MS for Screening

AC Research

Articles Anal. Chem. 1997, 69, 1683-1691

Immunoaffinity Ultrafiltration with Ion Spray HPLC/MS for Screening Small-Molecule Libraries Ray Wieboldt, Jerry Zweigenbaum, and Jack Henion*

Analytical Toxicology, New York State College of Veterinary Medicine, Cornell University, 927 Warren Drive, Ithaca, New York 14850

A solution-phase screening method for libraries of pharmaceutically relevant molecules is presented. The technique is applicable to screening combinatorial libraries of 20-30 closely related molecules. In this report, individual benzodiazepines are selected from a multicomponent library mixture by formation in solution of noncovalent immunoaffinity complexes with antibodies raised to therapeutically proven drugs such as nitrazepam, temazepam, or oxazepam. Captured compounds are separated from nonspecifically bound library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and analyzed by HPLC coupled with pneumatically assisted electrospray (ion spray) ionization mass spectrometric detection. Competition by the benzodiazepines for limited antibody binding sites is controlled by varying the stoichiometry of the complexation mixture. This procedure selects library components with the greatest affinity for a particular antibody. Specific capture of benzodiazepines is demonstrated by screening both a pool of structurally similar benzodiazepines and a more complex mixture of benzodiazepines with an additional set of unrelated compounds. Affinity ultrafiltration and electrospray mass spectrometry complement each other to enhance screening and identification of pooled drug candidates and potentially can be extended to other small-molecule combinatorial libraries and macromolecular receptor preparations. Applications of combinatorial library methods have the potential to significantly influence the pharmaceutical discovery process by facilitating the search for novel or functionally optimized drug * To whom correspondence should be addressed: Phone, (607)253-3971; FAX, (607)253-3973; E-mail, [email protected]. S0003-2700(96)01026-8 CCC: $14.00

© 1997 American Chemical Society

candidates.1,2 Increased use of combinatorial libraries provides an incentive to analytical chemists to explore faster, more efficient techniques for analysis and functional screening of these multicomponent mixtures. Libraries of structurally related compounds designed around a lead structure and surveyed by targeted selection procedures serve to efficiently identify molecules with optimal properties.3 Improvements in capabilities for parallel or simultaneous screening and analysis are expected to expedite the search for useful new drug molecules with combinatorial libraries.4 Strategies for screening combinatorial libraries include functional assays designed to test enzyme inhibition or receptorcoupled signaling processes, affinity-based binding assays, and many other high-throughput screening methods.5,6 Analysis of combinatorial synthesis products, either as spatially isolated pure compounds or as multicomponent mixtures demands effective and efficient analytical approaches. Mass spectrometry has been demonstrated to be useful for characterization of the composition and complexity of combinatorial libraries and has been applied in conjunction with analytical techniques such as HPLC and capillary electrophoresis for these purposes.7-12 The drug discovery process may be enhanced by choice of screening and analysis methods that complement each other. As examples, (1) Bevan, P.; Ryder, H.; Shaw, I. Trends Biotechnol. 1995, 13, 115-121. (2) Ecker, D. J.; Crooke, S. T. Bio/Technology 1995, 13, 351-360. (3) Wilson-Lingardo, L.; Davis, P. W.; Ecker, D. J.; He´bert, N.; Acevedo, O.; Sprankle, K.; Brennan, T.; Schwarcz, L.; Freier, S. M.; Wyatt, J. R. J. Med. Chem. 1996, 39, 2720-2726. (4) Bruce, J. E.; Anderson, G. A.; Chen, R.; Cheng, X.; Gale, D. C.; Hofstadler, S. A.; Schwartz, B. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1995, 9, 644-650. (5) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon, E. M. J. Med. Chem. 1994, 37, 1233-1251. (6) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gallop, M. A. J. Med. Chem. 1994, 37, 1385-1401. (7) Brummel, C. L.; Lee, I. N. W.; Zhou, Y.; Benkovic, S. J.; Winograd, N. Science 1994, 264, 399-401. (8) Brummel, C. L.; Vickerman, J. C.; Carr, S. A.; Hemling, M. E.; Roberts, G. D.; Johnson, W.; Weinstock, J.; Gaitanopoulos, D.; Benkovic, S. J.; Winograd, N. Anal. Chem. 1996, 68, 237-242.

