AC Research
Anal. Chem. 1998, 70, 3339-3347
Accelerated Articles
Simultaneous On-Line Characterization of Small Organic Molecules Derived from Combinatorial Libraries for Identity, Quantity, and Purity by Reversed-Phase HPLC with Chemiluminescent Nitrogen, UV, and Mass Spectrometric Detection Eric W. Taylor,* Mark G. Qian, and Gavin D. Dollinger
Small Molecule Drug Discovery, Chiron Technologies, Chiron Corporation, 4560 Horton Street, Emeryville, California 94608
In the ongoing quest for ever more rapid techniques to quantify small organic molecules, we have evaluated a chemiluminescent nitrogen detector (CLND) as a universal quantitation tool for nitrogen-containing molecules. By flow injection analysis (FIA) and in conjunction with reversed-phase (RP) chromatography using gradient elution, the CLND produced a linear response from 25 to 6400 pmol of nitrogen that was equivalent for a set of chemically and structurally diverse compounds. Over the entire linear range, the absolute response exhibited an average error of approximately (10% among the compounds. In addition, the response was independent of mobile-phase composition. These results demonstrate that the CLND can be used with FIA or on-line with RPHPLC for rapid and accurate quantitation down to lowpicomole levels, using a single external standard. We also used the CLND in combination with a UV detector and a mass spectrometer (MS) during RP-HPLC (LC/UV/N/ MS) to characterize several samples containing small organic compounds synthesized by both standard and combinatorial methods. The identity, quantity, and purity of compounds of interest were assessed from a single HPLC injection of each sample. These results show this technique (LC/UV/N/MS) to be a widely applicable, generic method for the pharmaceutical industry to rapidly identify, quantify, and determine the purity of small organic compounds. S0003-2700(98)00402-8 CCC: $15.00 Published on Web 07/18/1998
© 1998 American Chemical Society
Recent advances within the current paradigm for smallmolecule drug discovery have made the process increasingly efficient. Combinatorial chemistry has significantly increased the rate of finding pharmacophores by means of the synthesis of smallmolecule libraries composed of mixtures of compounds and a “deconvolution” strategy to identify the pharmacologically active compounds in the mixtures.1-4 Developments in computational chemistry (structure-based design) and solid-phase synthetic chemistries have made this possible.5,6 In addition, libraries containing >105 compounds can now be synthesized in a matter of days with robotic and semiautomated tools that have been designed to speed synthesis processes.7 The production rate of new libraries and the structural diversity within a collection of libraries are increasing dramatically, creating a rich source of small organic molecules to screen for new drug candidates. * Corresponding author: (e-mail) eric•
[email protected]; (fax) (510) 9234115. (1) Moos, W. H.; Green, G. D.; Pavia, M. R.. Annu. Rep. Med. Chem. 1993, 28, 315-24. (2) Zuckermann, R. N.; Martin, E. J.; Spellmeyer, D. C.; Stauber, G. B.; Shoemaker, K. R.; Kerr, J. M.; Figliozzi, G. M.; Goff, D. A.; Siani, M. A.; Simon, R. J.; Banville, S. C.; Brown, E. G.; Wang, L.; Richter, L. S.; Moos; W. H. J. Med. Chem. 1994, 37, 2678-85. (3) Plunkett, M. J.; Ellman, J. A. Sci. Am. 1997, 276, 68-73. (4) Hogan, J. C., Jr. Nat. Biotechnol. 1997, 15, 328-30. (5) Martin,. E. J.; Blaney, J. M.; Siani, M. A.; Spellmeyer, D. C.; Wong, A. K.; Moos, W. H. J. Med. Chem. 1995, 38, 1431-6. (6) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B.; Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646-7. (7) Zuckermann, R. N.; Kerr, J. M.; Siani, M. A.; Banville, S. C. Int. J. Pept. Protein Res. 1992, 40, 497-506.
