Size Exclusion Chromatography−Chemical Reaction Interface Mass

Jun 12, 1999 - Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alte...
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Anal. Chem. 1999, 71, 2951-2955

Size Exclusion Chromatography-Chemical Reaction Interface Mass Spectrometry: “A Perfect Match” Paolo Lecchi* and Fred P. Abramson

Department of Pharmacology, RE6640, School of Medicine and Health Sciences, The George Washington University, 2300 I Street, NW, Washington D.C. 20037

Size exclusion chromatography (SEC) has been coupled to chemical reaction interface mass spectrometry (CRIMS) for the analysis of biopolymers. This innovative combination allows the analysis of biopolymers with no limitation on the molecular weight and chemical composition of the species under investigation. With SEC-CRIMS we have examined different classes of biopolymers including polynucleotides, proteins, and polysaccharides. Moreover, CRIMS allows the simultaneous detection of multiple organic elements and their stable isotopes. When SEC is interfaced to CRIMS, further information (elemental detection) is obtained, enhancing the analytical capability of SEC. These features have been applied to the detection of a labeled protein (13C-rat growth hormone) in plasma and to the characterization of heparin and low molecular weight heparin from different sources. Size exclusion chromatography (SEC) is a separation technique able to sort analytes according to their hydrodynamic volumes. The principle behind SEC is the existence of size-defined microcavities within a porous stationary phase. Molecules that are smaller than the pore size enter the cavities and travel through the chromatographic column more slowly then larger molecules and therefore are eluted later. The introduction of packings with superior mechanical and chemical stability has allowed the development of high-performance size exclusion chromatography (HPSEC).1 HPSEC columns feature high reproducibility, efficiency, stability, short times per analysis, and little interaction with the analytes. In SEC the pore volume is (ideally) the only parameter that sets the ability of a column to separate molecules by size. Practically, non-size exclusion effects (e.g., hydrophobic and electrostatic interactions between analytes and the stationary phase) limit the separation. To achieve successful separations, it is necessary to overcome these non-size exclusion effects by optimizing the chromatographic protocols. In this regard, several parameters, e.g., solubility, ionic strength, and pH, have to be considered to obtain a suitable mobile phase.1-3 * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (202) 994-2870. (1) Mant, C. T.; Hodges, R. S. High Performance Liquid Chromatography of Peptides and Proteins: Separation Analysis and Conformation; CRC Press: Boca Raton, FL, 1991; Section III. (2) Mant, C. T.; Parker, J. M.; Hodges, R. S. J. Chromatogr. 1987, 397, 99112. 10.1021/ac981098r CCC: $18.00 Published on Web 06/12/1999

© 1999 American Chemical Society

When optimized, SEC is not limited by the molecular weight (MW) or by the chemical composition of the analyte. However, a complete exploitation of SEC requires a detection system that, in principle, has the same properties, i.e., applicability over a broad range of MW and chemical compositions. Most detectors interfaced to SEC measure a spectroscopic parameter of the analyte: UV absorption, light scattering, or refractive index.4-6 Such detectors are not linear in the wide range of MW covered by SEC. Moreover, they are usually not selective enough to track a single compound within a mixture, unless it contains a specific moiety with specific spectroscopic properties. Interfacing reversed-phase high-peformance liquid chromatography (HPLC) to mass spectrometry (MS) has become a relatively easy task to accomplish.7 Interfacing MS to SEC to separate molecules in a predictable size-dependent way has the potential to be a valuable analytical strategy.8 Although MS might seem to fulfill the requirements of universality and selectivity for yielding a perfect combination with SEC, two major problems exist. First is the general incompatibility between the involatile mobile phases used for SEC and most MS instruments. Only volatile buffers and solvent modifiers are acceptable with mass spectrometry. Second, despite the wide range of molecules that are amenable to mass spectrometry in general, a large variety of molecules are incompatible with any given type of MS. Thus, the universality of SEC will be nearly impossible for MS to exploit. SEC is a common step during the purification of biopolymers9 and has been coupled off-line to MS for the analysis of proteins10,11 and oligosaccharides in human milk.12 Clearly, an on-line method would be preferable to an off-line one, but except for the detection of inorganic elements in biological samples by inductively coupled plasma mass spectrometry,13 few SEC-MS on-line analyses of (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Ricker, R. D.; Sandoval, L. A. J. Chromatogr. A 1996, 743, 43-50. Stuting, H. H.; Krull, I. S. J. Chromatogr. 1991, 539, 91-109. Wen, J.; Arakawa, T.; Philo, J. S. Anal. Biochem. 1996, 240, 155-166. Cook, S. D.; Sible, V. S. Eur. Polym. J. 1997, 33, 163-168. Niessen, W. M. A. J. Chromatogr. A 1998, 794, 407-435. Lecchi, P.; Abramnson, F. P. J. Chromatogr. A 1998, 828, 509-513. Preneta, A. Z. In Protein Purification Methods; Harris, H. L. V., Angal, S., Eds.; IRL Press: Oxford, U.K., 1989; Chapter 6. Kisselev, A. F.; Akopian, T. N.; Goldberg, A. L. J. Biol. Chem. 1998, 273, 1982-1989. Kriwacki, R. W.; Wu, J.; Tennant, L.; Wright, P. E.; Siuzdak, G. J. Chromatogr. A 1997, 777, 23-30. Stahl, B.; Thurl, S.; Zeng, J.; Karas, M.; Hillenkamp, F.; Steup, M.; Sawatzky, G. Anal. Biochem. 1994, 223, 218-226. Yoneda, S.; Suzuki, K. T. Biochem. Biophys. Res. Commun. 1997, 231, 7-11.