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peptide library components preferentially phosphorylated by a kinase were readily identified by mass spectrometry.13 Affinity capillary electrophoresis/mass spectrometry was used to select combinatorially synthesized peptides that bind vancomycin.14 And finally, immunoaffinity techniques combined with mass spectrometry have been used successfully for epitope mapping and screening with peptide libraries.15,16 Affinity methods utilize the interaction between ligands in the compound library and the activities of biological surfaces such as enzymes, receptors, or antibodies.17,18 Compounds with affinities similar to a target receptor are also likely to share pharmacological properties. Thus, the affinity characteristics for a series of related molecules provide a delineation of the pharmacophore that is responsible for their biological activity. Several ingenious screening methods have been reported which link affinity selection with mass spectrometric analysis of selected library components.19-26 In these applications, mass spectrometry provides the capability to directly identify bound species in the affinity capture. In this study, we present a method for affinity selection of compound components in small-molecule libraries. A series of known and unknown benzodiazepines were chosen as test libraries to closely approximate benzodiazepine libraries prepared by combinatorial synthesis.27-31 In earlier work, on-line electrospray mass spectrometry coupled with immunoaffinity chromatography (9) Geysen, H. M.; Wagner, C. D.; Bodnar, W. M.; Markworth, C. J.; Parke, G. J.; Schoenen, F. J.; Wagner, D. S.; Kinder, D. S. Chem. Biol. 1996, 3, 679688. (10) Dunayevskiy, Y. M.; Vouros, P.; Carell, T.; Wintner, E. A.; Rebek, J., Jr., Anal. Chem. 1995, 67, 2906-2915. (11) Dunayevskiy, Y. M.; Vouros, P.; Wintner, E. A.; Shipps, G. W.; Carell, T.; Rebek, J., Jr., Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6152-6157. (12) Metzger, J. W.; Kempter, C.; Wiesmu ¨ ller, K.-H.; Jung, G. Anal. Biochem. 1994, 219, 261-277. (13) Till, J. H.; Annan, R. S.; Carr, S. A.; Miller, W. T. J. Biol. Chem. 1994, 269, 7423-7428. (14) Chu, Y.-H.; Kirby, D. P.; Karger, B. L. J. Am. Chem. Soc. 1995, 117, 54195420. (15) Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keogh, T. J. Am. Chem. Soc. 1995, 117, 3900-3906. (16) Zhao, Y.; Muir, T. W.; Kent, S. B. H.; Tischer, E.; Scardina, J. M.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4020-4024. (17) Kenan, D. J.; Tsai, D. E.; Keene, J. D. Trends Biochem. Sci. 1994, 19, 5764. (18) Zuckermann, R. N.; Kerr, J. M.; Siani, M. A.; Banville, S. C.; Santi, D. V. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4505-4509. (19) Hutchens, T. W.; Yip, T. Rapid Commun. Mass Spectrom. 1993, 7, 576580. (20) Nakanishi, T.; Okamoto, N.; Tanaka, K.; Shimizu, A. Biol. Mass Spectrom. 1994, 23, 230-233. (21) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-2613. (22) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (23) Huebner, V. D.; Kaur, S.; McGuire, L.; Tang, D.; Dollinger, G. In Proceedings of the 44th Conference on Mass Spectrometry and Allied Topics; Portland OR, May 12-16, 1996; p 748. (24) van Breemen, R. B.; Huang, C.-R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y.Z.; Venton, D. L. In Proceedings of the 44th Conference on Mass Spectrometry and Allied Topics; Portland OR, May 12-16, 1996; p 749. (25) van Breemen, R. B.; Huang, C.-R.; Nikolic, D.; Woodbury, C. P.; Zhao, Y.Z.; Venton, D. L. In Proceedings of the 44th Conference on Mass Spectrometry and Allied Topics; Portland OR, May 12-16, 1996; p 1031. (26) Kelly, M. A.; Liang, H.; Sytwu, I.-I.; Vlattas, I.; Lyons, N. L.; Bowen, B. R.; Wennogle, L. P. Biochemistry 1996, 35, 11747-11755. (27) DeWitt, S. H.; Kiely, J. S.; Stankovic, C. J.; Schroeder, M. C.; Reynolds Cody, D. M.; Pavia, M. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6909-6913. (28) DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res. 1996, 29, 114-122. (29) Bunin, B. A.; Ellman, J. A. J. Am. Chem. Soc. 1992, 114, 10997-10999. (30) Bunin, B. A.; Plunket, M. J.; Ellman, J. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4708-4712. (31) Ellman, J. A. Acc. Chem. Res. 1996, 29, 132-143.

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and HPLC was used to screen similar benzodiazepine libraries.32 Here, affinity screening of library mixtures is accomplished by solution-based complexation of library constituents with antibodies raised against representative benzodiazepines. This is followed with an ultrafiltration separation to isolate the affinity complexes from unbound molecules. The complexes are disrupted while residing on the filters, and the filtrates containing the selectively captured benzodiazepines are analyzed by LC/MS techniques. The method is potentially useful for selection of compounds with optimized affinity properties toward a target receptor from libraries composed of structurally similar molecules. The experiments described here provide a means not only to screen combinatorial libraries of small drug-like molecules but also to rapidly evaluate potential receptor candidates. EXPERIMENTAL SECTION Materials. Centrifugal ultrafiltration was performed with 400 µL Microcon microconcentrators (Amicon, Berverly MA). The filters have a nominal molecular weight cutoff of 50 000 and are used without preconditioning. Benzodiazepine reference standards were purchased from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI), and HPLC buffer components were obtained from Fisher Scientific (Pittsburgh, PA). Polyclonal sheep IgG’s for carbamazepine, flunitrazepam, nitrazepam, oxazepam, and temazepam were obtained as neat sera from Biodesign International (Kennebunk, ME). These anti-sera were used as supplied without further purification and had a nominal concentration of 1 mg/mL. In addition, polyclonal sheep anti-nitrazepam and purified sheep anti-flunitrazepam were also obtained from The Binding Site (San Diego, CA). Binding specificities toward components of the test benzodiazepine mixture between the latter antibodies and those from Biodesign were similar. Stock solutions of the antibodies were prepared as 100 µg/mL dilutions in assay buffer and stored at 4 °C for up to four weeks. Unless indicated otherwise, the assay buffer was 10 mM ammonium acetate adjusted to pH 5.0 with ammonium hydroxide. Benzodiazepine Test Solutions. A pooled mixture of the benzodiazepines was prepared in 10 mM ammonium acetate buffer at pH 5.0; the composition is summarized in Table 1. This solution is referred to in the text as the “BDZ test mix”. The concentration of each compound in the mixture was 10 µM and the solution additionally contained 0.035% (v/v) acetonitrile arising from concentrated stock solutions used in preparation of the mix (75% acetonitrile aided dissolution of the generally hydrophobic benzodiazepines). Libraries of unknown benzodiazepines similar to but not identical with the clinically used compounds of the test mix were provided as a gift from Hoffmann-La Roche (Nutley, NJ). The unknowns were supplied as mixtures of 20 compounds in powdered form, and solutions of the unknowns were prepared in a manner similar to that used for the benzodiazepine standards. Initial dissolution of the powdered mixture was performed in 75% acetonitrile to solubilize the sample followed by 1000-10 000-fold dilution into 10 mM ammonium acetate buffer. Immunoaffinity Extraction. Antibody and compound library mixtures are combined in different stoichiometric proportions in 100 µL of 10 mM ammonium acetate buffer at pH 5.0. The binding is accomplished by incubation of the mixture at room temperature for 1 h. The length of the incubation did not significantly influence (32) Nedved, M. L.; Habibi-Goudarzi, S.; Ganem, B.; Henion, J. D. Anal. Chem. 1996, 68, 4228-4236.

