of the two protein hydrolysates. At the end of the gradient, at the retention times 18.3 (m/z 299), 21.9, and 22.9 min (m/z 399), three chromatographic peaks appear

specifically in the case of the Oxyglobin hydrolysate. These m/z 299 and 399 ions were further studied using LC combined with sequential MS3 experiments because of their higher abundances and their previous observation in the ESI mass spectrum of the hydrolysates directly infused (Figure 3). LC combined with sequential MS3 experiments were performed on m/z 140 and 240 ions (Figure 4). As already observed for their parent ions at m/z 299 and 399, those ions are very stable toward CID processes and their fragmentations in the ion trap mass spectrometer appeared somewhat difficult under conventional resonant exciting conditions. Parameters concerning the isolation of these precursor ions have to be modified to make sure the ions are highly stabilized by the rf voltages. Nevertheless, the CID spectrum of m/z 140 shows a fragment ion at m/z 84 that could be a fragment ion derived from a modified lysine residue.41-43 However, sequential MS3 experiments of m/z 240 and 140 ions do not give enough information to draw conclusions about their structures and to determine whether they correspond to modified lysine residues, modified NH2-terminal residues, or even other species. Screening Method Applied on Human Serum Spiked with Oxyglobin. ESI mass spectra by direct infusion displayed no differences between the spiked serum hydrolysate and the blank serum hydrolysate (data not reported). But since the two Oxyglobin-specific ionssm/z 299 and 399sobserved for pure product hydrolysate analysis are both able to lose the common neutral, constant neutral 159 u loss experiments on serum samples have been conducted on the triple quadrupole mass spectrometer. The constant 159 u neutral loss spectra of all hydrolysate solutions display the same dipeptide ions at m/z 231 (VL), 245 (LL), 269 (HL), 279 (FL), 295 (YL), and 330 (tripeptide) (Figure 5). For each dipeptide, a leucine residue can be replaced by an isoleucine residue; even if isoleucine is absent in the sequence of bovine hemoglobin, the contribution of other serum proteins must be considered. In this case, the loss of 159 u corresponds to the

Figure 3. Total ion current chromatograms (ESI/IT) of acid hydrolysis products of (a) bovine hemoglobin and (d) Oxyglobin. Reconstructed ion chromatograms of (b) and (e) m/z 299 and (c) and (f) m/z 399 ions. The chromatographic peaks at 18.3 (m/z 299), 21.9, and 22.9 min (m/z 399) are detected in the case of Oxyglobin and do not appear in the chromatogram of acid hydrolysis products of bovine hemoglobin. Note that the two chromatographic peaks in (f) contain twice m/z 399 that may be isomeric forms. 6632 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

Figure 4. Second-generation mass spectrum (MS3) of (a) m/z 240 (from m/z 399) and (b) m/z 140 (from m/z 299) performed with an ion trap mass spectrometer. The isolation width was set at 10 Th, and laboratory energies and excitation cutoff were respectively of 1.30 Vp-p and m/z 90 for the m/z 240 ion and 1.15 Vp-p and m/z 48 for the m/z 140 ion.

Figure 5. Constant neutral loss 159 u spectra of (a) blank serum and (b) Oxyglobin-spiked serum at 4 g‚L-1 acid hydrolysates performed with triple quadrupole experiments under pressure of collision gas (argon) of 1.5 × 10-4 mbar and laboratory collision energy of 30 eV. The cone voltage was set at 30 V.

consecutive losses of a leucinesor isoleucinesresidue, a carbon monoxide molecule, and a molecule of water. In addition to these common signals, the constant neutral loss spectrum of Oxyglobincontaining serum hydrolysate solution displays the precursor m/z 299 and 399 ions with a sufficient signal-to-noise ratio (S/Nm/z 299 ) 20.5 ( 2.5, S/Nm/z 399 ) 6.4 ( 1.7, number of experiments: n ) 4) for the serum sample containing 4 g‚L-1 of Oxyglobin. Constant neutral loss of 159 u experiment on serum hydrolysates leads to quick and highly specific response even (36) Shang-gui, D.; Zhi-ying, P.; Fang, C.; Ping, Y.; Tie, W. Food Chem. 2004, 87, 97-102. (37) Candito, M.; Bedoucha, P.; Mahagne, M. H.; Scavini, G.; Chatel, M. J. Chromatogr., B 1997, 692, 213-216. (38) Zhao, Q.; Sannier, F.; Garreau, I.; Lecoeur, C.; Piot, J. M. J. Chromatogr., A 1996, 723, 35-41. (39) Lozanov, V.; Petrov, S.; Mitev, V. J. Chromatogr., A 2004, 1025, 201-208. (40) Salchert, K.; Pompe, T., Sperling, C.; Werner, C. J. Chromatogr.,. A 2003, 1005, 113-122. (41) Fenaille, F.; Tabet, J.-C.; Guy, P. A. Rapid Commun. Mass Spectrom. 2004, 1, 67-76. (42) Rogalewicz, F.; Hoppilliard, Y.; Ohanessian, G. Int. J. Mass Spectrom. 2000, 195/196, 565-590. (43) Yalcin, T.; Harrison, A. G. J. Mass Spectrom. 1996, 31, 1237-1243.