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biopolymers have been reported, and they are limited to the analysis of small peptides14,15 using electrospray ionization. During the past few years, an innovative mass spectrometric technique named chemical reaction interface mass spectrometry (CRIMS) was developed in our laboratory and coupled to HPLC.16 In brief, CRIMS allows the analyst to monitor the elemental and isotopic composition of any organic molecule after its quantitative transformation into a set of specific low-MW reaction products. Because the analyte does not enter the mass spectrometer intact, but after decomposition by a chemical reaction, CRIMS does not provide information about the molecular weight of the intact species. The benefit of this decomposition strategy is that any organic molecule of any size that enters the chemical reaction interface is detected. In doing so, CRIMS provides a set parameters for the characterization of the analyte, (e.g., 13C/12C isotope ratio) or the presence of an unusual element (S, Cl) or isotope (2H, 13C, 15N) different from that of most MS methods.17 Unlike other MS techniques, the CRIMS response is proportional to the amount of a specific organic element present in biopolymers because the same product species (CO2, NO, etc.) is being detected from all analytes. This response is related to the number of the targeted species in, but not to the size or chemical nature of, the molecule under investigation. This report, which is the first example of on-line SEC-MS for the analysis of biopolymers of different chemical nature including proteins, polynucleotides, and polysaccharides, embodies the concept of interfacing a universal separation technique (SEC) with a universal detection system (CRIMS). The “perfect match” between SEC and CRIMS makes this method a very promising analytical tool for the study of biopolymers. EXPERIMENTAL SECTION Materials. Chemicals employed in this work were obtained from Sigma (St. Louis, MO). Low-MW heparin (Lovenox, RhonePoulenc) and unfractionated heparin from porcine intestine (Elkins-Sinn, Inc., Cherry Hill, NJ) were obtained from GWU Hospital Pharmacy (Washington, DC). Solvents for chromatographic separations were obtained from EM Scientific (Gibbstown, NJ) at the highest grade of purity available. Standard oligodeoxynucleotides (dT8, dT15, dT20, dT30, and dT50) were purchased from Synthegen (Houston, TX) and purified by HPLC with a 150 × 4.6 PRP-1 Hamilton column (Reno, NV) using 100 mM tetraethylammonium acetate at pH 8.5 (A) and acetonitrile (B) as mobile phases. The purification was done isocratically for the first 3 min (with 20% B) and with a linear gradient to reach 50% B in 15 min with a flow rate of 1 mL/min. Rat growth hormone (ratGH) uniformly labeled with 13C was produced in our laboratory from the rat adenoma cell line GH3 (ATCC, Manassas, VA) as described elsewhere.18 Chromatographic Conditions. SEC was performed with silica-based SynChropak columns. Either a GPC 300 (300 Å pore size), 250 × 4.6 mm i.d., 5 µm particle size column or a mixed(14) Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Senko, M. W.; McLafferty, F. W.; Ganem, B. J. Am. Chem. Soc. 1993, 115, 8409-8413. (15) Nylander, I.; Tan-No, K.; Winter, A.; Silberring, J. Life Sci. 1995, 57, 123129. (16) Abramson F. P. Mass Spectrom. Rev. 1994, 13, 341-356. (17) Song, H.; Abramson, F. P. Drug Metab. Dispos. 1993, 21, 868-873. (18) Osborn, B. L.; Abramson, F. P. J. Labelled Compd. Radiopharm. 1997, 39, 935-953.