Table 1. Components of the Benzodiazepine Test Mixture (BDZ Test Mix) with Masses of the Protonated Molecule Ionsa

j p a e h l o c f k g i n d m b

Table 2. HPLC Gradient Profiles for Both Benzodiazepine and Non-Benzodiazepine Separationsa gradient I

gradient II

benzodiazepine

(M + H)+ ( m/z)

retention time (min)

time (min)

component A (%)

time (min)

component A (%)

bromazepam flurazepam carbamazepine oxazepam alprazolam lorazepam triazolam nitrazepam chlordiazepoxide clonazepam temazepam flunitrazepam lormetazepam diazepam prazepam medazepam

316 388 237 287 309 321 343 282 300 316 301 314 335 285 325 271

3.8 3.8 4.2 4.6 4.7 4.9 4.9 5.0 5.3 5.3 6.1 6.1 6.5 7.7 9.8 10.3

0 5 10 14 14.1

25 50 100 100 25

0 5 12 16 16.1

0 50 100 100 0

a

Retention times are given for the chromatogram appearing in Figure 1.

the results of the affinity extraction. Comparison of extractions performed immediately after mixing the components of the binding reaction with incubations done overnight at room temperature showed few differences. However, incubation at 37 °C overnight increased the overall amount of each captured component and slightly altered the selection specificity as well. The solutions are then transferred to Microcon 50 centrifugal filters and centrifuged at 10000g for 8 min or until the filter appears dry. A small amount (3 to 5 µL) of solution is retained in the filter membrane as a built-in dead volume for the filtration device. For this reason, three wash steps are required to reduce the retention of unbound analytes to a minimum. Each wash is accomplished by addition of 200 µL of pH 5.0 ammonium acetate buffer to the filter with subsequent centrifugation at 10000g for 8 min or until dry. The eluates from the wash steps containing the unbound components of the mixture are discarded. Finally, disruption of the affinity complex collected on the filter and elution of the captured benzodiazepines is performed by addition of 30 µL of 1% trifluoroacetic acid in water to the filter. The released components are eluted by centrifugation into a fresh collection vessel; the antibody remains trapped on the filter. The eluate from this final extraction step is evaporated to dryness by centrifugation in vacuo (Speed Vac, Savant) and reconstituted by sonication in 3 µL of a 1:1 acetonitrile/water solution immediately before LC/ MS analysis. Amicon recommends preconditioning if the residual glycerine in the ultrafiltration membranes interferes with the assay. Glycerine does not interfere in this application and is removed in the initial wash steps. An unknown component was extractable with 1% TFA from the membranes and, in the chromatography, eluted with 100% acetonitrile after all the benzodiazepines. This component displayed an abundant ion at m/z ) 488, and its chromatographic peak did not interfere with the benzodiazepine analytes. Non-specific binding of benzodiazepines in the test mixtures was not observed in control filtrations performed for each experiment. However, retention of eluates in the membrane dead volume did occur as explained above and was minimized with multiple wash steps. Elution with 1% TFA was sufficient to completely desorb the most strongly bound compounds from the

a Gradient I was used for the chromatograms in Figures 1-6 while gradient II was applied in Figure 7. The gradient was formed from two components: component A was 100% acetonitrile and buffer B was 20% acetonitrile in aqueous 10 mM ammonium acetate at pH 5.0. Three to five minutes equilibration at initial buffer composition is applied between injections.

antibodies. The hydrophobic benzodiazepines had no affinity for the hydrophilic cellulose acetate ultrafiltration membranes and were eluted along with more hydrophilic components. Elution with a 20% acetic acid solution produced identical results. Chromatography. HPLC separations were accomplished with a 1 mm × 100 mm Betasil C18 analytical column (Keystone Scientific, Bellefonte PA); a 0.22 µm frit and a 1 mm Keystone C18 drop-in guard column were placed upstream of the HPLC microcolumn. A flow rate of ∼50 µL/min through the injector and analytical microcolumn to the mass spectrometer interface was controlled with a precolumn split-flow method. The HPLC pump effluent was divided between the analytical column and a 4.6 mm × 17 mm C18 guard column which was protected with a 0.22 µm stainless steel frit. The short reversed-phase guard column served to balance the flow in the two sections so that the flow rate to the interface was constant during each step of the gradient. The pump pressure varied from 750 to 250 psi as the gradient progressed from 40 to 100% acetonitrile. Gradient elution was performed with a Waters 600MS multisolvent delivery system at flow rates of 0.6-1 mL/min to the splitter using electronic pulse dampening (Waters SILK method). A rapid transition from low to high organic buffer composition was employed for the benzodiazepine separations shown in Figures 1-6; an extended gradient was used in Figure 7 for separation of the more diverse library mixture. The two gradient programs and the HPLC buffer compositions are described in Table 2. Sample introduction was performed by filling the 5 µL injection loop of a Rheodyne 7125 injector with 10 mM ammonium acetate followed by 1-3 µL of an analyte solution. This “sandwich” technique served to concentrate the benzodiazepines at the inlet of the microcolumn and thus improve the resolution of the eluted chromatographic components. Mass Spectrometry. The HPLC effluent was analyzed with an API 300 tandem mass spectrometer (PE-Sciex, Concord, ON, Canada) equipped with a pneumatically assisted electrospray (ion spray) interface.33 Full-scan data collection was performed to sample the inclusive range of masses expected for the BDZ test mix as well as unknown samples. A range of 200-500 Da scanned in 0.2 Da steps with a sampling dwell time of 1.0 ms was typically used, but the mass range was also broadened when compounds with higher or lower molecular weights were expected. The results reported here were acquired exclusively by observation (33) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 26422646.