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Simultaneous with the developments in synthesis have been advances in biomedical research and robotics, leading to highthroughput screening. Target-based screens have been developed, miniaturized, and automated using robotics.8 The ability to screen thousands of samples per day has created a prodigious appetite for new molecules to feed the screens. All of these increased efficiencies have led to a greater demand for analytical characterization of samples, ranging from chemistry validation, mixture characterization, and active compound characterization to structure-activity relationship (SAR) studies of a lead compound or series. Following the synthesis of a small organic molecule, an important early step in the characterization of the compound is to obtain an accurate concentration of the compound in solution in order to generate an accurate pharmacological activity measurement (potency). The most commonly used method for determining the concentration of a chemical product is the laborious process of purifying milligram quantities to a purity greater than 95%, drying the purified material, and weighing it as accurately as possible. A solution of known concentration is then prepared by adding a defined volume of solvent to the known mass of the compound. This method is too restrictive and slow to keep pace with the perpetual acceleration of the drug discovery process and is inaccurate in the presence of impurities, counterions, unevaporated solvents, and unidentified particulatessparticularly when compounds are available only in low-milligram amounts. For smaller scale syntheses (65 000 developmental and marketed drugs in the commercial database MDL Drug Data Report (MDDR) contain nitrogen, indicating that the CLND is widely applicable as a detector for analyzing pharmaceuticals. We began using and evaluating the CLND in 1995 for quantitation of small organic molecules derived from combinatorial libraries. However, our early work was problematic, as the detector was at first designed to operate for only a few hours at a time. The CLND has recently been redesigned and upgraded, and we have found the present model durable and reliable in everyday operations. We have thoroughly evaluated the CLND for routine on-line quantitation of organic compounds that contain nitrogen, extensively with low-molecular-weight compounds. In addition, we have successfully used the CLND in conjunction with a UV detector and a mass spectrometer following RP-HPLC (LC/UV/N/MS) for simultaneous compound identification, quantitation, and purity assessment to provide fast, accurate answers to the questions: What is it? How much is there? How pure is it? EXPERIMENTAL SECTION Materials. The set of compounds used to calibrate the CLND consisted of drug reference standards with a purity of >99%, diphenylalanine, angiotensin II, and bovine serum albumin (BSA) purchased from Sigma (St. Louis, MO). All other synthetic compounds were synthesized at Chiron Corp. (Emeryville, CA) as previously described2 and provided as solutions in dimethyl sulfoxide (DMSO). GC/MS grade methanol (MeOH) and HPLC grade 2-propanol (IPA) were purchased from Burdick and Jackson (Muskegon, MI). Water was purified through a Milli-Q plus TOC (12) Fujinari, E. M.; Courthaudon, L. O. J. Chromatogr., A 1992, 592, 209-14. (13) Bizanek, R.; Manes, J. D.; Fujinari, E. M. Pept. Res. 1996, 9, 40-4. (14) Taylor, E. W.; Borny, J.-F. A.; Dollinger, G. Abstract presented at Pittsburgh Conference, 1997, Abstr. 647P. (15) Fitch, W. L.; Szardenings, A. K.; Fujinari, E. M. Tetrahedron Lett. 1997, 38, 1689-92.
Figure 1. Schematic diagram of the chemiluminescent nitrogen detector. Effluent from the RP-HPLC is introduced into a heated pyrotube via a nebulizer using a mixture of oxygen and argon as the nebulizing gas. In the presence of high temperature (1050 °C) and oxygen, organic analytes that contain nitrogen are oxidized to form nitric oxide (•NO), carbon dioxide (CO2), water (H2O), and other oxides. Gases are split at the end of the pyrotube between waste, which is predominantly water, and a membrane dryer, which removes residual water. The gases are drawn into the membrane dryer by vacuum, and a needle valve at the end of the membrane dryer is used to adjust the split ratio. The dried gases are drawn into the reaction chamber by the vacuum and mixed with ozone (O3) where, under vacuum, the •NO reacts with the O3 to form nitrogen dioxide in the excited state (*NO2). The *NO2 rapidly decays to ground state and emits a photon. The photons emitted are detected using a photomultiplier tube and converted into an analog signal that is recorded by an integrator.