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Figure 1. SEC-CRIMS setup. An HPLC pump is used to deliver solvent analytes to an SEC column; the solution is then thermosprayed into the universal interface (UI), which efficiently removes the volatile solvents and solvent modifiers from the effluent stream. The dry particles in a stream of helium (He) pass through a momentum separator, which removes yet more vapor and a large portion of the helium gas carrier. Reactant gas (SO2 or NF3) is added to the gas stream prior to entering the alumina tube that passes through the microwave-powered cavity, which activates the chemical reaction interface (CRI). In the CRI, regardless of size or composition, analyte molecules are chemically transformed to stable specific lowMW products. These reaction products enter the mass spectrometer (MS) where they are monitored using selected ion recording. Analytical data are acquired and processed on-line.

bed (GPC linear, 100-1000 Å pore sizes) 250 × 4.6 mm i.d., 7 µm particle size column obtained from Keystone Scientific (Bellefonte, PA) was used in all the analyses. For the analysis of the nucleic acids, the two columns were connected in series. A solution of 100 mM ammonium acetate at pH 6.5 was used as the mobile phase for separation of nucleic acid species and polysaccharides. The mobile phase for protein separation was 1.0 M triethylammonium formate (TEAF) in water/tetrahydrofuran (THF) at pH 3.0. This mobile phase was prepared by adding triethylamine to formic acid to reach pH 3 and then adding THF to a final ratio water/THF of 2/1. Protein SEC was carried out at 40 °C. All other separations were done at room temperature. CRIMS Analysis. Figure 1 shows the HPLC-CRIMS instrumental setup. A detailed explanation of the instrument was published previously.19 Briefly, an HPLC instrument (Model 222C, Scientific Systems Inc., State College, PA) was connected to a quadrupole MS (Extrel C50/400, ABB Extrel, Pittsburgh, PA) via a Vestec universal interface (UI) (PerSeptive Biosystems, Framingham, MA), which removed the solvents employed for the chromatographic separation. Located after the UI and before the MS, a microwave-powered chemical reaction interface (CRI) transformed the analytes to a set of low-MW products by reaction with a specific gas, e.g., SO2 (Matheson Gas Products, East Rutherford, NJ) or NF3 (Air Products, Allentown, PA). Products of the CRIMS reaction entered the MS where they were ionized in the electron impact mode. The low-MW reaction products obtained derived from both the elemental composition of the analyte and the reactant gas employed in the chemical reaction. For example, with SO2 as the reactant gas, 12C atoms in the (19) McLean, M.; Vestal, M. L.; Teffera, Y.; Abramson, F. P. J. Chromatogr. A 1996, 732, 189-199.