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of positive ions, but negative ion detection was also monitored and provided some additional qualitative analytical information about the compounds tested. The benzodiazepines in the test mixture exhibited different responses to variation of the orifice and electrospray voltages; some benzodiazepines were especially labile to fragmentation in the nozzle-skimmer region of the API 300 ion spray interface. For this reason, a lower nozzle-skimmer voltage (typically 20-35 V) was employed as a compromise to produce optimal survey conditions for the LC/MS chromatograms. These conditions provided a reasonable signal-to-noise ratio in the LC/MS chromatogram at sample levels down to 0.1 ng but typically injected samples containing 1-5 ng, of each component were used. RESULTS AND DISCUSSION LC/MS Assay Development. The initial goal in development of the screening assay was to establish chromatographic conditions that would maximize chances of separating and detecting the components of the library mixture. The numerical size and chemical similarity of library constituents influence the selection of analytical conditions. We chose to optimize chromatographic conditions for groups of ∼20 compounds to initially test the capabilities of the affinity ultrafiltration. Also, this was the size of the unknown library mixtures provided by Hoffmann-La Roche. The conditions accommodate separation of compounds with a broad range of chromatographic characteristics. Also, it was anticipated that the affinity extraction would reduce the number of expected compounds in the ultrafiltration products so that resolution could be traded for speed of analysis. This compromise is acceptable when a mass spectrometer is the chromatographic detector because, in many cases, the spectrometer provides the ability to identify multiple coeluting components. For these reasons, the gradient elution protocol described in the Experimental section was selected. The general structure for 1,4benzodiazepines is given in Chart 1. The substituent groups are variable, but the molecules all contain secondary or tertiary amines which are amenable to positive ion detection with electrospray MS. Sensitivity of the full-scan LC/MS data was also a concern since the centrifugal ultrafiltration devices limit the amount of antibody used in an assay to ∼100 µg per trial. The upper limit for the total number of captured molecules in an assay is ∼10-9 mol apportioned among all species captured. Choice of a 1 mm reversed-phase HPLC microcolumn provided the required sensitivity. Figure 1 shows an example of a full-scan (m/z 200-500 Da) LC/MS total ion current (TIC) chromatogram for the BDZ test mix corresponding to an injection of 10 pmol of each of the mix components. Even though some of the compounds coelute, each benzodiazepine in the test mix can be uniquely assigned to a peak in the chromatogram on the basis of mass or isotopic ratio patterns of halogenated species. The retention times for each of the 16 components in the BDZ test mixture are given in Table 1. Using the analytical conditions described above, ∼75% of the components of the BDZ test mix were found in a manual singlepass examination of the ion current profile shown in Figure 1. The remaining components were located by using knowledge of the expected masses to create individual extracted ion profiles. Masses of impurities and degradation peaks were also identified. More sophisticated data reduction procedures are required to identify all components in the mixture for routine interpretation of LC/MS data of an unknown library. For example, procedures 1686


Figure 1. LC/MS TIC chromatogram of the BDZ test mix using the protocol given in the Experimental Section. Assignment of the peaks is given in Table 1. The gradient program for the chromatographic separation is given in Table 2. The effluent from the RP-HPLC microcolumn was connected directly to the ion spray interface of the mass spectrometer. Approximately equal amounts (10 pmol) of each component were present in the 1 µL of injected sample, and the differences in peak intensity reflect the variable mass spectrometer response of each benzodiazepine under ion spray conditions.

Chart 1. General 1,4-Benzodiazepine Structure

that use mass spectrometric data to locate coeluting peaks and discriminate small signals from the chemical noise background will enhance the efficiency of mass spectrometric characterization of complex mixtures.34,35 Qualitative Results of Immunoaffinity Ultrafiltration. A demonstration of the immunoaffinity selection of a subset of the benzodiazepines in the BDZ test mix is given in Figure 2. Binding solutions consisting of sheep polyclonal antisera for flunitrazepam and nitrazepam in the presence of an ∼10-fold molar excess of the benzodiazepines in the BDZ test mix were processed using the ultrafiltration protocol. The concentration of the benzodiazepines was 1 µM while the nominal concentration of the antibodies was 100 nM in the complexation solution. Figure 2c displays a full-scan reference TIC chromatogram of the 16-component BDZ test mix. The flow rate through the analytical column was faster in these trials because of a small change in the precolumn split ratio. The compounds therefore elute earlier when compared with the retention times shown in Figure 1; the order of elution is identical. Figure 2a shows the LC/MS trace of the products from extraction with the anti-nitrazepam antibody. Four components were preferentially captured: alprazolam (3.9 min), nitrazepam (4.1 min), diazepam (6.7 min), and prazepam (8.8 min). The extraction with anti-flunitrazepam shown in Figure 2b captured (34) Windig, W.; Phalp, J. M.; Payne, A. W. Anal. Chem. 1996, 68, 3602-3606. (35) Abbassi, B. E.; Mestdagh, H.; Rolando, C. Int. J. Mass Spectrom. Ion Processes 1995, 141, 171-186.