when the protein concentration is lower than the therapeutic dose. Therefore, such methodology seems to be a sensitive method to easily and rapidly detect the presence of Oxyglobin in human blood in fast screening and can be included into a high-throughput approach for doping control. To confirm these data, LC-MS experiments have been conducted on serum hydrolysates under the same experimental conditions as described for pure product hydrolysates. Total ionic current chromatograms (Figure 6) display the chromatographic peaks corresponding to amino acids, dipeptides, and tripeptides at the beginning of the solvent gradient. Reconstructed ion chromatograms of m/z 299 and 399 ions show chromatographic peaks at the same retention times as for the pure product hydrolysate with an experimental shift of 2%. Moreover, LC combined with sequential MS2 and MS3 experiments showed that the fragmentation patterns of these two ions are identical to those observed on the pure product hydrolysate. Therefore, multiple reaction monitoring (MRM) experiments on the m/z 299 and 399 ions could be a way to detect and quantify the amount of Oxyglobin in human plasma and also lower the detection threshold. LC-MS (and LC-MSn) experiments constitute a good confirmation method for the detection of Oxyglobin in the human serum with a detection limit of 4 g‚L-1, which is far less than the expected concentration of Oxyglobin in blood under therapeutic conditions. Complementary experiments. To further demonstrate the specificity of the current methodology for glutaraldehyde crosslinked hemoglobins and also its wider applicability, the same experiments were carried out on a glutaraldehyde cross-linked human hemoglobin (PolyHeme, Figure 7). Monitoring the constant neutral loss of 159 u enables the visualization of the same diagnostic ions at m/z 299 and 399 as seen previously for Oxyglobin (Figure 5). Moreover, it must be emphasized that these two ions were not observed when polyoxyethylene cross-linked human hemoglobin was analyzed (data not shown). Although molecular structures of the different species involved are still to be established, these complementary results demonstrate that the m/z 299 and 399 ions are specific of glutaraldehyde cross-linked hemoglobins and therefore could be used for doping control. To our knowledge, this work describes the first methodology that enables the detection of glutaraldehyde cross-linked hemoglobins from both bovine and human origin, which is of high interest for the field. CONCLUSION The detection of specific ions of a bovine hemoglobin-based oxygen therapeutic after an acid hydrolysis procedure was performed using a triple quadrupole mass spectrometer from a mass spectrum and from product ion scan modes under CID conditions. Hydrochloric acid hydrolysis followed by mass spectrometry analysis provides at least two specific ions, m/z 299 and 399, both able to yield a neutral 159 u release. Because of its enhanced sensitivity from product ion scan experiments and the possibility to conduct multiple-stage MSn experiments, an ion trap mass spectrometer was used to perform LC-MSn experiments. HPLC/ITMS experiments brought confirmation of the presence of two Oxyglobin-specific species. The corresponding m/z 299 and 399 ions underwent multiple-stage MSn fragmentations into the ion trap. Our present study gives an indication that they could Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Figure 6. Total ion current chromatograms (ESI/IT) of acid hydrolysis products of (a1) blank serum, (b1) serum spiked with Oxyglobin 4 g‚L-1, and (c1) serum spiked with Oxyglobin 10 g‚L-1. Reconstructed ion chromatograms from mass spectra of (a2), (b2), (c2) m/z 299 and (a3), (b3), (c3) m/z 399 ions in these hydrolysate solutions.

Figure 7. Constant neutral loss mass spectra of (a) human hemoglobin and (b) PolyHeme acid hydrolysates performed with a triple quadrupole instrument under pressure of collision gas (argon) of 1.4 × 10-4 mbar and laboratory energy of 50 eV. The cone voltage was set at 30 V. The m/z 299 and 399 ions are specifically detected in the case of glutaraldehyde cross-linked hemoglobin.

contain a lysine residue. However, the unambiguous structures of these ions are not established, and structural elucidations are in progress. Nevertheless, we developed a simple and rapid method based on a constant 159 u neutral loss spectrum to detect the precursor m/z 299 and 399 ions, considered as Oxyglobin-specific ions in human serum samples using the triple quadrupole mass spectrometer. Screening the presence of cross-linked or modified hemoglobin in the serum in comparison with blank serum samples

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becomes possible this way. In addition, LC-MS experiments on an ion trap mass spectrometer permit detection of the same ions in spiked serum samples. This procedure could provide an alternative method to constant neutral loss experiments. We thus developed two mass spectrometry-based methods with good specificity and a low-consuming time of hydrolysis. Quick mass spectrometry studies have been performed thanks to direct injection into the mass spectrometer and when coupled to liquid chromatography. Results can be obtained rapidly, and the sensitivity threshold is acceptable. Yet, the procedure can be improved by optimizing the experimental conditions (e.g., times for LC) or by developing a MRM method that could allow quantification in MS. Further investigations should concern the detection of several oxygen therapeutics (either polymerized with glutaraldehyde or not) into natural matrixes. Another aim of our studies would be to get the precise and unambiguous structures of the specific ions detected. ACKNOWLEDGMENT We thank French Laboratoire de Depistage du dopage for financial support and for providing hemoglobin-based products and human serum samples and Remi Lemaire for his fruitful advice.

Received for review May 18, 2004. Accepted August 23, 2004. AC049275D