analytes were detected as 12CO2+ ions (m/z 44), whereas if NF3 was used, 12C atoms were detected as 12CF3+ (m/z 69). CRIMS completely decomposed NF3 to F2 and N2. The F2 was chemically aggressive toward many parts of the mass spectrometer. To prevent damage to the instrumentation, we used a fluorine scrubber made of 316 stainless steel (6.35 mm (1/4 in.) o.d. × 150 mm long) filled with 40 mesh copper powder (Sigma) and heated to 325 °C downstream of the CRI and before the MS. The high reactivity of F2 toward copper at this temperature was sufficient to completely remove the reactive fluorinated species HF and F2 so that they were no longer seen in the mass spectrum. After weeks of use, no problems were noticed in the instrumental setup using the copper scrubber. Data from the MS were processed by a Vector-2 data system (Teknivent, Maryland Heights, MO). The UI was designed to give the best performance at a flow of 1 mL/min. However, analytical SEC separations were better if carried out at relatively low flow rates.2,3 To achieve efficient chromatographic separations without compromising the UI, we performed SEC at 0.2-0.3 mL/min and added water to the column eluate through a three-way mixer (Alltech, Deerfield, IL) up to the optimal flow rate of 1 mL/min using another HPLC pump. RESULTS AND DISCUSSION Interfacing SEC to CRIMS. The major limitation in interfacing SEC to MS is the nature of the mobile phases used for SEC. As described in the literature,20 these mobile phases usually contain nonvolatile salts (e.g., KH2PO4, NaCl, or Na2SO4); therefore, they are not suitable for MS. To pursue our goal of connecting SEC to MS, we tested different mobile phases and chromatographic conditions. With the column that we used, a buffer solution containing the volatile salt ammonium acetate worked for the analysis of polysaccharides (dextrans, heparin) or nucleic acid polymers (RNA, oligonucleotides). For the analysis of proteins, we found that 2/1 TEAF/THF was an excellent mobile phase. It is completely volatile and well tolerated by the instrument. Months of use did not present any problem for the column or for any part of the MS. Analysis of Biopolymers by SEC-CRIMS. The universality of SEC-CRIMS was tested by examining biopolymers of different chemical classes and different MW ranges. All these, obviously, contain carbon. Hence, in this set of analyses, CRIMS was employed as a nonspecific carbon detector by monitoring 12CO2+ at m/z 44. Figure 2 shows the SEC-CRIMS analysis of nucleic acid species. 23S rRNA (3566 bases, ∼1100 kDa), 16S rRNA (1776 bases, ∼600 kDa), tRNA (average molecular mass 25 kDa), and uridine monophosphate (UMP, 324 Da) were successfully separated and detected as individual peaks. This chromatogram was generated by injecting 5 µg of each material and then separated with a tandem arrangement of a GPC linear and a GPC 300 column. The mobile phase flow was 0.2 mL/min, with 0.8 mL/ min of water added postcolumn. Reproducibility was tested by injecting each compound several times and measuring the retention time. Most common SEC protocols for the analysis of proteins are not suitable for mass spectrometric detection. Those acceptable (20) Boyes, B.; Alpert, A. In Practical HPLC Method Development; Snyder, L. R., Kirkland, J. J., Glajch, J. L., Eds.; Wiley and Sons Inc.: New York, 1997; Chapter 11.

Figure 2. SEC-CRIMS analysis of nucleic acids. A ) 23S rRNA (3556 b, ∼1100 kDa); B ) 16S rRNA (1776 b, ∼600 kDa); C ) tRNA (75-85 b, ∼25 000); D ) uridine monophosphate (362 Da). Five microgram samples of each were separated by a GPC linear and a GPC 300 column connected in series. The mobile phase was ammonium acetate, 100 mM, pH 6.5, at a flow rate of 0.2 mL/min with water, 0.8 mL/min, added postcolumn.

protocols that use volatile mobile phases (i.e., 0.1% TFA)14,15,21 did not give adequate results with the columns that we used. Interactions between proteins and the column led to unacceptable chromatographic separation. To find a suitable mobile phase, several parameters had to be considered to achieve a sizedependent separation of proteins and to overcome nonspecific interactions with the stationary phase. Therefore, we tested different chromatographic conditions, taking into consideration ionic strength, protein solubility, and volatility of the solvent. As result of this research, we introduced a mobile phase consisting of 1 M TEAF in 2/1 water/THF at pH 3.0. This mobile phase could dissolve and elute all the standard proteins so far tested, with results comparable to those of SEC using a conventional involatile modifier in the mobile phase (e.g., 50 mM, K2HPO4 and 200 mM NaCl at pH 6.5; as seen with UV detection (data not shown)). Proteins that were successfully analyzed by SECCRIMS included thyroglobulin, catalase, collagen, transferrin, albumin, carbonic anhydrase, lysozyme, and insulin. Figure 3 shows the analysis of a mixture of standard proteins (each 5 µg), separated by the GPC 300 column at 40 °C, including human placental collagen (180 kDa), bovine serum albumin (66.5 kDa), and chicken lysozyme (14.5 kDa), along with tryptophan (204 Da). Each species appears as a single well-defined peak in the chromatogram obtained by monitoring the CO2+ trace at m/z 44. Figures 2 and 3 display the wide MW capabilities of CRIMS as a SEC detector. SEC-CRIMS has no MW limitation because macromolecules such as polynucleotides, proteins, or dextran polysaccharides (data not shown) are completely transformed by CRIMS and detected as simple low-MW compounds. Using volatile mobile phases instead of those described in conventional SEC protocols did not change the separation ability of the chromatographic columns as seen with UV detection (data not shown). When CRIMS is used as a nonspecific detector (e.g., to monitor carbon at m/z 44), the presence of low-MW carbon-containing sample contaminants leads to unexpected peaks or to a peak overlapped with a low-MW analyte. These contaminants have (21) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283-2291.