Figure 2. Immunoaffinity extraction with anti-nitrazepam and antiflunitrazepam. Approximately 10 pmol of antibody was incubated with the 100 pmol each of the benzodiazepines from the BDZ test mix in a total volume of 100 µL of 10 mM ammonium acetate buffer at pH 5.0 and room temperature. Unbound benzodiazepines were separated from the immunoaffinity complex by ultrafiltration. The captured products from the anti-nitrazepam and anti-flunitrazepam assays are shown in the respective LC/MS traces a and b. The lower trace c is an LC/MS reference TIC chromatogram of a 1 µL injection of the 10 µM BDZ test mix. Chromatographic conditions for the LC/MS separation are the same as in Figure 1. The ion current of the largest displayed signal in each trace is given in units of (counts per s)/106 (Mcs).

two of the same components, diazepam and prazepam, and three additional benzodiazepines. These include triazolam (4.1 min), flunitrazepam (5.2 min), and lormetazepam (5.6 min). The identities of the captured components are determined both by comparison with the reference chromatogram of the BDZ test mix in Figure 2c and by the masses of the components associated with each chromatographic peak. LC/MS analysis provides unambiguous identification of closely eluting components such as alprazolam and nitrazepam and facilitates characterization of complex mixtures that may result from combinatorial synthesis methods. The results shown in Figure 2 illustrate important features about immunoaffinity extraction performed with ultrafiltration on microcentrifuge concentrators. First, capture with 10 pmol of the antibodies provides a sufficient yield of product molecules for fullscan LC/MS detection. Full-scan mass spectrometric detection is not as sensitive as chemically amplified immunoassays such as ELISA or EMIT, which are typically employed for high-throughput library screening. However, this experiment shows that the signal level of captured benzodiazepines using moderate amounts of antibodies for the extraction is high enough to take advantage of the additional qualitative information provided by LC/MS detection. Polyclonal antibodies raised against nitrazepam and flunitrazepam employed in the ultrafiltration assay as affinity reagents in Figure 2 produce distinctly different patterns of affinity. The antibodies not only capture their nominal antigenic compound but also show cross-reactivity with other benzodiazepines. In particular, diazepam and prazepam exhibited strong affinity for each of the tested benzodiazepine antibodies. They were also found to bind to nonbenzodiazepine antibodies (data not shown) but with weaker affinity. This observation raises the possibility that the unpurified polyclonal antibody preparations used here contain additional sites with affinity for these benzodiazepines unrelated to those associated with their nominal antigenic determinants. The

Figure 3. Extraction recovery and antibody concentration. The amount of each benzodiazepine extracted in the affinity ultrafiltration assay is directly related to the quantity of anti-flunitrazepam antibody. Compositions of the extraction media were identical to those used in Figure 2b except that the antibody level was varied as explained in the text. Trace a is a repeat of the anti-flunitrazepam extraction in Figure 2b. Relative level of antibody in each of the other extractions: (a) 100, (b) 50, (c) 15, (d) 5, and (e) 0%. Trace f is a reference chromatogram of the BDZ test mix, and all traces are plotted on the same vertical scale.

extractions in Figure 2 show that the antibodies reject both the late-eluting hydrophobic medazepam and the early-eluting hydrophilic bromazepam and flurazepam. Therefore, the strong affinity to these antibodies shown by diazepam and prazepam cannot be explained solely by their higher hydrophobicity and nonspecific complexation with the antibody. Relation between Antibody Concentration and Extraction Efficiency. Figure 3 shows the effects of varying the amount of antibody in the extraction (in this case anti-flunitrazepam was used) while the concentration of the benzodiazepines remains constant. In each complexation solution, the concentration of each of the benzodiazepines from the test mixture was 1 µM and the nominal concentration of the antibodies ranged from 5 to 100 nM. The stoichiometric ratios of each benzodiazepine to the antibody are respectively 10:1, 20:1, 65:1, and 200:1 in Figure 3a-d, respectively. No antibody was included in the extraction corresponding to the trace in Figure 3e, which therefore serves as a control reference for the ultrafiltration and wash steps. Figure 3f is an LC/MS chromatogram of the BDZ test mix. The signal level of the extracted components is correlated to the amount of antibody present. Larger amounts of antibody yield LC/MS chromatograms with improved signal-to-noise ratios. However, a practical limit to the amount of antibody usable with the microcentrifuge concentrators is reached at ∼100 µg of protein per filter. Loading levels of antibody above 100 µg impede flow through the filter, making it impossible to successfully wash off nonspecifically bound analytes. Effect of Competition for Antibody Binding Sites between Analytes. An excess of one ligand in the affinity assay appears to effectively compete for binding sites on the antibody and excludes capture of other library components. In the ultrafiltration extractions shown in Figure 4, the amount of antibody is again the limiting reagent with a 5-fold molar excess of each benzodiazepine from the BDZ test mix relative to the antibody. The concentration of the benzodiazepines was 1 µM while that of the antibodies was 200 nM. Figure 4a is the LC/MS TIC profile of the test mix and is included as a reference. Incubation of the Analytical Chemistry, Vol. 69, No. 9, May 1, 1997

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Figure 4. Effect of excess temazepam in affinity extractions of the BDZ test mix with sheep anti-temazepam. Temazepam competes with other captured components and provides a measure of relative binding affinities. Trace a is an LC/MS reference chromatogram of the BDZ test mix while b-d are results of ultrafiltration extractions with the antibody. The extractions were performed with a fixed 1:5 molar ratio of antibody to each library component. Trace b shows products captured by the antibody before addition of any excess temazepam. Traces c and d show the competitive effects of a 2- and 10-fold excess addition of temazepam to the binding reaction. Instrumental conditions are identical to those used in Figure 1. The ion current for each diazepam peak measured at the peak maximum for comparison are labeled in traces b-d.