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Figure 3. SEC-CRIMS analysis of proteins. A ) collagen (soluble fraction from human placenta, ∼180 kDa); B ) bovine serum albumin (66.5 kDa); C ) lysozyme (from chicken, 14.5 kDa); D ) tryptophan (204 Da). A mixture containing 5 µg of each material was applied to a GPC 300 column and separated at 40 °C using 1 M TEAF, pH 3, in 2/1 water/THF as the mobile phase. The flow rate was 0.25 mL/ min; water was added postcolumn to reach a final flow rate of 1 mL/ min.

complete access to the entire volume of the pores. Thus, the SEC column is unable to separate the contaminants from the analytes with a MW below the analytical MW range of the column. This is evident from the larger than anticipated peaks for UMP (peak D, in Figure 2) and tryptophan (peak D, Figure 3) or from an additional peak at low MW in the analysis of heparin.8 Peaks for low-MW contaminants are not present or are drastically reduced when RP-HPLC-purified samples are analyzed. Analysis of a Stable-Isotope-Labeled Protein by SECCRIMS. When a molecule containing 13C is analyzed by CRIMS using SO2 as the reactant gas, the product of the reaction is 13CO2+, detected at m/z 45. Subtracting the natural 13C abundance (1.119% of the 12C) from the signal at m/z 45 results in only 13C-labeled molecules giving a signal. The result of this subtraction generates an “enrichment-only chromatogram” (EOC).22 The limit of detection in this EOC is a few nanograms of 13C within any given biopolymer. For our experiments, we used labeled ratGH spiked into horse serum and analyzed by SEC-CRIMS as described above for proteins. Figure 4 shows the results of this analysis: the solid line is the trace at m/z 44 with the predominant peak at a retention time corresponding to 65 kDa (the molecular mass of serum albumin); the dotted line shows the EOC where the only peak detected corresponds to the molecular mass of ratGH (∼21 kDa). The ability of CRIMS to detect isotopically labeled molecules should make SEC-CRIMS a particularly valuable tool for the study of the metabolic fate of biopolymers labeled with stable isotopes (e.g., 13C, 15N, and 2H). Analysis of Heparin by SEC-CRIMS. Heparin, a widely used anticoagulant, is a mixture of partially sulfated polysaccharides that may be isolated from a variety of mammalian tissues. The two monomeric units of heparin are hexuronic acid and glycosaminoglycan sugars connected by several linkages and with various degrees of sulfation.23 The molecular masses of unfractionated heparins average between 5 and 25 kDa, depending on source and method of determination. Several reports dealing with (22) Chace, D. H.; Abramson, F. P. Anal. Chem. 1989, 61, 2724-2730. (23) Lasker, S. E. Fed. Proc. 1977, 36, 92-97.

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Figure 4. SEC-CRIMS analysis of 13C-labeled ratGH spiked in serum: solid line, 12C trace at m/z 44; dotted line, 13C enrichment only chromatogram. Labeled ratGH was added to serum and analyzed by SEC-CRIMS as described for detection of proteins in the Experimental Section.

the difficult task of heparin structure characterization and its correlation with pharmacological activity have been published.24-26 Recent clinical studies show that low molecular weight heparin (LMWH) has comparable efficacy but less side effects and a more predictable anticoagulant response than unfractionated heparin.27 However, the challenges associated with the characterization of heparin have complicated the regulatory approval process of heparin derivatives despite their superior pharmacological properties.28 We have used SEC-CRIMS to examine both the MW distribution and the variation in the sulfur/carbon ratio (S/C) as a function of MW in different heparins. For this analysis, the reactant gas employed was NF3, which gives CF3+ (m/z 69) as a product of carbon and SF5+ (m/z 127) as a product of sulfur. Before measuring the MW distribution of the heparin, we calibrated the SEC column by multiple analysis of HPLC-purified oligodeoxynucleotides with different lengths (dT8, dT15, dT20, dT30, dT50). With the ammonium acetate mobile phase, the excellent linearity of the log MW vs retention time calibration curve (r2 ) 0.995) in the range considered (2-15 kDa) allowed a good estimate of the MW distribution of each heparin product. Figure 5 shows the chromatogram obtained from the analysis of four oligonucleotides (expected molecular masses: 15 148, 9064, 4501, and 2372 Da, respectively) superimposed on the chromatogram obtained from a pharmaceutical LMWH (Lovenox). The molecular mass distribution of carbon content for Lovenox (as obtained from SECCRIMS analysis) was 10% in the range 1-2 kDa, 81% in the range 2-8 kDa, and 9% above 8kDa; the mean molecular mass was 4500 Da. These parameters were calculated using the calibration curve obtained from the SEC-CRIMS analysis of MW standard compounds. These results obtained agree with those reported by the manufacturer, i.e., molecular mass distribution of 20% below 2 kDa, 68% in the range 2-8 kDa, and 15% above 8 kDa, with a mean molecular mass of 4500 Da. (24) Hemker, C. H.; Beguin, S.; Kakkar, V. V. Haemostasis 1996, 26, 117-126. (25) Chai, W.; Luo, J.; Lim, C. K.; Lawson, M. A. Anal. Chem. 1998, 70, 20602066. (26) Knobloch, J. E.; Shaklee, P. N. Anal. Biochem. 1997, 245, 231-241. (27) Wood, A. J. J. N. Engl. J. Med. 1997, 10, 688-698. (28) Linhardt, R. J.; Hileman, R. E. Gen. Pharmacol. 1995, 26, 443-451.