polyclonal sheep anti-temazepam antibody with the BDZ test mix produces the extracted compounds in the LC/MS TIC shown in Figure 4b. The anti-temazepam antibody is less discriminating than the anti-nitrazepam and anti-flunitrazepam antibodies used for extractions in Figures 2 and 3. Under the conditions used here, anti-temazepam captures more components from the mix. The benzodiazepines appearing in Figure 4b captured by antitemazepam in the ultrafiltration assay include alprazolam, triazolam, temazepam, lormetazepam, diazepam, prazepam, and medazepam. When the concentration of temazepam in the complexation mixture is doubled while keeping the concentrations of both the antibody and the other benzodiazepines fixed, medazepam is effectively competed off the antibody (Figure 4c). Effects on the binding of the other captured components are small. In Figure 4d, the concentration of temazepam in the assay is increased to a 10-fold excess relative to the others (the final concentration of temazepam was 10 µM). At this higher ratio, the extracted amounts of the other benzodiazepines found in Figure 4b are reduced. Characterization of Library Compounds with Their Apparent Affinites to Antibodies As Determined by Ultrafiltration LC/MS. The experiments shown in Figures 2-4 provide an illustration of the information available from immunoaffinity screening of libraries followed by LC/MS analysis of the ultrafiltrate. First, cross-reactivity between the components of the library, appearing as a unique multicomponent capture pattern for each antibody, suggests that those components share a common antigenic determinacy. Cross-reactivity thus provides an affinity fingerprint for each antibody and establishes a basis for a relationship between the library compounds. These patterns may also correlate with other properties or pharmacological functions of the compounds. Second, competition for antibody binding sites between components of the mixture provides a means to assign the relative 1688


affinity of captured compounds for the antibody. Concentrations of the benzodiazepines in the test mixture are in the micromolar range, which is much higher than the typical nanomolar dissociation constants expected for the benzodiazepine-antibody interaction. This disparity prevents assignment of absolute affinity constants from the results of the ultrafiltration. The apparent relative affinities determined here are dependent on experimental conditions which include interaction of multiple classes of antibody binding sites and competition by the strong and weak ligands in the complexation mixture. In Figure 4, medazepam is the first component eliminated when the concentration of temazepam is increased. Medazepam appears to have the lowest affinity for the antibody of all those shown to be captured (of course, the mixture components that do not appear in Figure 4b have even lower affinity). Thus, competition experiments provide a method for qualitatively ranking affinities of the different components in the library. Derivation of quantitative information about the interaction between an antibody and a library of compounds is dependent on accurate measurement of library component concentrations and prior characterization of all binding sites on the antibody affinity reagent. Affinity constants are not directly available from the experiment in Figure 4 because of the presence of competing benzodiazepines besides temazepam. There is therefore no simple expression for the IC50 as a function of temazepam concentration; the expression must account for all competing equilibria. The possible presence of multiple high- and low-affinity binding sites associated with the antibody preparation also complicates the analysis. The polyclonal antibodies used in the experiments here are expected to have some heterogeneity in their binding affinities for the antigens used in their production. For these reasons, the practical information from competition experiments with libraries of pooled compounds is limited to classification of cross-reactivities and relative binding affinities. Isolation of Affinity Products from an Unknown Benzodiazepine Mixture. The techniques illustrated above form the basis for practical evaluation and screening of small-molecule combinatorial libraries. Twenty-component libraries of benzodiazepines were provided as dry powdered mixtures as a gift from Hoffmann-La Roche. The identities of the components of the mixtures were not disclosed prior to the analysis in order to simulate unknown combinatorial libraries as closely as possible. The structurally similar benzodiazepines are analogous to those in combinatorial libraries that have been produced by spatially discrete combinatorial synthesis.27,31 Dilutions of the unknown pools were made in 10 mM ammonium acetate at pH 5.0. Initial dissolution of the generally hydrophobic molecules was performed in 75% acetonitrile with 25% of either 5% acetic acid or 5% ammonium hydroxide as needed. From these stock solutions it was possible to solubilize the solids with the aqueous buffer by diluting them into it 1000-10 000 times. For the extractions presented in Figure 5, the concentrations of antibodies and benzodiazepines were set to be approximately equimolar (the nominal concentration of each component in the 100 µL complexation solution was 5 µM) to maximize the capture by the antibody and to minimize effects of competition between the library constituents. The first point to be made with this experiment is that mass spectrometric detection provides both an indication of the complexity of the library and molecular weights and qualitative

Figure 5. Selective capture of components of a library of benzodiazepine unknowns by three different antibodies. Traces a-c are full-scan (200-400 Da) LC/MS ion current chromatograms of the products of affinity ultrafiltration extractions performed with antibodies to flunitrazepam, oxazepam, and temazepam, respectively. The concentration of the library components and the antibody in each complexation solution was ∼5 µM. Masses of prominent protonated molecule ions are labeled and are determined by comparison between selected ion current profiles from chromatograms of the extractions and of the unknown mix. The positive ion TIC LC/MS trace of the 20-component mixture of unknown benzodiazepines used as the reference is shown in (d). The instrumental and display parameters are equivalent to those described for the experiments in the previous figures.

information about unknown library constituents.11,12 Retention times, positive and negative ion mass spectra, ion intensities, and isotope ratios available from the LC/MS data provide qualitative and semiquantitative elucidation of the library composition. This information can be used to verify products of a combinatorial synthesis and identify reaction byproducts and impurities. The components found in the reference chromatogram of the unknown mixture (shown in Figure 5d) are not completely resolved by the rapid gradient elution used in these experiments. However, examination of the full-scan mass spectra associated with each peak unmasks components that would be overlooked in the TIC profile and with conventional HPLC/UV detection. For example, the spectrum of the peak eluting at 4.1 min in Figure 5d was found to consist of three ions with m/z 255, 312, and 328. This peak therefore contains at least three unresolved components. The second point to be made with the experiment shown in Figure 5 is that affinity patterns can be used as a screening procedure to classify library constituents. Parts a-c of Figure 5 display the LC/MS total ion current chromatograms of the products from separate affinity ultrafiltration assays by antiflunitrazepam, anti-oxazepam, and anti-temazepam. Each of the antibodies selects a different set of components from the library pool. The anti-flunitrazepam antibody selects ions with m/z 294, 266, 315, and 285 (Figure 5a); anti-oxazepam selects m/z 255, 328, 273, and 315 (Figure 5b); and anti-temazepam selects m/z 312, 315, 285, 266, 294, and 347 (Figure 5c) from the library of unknown benzodiazepines. The different patterns of affinity selection suggest that there are structural relationships between some of the compounds which could be used to classify the library constituents into subgroups. Structural similarity implies that the captured molecules may also share functional properties. Also, the affinities of a set of different antibodies for a test library could be used to select and characterize antibodies with similar properties. Other macromolecular receptors used in place of the antibodies in the ultrafiltration screening protocol are likely to

Figure 6. Effect of excess temazepam in affinity extractions of the unknown benzodiazepine library. Addition of free temazepam to the affinity extraction assay competes with binding by components of the unknown benzodiazepine library. This experiment parallels the one described in Figure 4 with a library of benzodiazepine unknowns substituted for the BDZ test mix. Trace e is the LC/MS TIC chromatogram of sample from the unknown benzodiazepine library using the same instrumental conditions as in Figure 2. Trace a shows extraction products of the affinity ultrafiltration assay using antitemazepam antibody on the unknown benzodiazepine library in the absence of any added temazepam. Temazepam at concentrations of 2, 10, and 20 µM was spiked into the complexation assays used to produce traces b-d. The concentration of unknown benzodiazepines were ∼5 µM while the anti-temazepam antibody was also 5 µM. Traces a-c are plotted on the same vertical scale, and approximate comparison of like-benzodiazepine signal intensities between traces is possible. The ion current at peak maximum is labeled in each trace for the m/z 315 peak. Capture of the unknowns is reduced as the temazepam concentration increases.

generate unique affinity capture patterns with appropriate libraries. Thus, affinity ultrafiltration with mass spectrometric detection may provide a general method useful in searches for receptor agonists and antagonists. Finally, effects of interference between components of a mixture are recognized as a potential problem for other assays used for high-throughput screening of compound mixtures.2,3,36-38 Some library components may suppress or even potentiate the affinity response of other compounds. This interference must be considered in interpretation of the capture results. Adverse interactions increase when affinity-based screening methods are applied to numerically larger libraries, especially if the libraries contain similar compounds and when there are uncertainties in concentrations of the library components. The LC/MS chromatograms of extraction products shown in Figure 5 were performed in the presence of a 1:1 proportion of antibody to library components in order to both ensure capture of representative unknown benzodiazepines and to minimize effects of intercomponent interference. Competition for antibody binding sites between library components becomes more significant when the antibody is the limiting reagent. Comparison of Affinity Properties of Temazepam and Unknown Benzodiazepines. The effects of competition between library constituents can be used as explained above to determine relative affinities of library constituents. In Figure 6, the library (36) Konings, D. A. M.; Wyatt, J. R.; Ecker, D. J.; Freier, D. J. J. Med. Chem. 1996, 39, 2710-2719. (37) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J.; Steele, J. Tetrahedron 1995, 51, 8135-8173. (38) Felder, E. R. Chimia 1994, 48, 531-541.


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of unknown benzodiazepines was screened with anti-temazepam antibody in the affinity ultrafiltration assay in the presence of different concentrations of added temazepam. No temazepam is found in the unknown library mixture as shown in the LC/MS reference chromatogram in Figure 6e. The products of affinity ultrafiltration with the anti-temazepam antibody are displayed in Figure 6a. The concentration of added temazepam is incrementally increased in each of the next traces, and the relative amount of captured unknowns decreases (Figure 6b-d). The components with ions at m/z 312 and 315 are the last to be eliminated as the competition stringency increases. Therefore, these ions are likely to correspond to components in the unknown library having affinity properties with the greatest similarity to those of temazepam. They are also the most likely library components to share temazepam’s molecular and pharmacological properties. Effect of Library Complexity on Affinity Ultrafiltration Extraction. As discussed above, library mixtures may exhibit considerable chemical diversity with variation of the relative concentrations of each component. Library compounds may be structurally similar, as are the benzodiazepine libraries, or represent a more diverse range of molecular structures. These library characteristics may influence results of the affinity extraction. Affinity ultrafiltration screening with the anti-flunitrazepam antibody on a library consisting of both benzodiazepines and nonbenzodiazepines was performed to evaluate the effects produced by the presence of other compounds on the extraction. A new library mixture was prepared in which the nonbenzodiazepine compounds were included in a 5-fold molar excess relative to the benzodiazepines. The nonbenzodiazepines in the library are as follows: 17-R-methyltestosterone, 19-nortestosterone, 6-hydroxydopamine, isoproterenol, oxytetracyline, naproxen, propranolol, kynurenine, carbofuran, neostigmine bromide, clofibric acid (which does not appear in the positive ion LC/MS chromatogram), methotrexate, chlorpromazine, and epinephrine. The included benzodiazepines were as follows: diazepam, nitrazepam, flunitrazepam, alprazolam, triazolam, carbamazepine, medazepam, and prazepam. A list of the nonbenzodiazepine compounds with masses of their characteristic ions and retention times is given in Table 3. The molar ratio of the benzodiazepine components to the anti-flunitrazepam antibody in this test was ∼2:1 (the concentration of the nonbenzodiazepine components was 5 µM, the benzodiazepines were 1 µM, and the antibody was nominally 500 nM). The results of the affinity ultrafiltration assay performed with the more complex library are given in Figure 7. The LC/MS total ion current profile (scan range of 150-500 Da) for the mixture is displayed in Figure 7d. The gradient program used for the chromatographic separation is described in Table 2 and was chosen to accommodate separation of the early-eluting nonbenzodiazepine library components. For this reason, retention times are not directly comparable to those of the previous figures. However, the order of elution of the benzodiazepines is the same as that in Figure 1. Detection of antibody-specific capture of benzodiazepines shown in Figure 7 is enhanced by selecting narrower mass ranges for the selected ion current profiles. In Figure 7a, the full LC/ MS scan range (150-500 Da as compared to the 200-400 Da range in Figure 1) displays higher chemical noise in the TIC profile, and reduces the ability to observe the captured benzodiazepine signals in the TIC. Parts b and c of Figure 7 display 1690 Analytical Chemistry, Vol. 69, No. 9, May 1, 1997