Figure 5. SEC-CRIMS analysis of heparin: solid line, low-MW heparin (Lovenox); dotted line, molecular mass reference oligodeoxynucleotides (dT50, dT30, dT15, and dT8, respectively: 15 148, 9064, 4501, and 2372 Da). Both Lovenox and the oligonucleotide mixture were separated with a GPC linear column and ammonium acetate, 100 mM, pH 6.5, with a flow rate of 0.25 mL/min. Shown is the CF3+ trace at m/z 69.

Besides the MW information, SEC-CRIMS offers the possibility of simultaneous detection of elements other than carbon, and this property was used to evaluate the relative sulfur content in different heparin products. The sulfation degree of the polysaccharidic chains in heparin has been related to heparin-associated thrombocytopenia, the most frequent drug-induced immune thrombocytopenia,29 so measuring the S/C ratio may be an important parameter for the characterization of different heparin products. The S/C ratio obtained from the CRIMS analysis of unfractionated heparin from porcine mucosa was normalized to the absolute S/C value obtained from elemental analysis of the same compound.23 The normalized S/C was set equal to 100 and used as the reference value. In Figure 6 we report the S/C for two LMWHs (i.e., Lovenox and LMWH from bovine intestine, Sigma) and two unfractionated heparins (i.e., heparin for injection from porcine intestine and Sigma heparin from bovine intestine); each bar represents the S/C in a different range of molecular mass. These results show that, in all preparations tested, the S/C ratio gradually decreases as polymer size increases (higher molecular mass). However, while comparable ratios were obtained at 4-8 and 8-16 kDa for LMWH and unfractionated heparin from bovine intestine (B and C), Lovenox shows a much lower ratio with respect to unfractionated heparin from porcine gut mucosa. This difference may be from different preparation procedures and could be diagnostic. (29) Greinacher, A.; Alban, S.; Dummel, V.; Franz, G.; Muller-Eckhardt, C. Thromb. Haemostasis 1995, 74, 886-892.

Figure 6. Normalized sulfur carbon ratio (S/C) in different molecular mass ranges for heparin preparations: A ) Lovenox; B ) low-MW heparin (from bovine intestine); C ) unfractionated heparin (from porcine intestine); D ) unfractionated heparin (from bovine intestine). SEC was performed as described in Figure 5. For carbon detection, the monitored species was CF3+ (m/z 69); for sulfur detection, SF5+ (m/z 127) was monitored.

CONCLUSIONS We describe the combination between a universal chromatographic technique (SEC) and a universal detection system (CRIMS) that enables the analysis of biopolymers without limitation in terms of molecular mass or chemical composition. For the analysis of biopolymers the two techniques are universal, compatible, and complementary, with each supplying a certain type of molecular information. If this system has a limitation, it lies in the chromatography. One may not find acceptable solvents to elute specific molecules from a given column. The ability of CRIMS to detect isotopically labeled molecules makes SEC-CRIMS a particularly valuable tool whenever the metabolic fate of stableisotope-labeled biopolymers is a major research interest, as it is in this laboratory. In the same way, CRIMS offers the possibility for analyzing the distribution of a specific element within a heterogeneous material. The analysis of heparin and LMWH provides an example of multiple elemental detection for the characterization of biopolymers. ACKNOWLEDGMENT Financial support for this research by the National Institute of Health (Grant R01-GM36143) is gratefully acknowledged. Preliminary results of this work were presented at the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 1-5, 1998.

Received for review October 6, 1998. Accepted April 9, 1999. AC981098R

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