Table 3. Components of the Mixed Library of Benzodiazepines and Non-Benzodiazepines Used for the Affinity Ultrafiltration Extraction in Figure 7a compound

(M + H)+ (m/z)

retention time (min)

kynurenine epinephrine 6-hydroxydopamine methotrexate isoproterenol neostigmine bromide oxytetracyline clofibric acid carbamazepine propranolol alprazolam nitrazepam triazolam naproxen carbofuran flunitrazepam 19-nortestosterone chlorpromazine diazepam 17-R-methyltestosterone prazepam medazepam

209 184 170 455 212 223 461 213b 237 260 309 282 343 231 222 314 275 319 285 303 325 271

1.2 1.4 1.5 1.9 1.9 2.5 3.9 5.2 5.2 6.6 6.7 6.9 7.1 8.0 8.5 8.6 8.9 9.8 9.9 10.2 12.0 12.6

a Retention times are reported for the extended gradient conditions described in the text. b For (M - H)-.

Figure 7. Effect of non-benzodiazepine compounds on affinity extractions with an anti-benzodiazepine antibody. The complexity of the library mixture affects the ability of an antibody to isolate specific ligands. The conditions of the assay are given in the text. In the presence of a 5-fold molar excess of the nonbenzodiazepine molecules relative to the level of benzodiazepines, sheep anti-flunitrazepam shows a pattern of affinity similar to that seen with the BDZ test mix in Figure 2. Trace d is the full-scan (150-500 amu) LC/MS chromatogram of the diverse small library and (a) shows the products of affinity ultrafiltration displayed with the same mass range. Trace b is a 200-500 Da extracted ion current profile from the same data as in (a). The limited range corresponds to that used in Figure 2 and was chosen to reduce the chemical noise of the background. Trace c further restricts the mass extraction range to 280-350 amu to emphasize the peaks of the captured benzodiazepines. The restricted ranges exclude some of the masses of mixture components but serve to enhance detection of the signals that do fall in the reduced range.

extracted ion current chromatograms with ranges of 200-500 and 280-350 Da, respectively. The narrower extracted ion current profile ranges reduce chemical noise in the ion current chromatograms and improve detection of the captured benzodiazepines. The specificity of the extraction with anti-flunitrazepam in Figure 7 is similar to that observed in Figure 2. Triazolam,

flunitrazepam, diazepam, prazepam, and medazepam are all captured in the presence of the other compounds. With the exception of medazepam, other benzodiazepines are rejected as before. A possible explanation for the capture of medazepam in this experiment and the absence of binding in Figure 2b is that the antibody-to-benzodiazepine ratio was greater in the extractions performed for the data shown in Figure 7. The presence of more antibody in the complexation medium causes less competition for available antibody binding sites between the benzodiazepine components of the mixture. In summary, when complex libraries are screened with an affinity-based technique, interpretation of the capture results should be carefully qualified with regard to the conditions of the binding reaction. The LC/MS technique facilitates interpretation of these results by providing more specific information than is available from some other common analytical methods such as HPLC with optical detection. In addition to the benzodiazepines, the LC/MS chromatogram of the extraction products from the assay shown in Figure 7b contain a weak signal for propranolol eluting at 6.8 min. None of the other nonbenzodiazepine molecules are captured by the antiflunitrazepam antibody. This raises the possibility that there is some configurational similarity between propranolol and the epitope which defines the specificity of the anti-flunitrazepam antibody. However, the interaction between the antibody and propranolol does not necessarily involve binding at the flunitrazepam recognition site. The polyclonal antibody preparation may have binding sites unrelated to those specific for flunitrazepam. CONCLUSIONS Immunoaffinity selection with ultrafiltration provides a method for elucidation of molecular recognition properties for mixtures of related compounds and antibodies. This method could be extended to include other types of receptors and the products of combinatorial library synthesis. Ultrafiltration membranes are

currently available with molecular weight cutoffs ranging from 500 to 500 000, daltons so it is possible to choose experimental configurations appropriate for many macromolecules and biological receptors. Auspicious choice of the biomolecule receptor used for the affinity capture could provide the means to explore libraries of molecules for new therapeutic applications and lead drug candidates. Further, compound screening in free solution has an advantage over libraries synthesized on beads because the assay is uncomplicated by possible interferences from the solid support. The choice of LC/MS as an analytical tool for combinatorial library screening is appropriate both for libraries generated by pooling components and for those produced by multiproduct combinatorial synthesis. Even with libraries created by pooling known molecules, the existence of degradation products, unknown impurities, or unanticipated reactions in the mixture alter the library composition unpredictably. LC/MS provides insight into the diversity of a library by effective identification of its components. Also, analytical information from LC/MS/MS experiments should be helpful for further structural elucidation of the products of a selection assay. Results from multiple library screenings such as this may be useful for isolating sets of compounds with similar properties and also for defining the epitope associated with an antibody. ACKNOWLEDGMENT We thank PE-Sciex for the API 300 mass spectrometer, Waters for use of their 600 MS HPLC gradient controller, and HoffmannLa Roche for the gift of pooled benzodiazepine analogs employed here as a combinatorial library. Received for review October 7, 1996. Accepted February 13, 1997.X AC9610265 X

Abstract published in Advance ACS Abstracts, April 1, 1997.


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