water purification system (Millipore Corp., Bedford, MA). Sequencing grade trifluoroacetic acid (TFA) was purchased from Pierce Chemical (Rockford, IL). Instruments. A modular, automated HPLC system was used. The system comprised an Eldex MicroPro dual-syringe highpressure pump equipped with 2-mL syringes and a 15-µL dynamic mixer (Napa, CA), a Shimadzu SIL-10A autosampler (Columbia, MD), a Linear Instruments model 205 dual-wavelength UV/visible detector (Fremont, CA) equipped with a 0.3-µL microbore flow cell (Michrom BioResources, Auburn, CA), and 0.003-in.-internal diameter (i.d.) PEEK tubing throughout the system. Following UV detection, the eluate was either connected directly to an HPLC CLND model 7060 (Antek Instruments, Inc., Houston, TX) or split 4:1 by a microsplitter (Michrom BioResources, Inc.) between the CLND and a Finnigan LCQ ion trap mass spectrometer equipped with an electrospray interface (San Jose, CA), respectively. Data from the UV detector and the CLND were collected on a computer using the EZChrom chromatography data system (Scientific Software, Inc., San Ramon, CA). Flow Injection Analysis of Calibration Compounds. The solvent and the diluent for preparation of samples for flow injection analysis (FIA) was 10% (v/v) MeOH, 0.1% (v/v) TFA. Calibration compounds were dissolved and then each compound was diluted to yield a solution with a nitrogen concentration of 2 mM. These solutions were further diluted to a nitrogen concentration of 1.28 mM and then by 2-fold serial dilutions to 2.5 µM. A 15-µL aliquot of each sample was loaded into a 5-µL sample loop (a 3-fold overfilling) and infused into the CLND at a rate of 50 µL/min in an infusion buffer of 10% MeOH, 0.1% TFA. Because the CLND is nitrogen-sensitive, a MeOH/water-based infusion buffer was chosen. The parameters on the CLND were optimized and set as follows: a TL-HEN-120-AA glass nebulizer (J. E. Meinhard Associates, Santa Ana, CA) was used to introduce the sample into
the furnace. Argon and pyro-oxygen lines were linked by a tee to mix them before entering the nebulizer. The pyro-oxygen flow was set to ∼25 cm3/min, and the argon flow was adjusted to generate a back pressure in the nebulizer of ∼20 psi. The photomultiplier voltage was set at 750 V, and the temperature of the photomultiplier tube was maintained at ∼-13 °C. Furnace and membrane dryer oven temperatures were set to 1050 and 97 °C, respectively. A PEEK needle valve (Upchurch Scientific, Temecula, CA) was put at the outlet end of the membrane dryer to adjust the split at the outlet of the pyrotube such that >90% of the gas flow went into the membrane dryer. This split ratio resulted in a vacuum of ∼20 Torr in the reaction chamber. Ozone-oxygen flow into the reaction chamber was set to ∼25 cm3/min. The same peak integration parameters were used for all samples, and calibration curves were fit using linear regression with 1/x2 weighting. To assess the precision of the CLND response by FIA, one sample from each dilution series of compound was injected in triplicate and peak areas were measured; the relative standard deviations (RSD) calculated for each set of triplicates were all e2%. RP-HPLC of Calibration Compounds. RP-HPLC was carried out using a BetaBasic C18 column, 1 × 150 mm, 3-µm particle size (Keystone Scientific, Bellefonte, PA). A 5-µL aliquot of each sample of calibration compound from above was injected onto the column using the same 3-fold sample loop overfilling procedure as described above. The column was eluted at 50 µL/min with a linear gradient from 5 to 95% buffer B in 15 min. Buffer A was 0.1% (v/v) TFA in water, and buffer B was 0.08% (v/v) TFA in 75% (v/v) MeOH, 25% (v/v) IPA. The same peak integration parameters were used for all samples, and calibration curves were fit using linear regression with 1/x2 weighting. To measure the precision of the CLND response, 5 µL of each calibration compound at a nitrogen concentration of 2 mM was mixed and Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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diluted to yield a solution containing each compound at a final nitrogen concentration of 0.4 mM. A 20-µL aliquot of this solution was injected in quadruplicate onto the column. The column was eluted at 50 µL/min with a linear gradient from 5 to 65% buffer B in 30 min. Peak areas were measured, and the RSD for the quadruplicate peaks of each compound was reported as the precision of the CLND response. To evaluate the effect of mobile-phase composition on the response of the CLND, a stock solution of diphenylalanine was diluted 1:100 into each of 10, 30, 50, 70, and 90% buffer B to yield 0.2 mM solutions. A 5-µL aliquot of each of these solutions was analyzed by FIA using an infusion solvent consisting of the same percentage of buffer B as in the diluent. Each solution was injected in triplicate. Peak areas were measured; the RSDs calculated for each set of triplicates were all e2%. To demonstrate that the CLND response was mass-dependent, 5 µL of a 0.2 mM solution of diphenylalanine in 10% MeOH, 0.1% TFA was injected onto 0.5-, 1-, and 2-mm-i.d. Keystone BetaBasic C18 columns (all 150 mm in length) and eluted at flow rates of 12.5, 50, and 100 µL/min, respectively, with a 15-min linear gradient from 5 to 95% buffer B in 15 min. For the 2-mm column, 10-mL syringes and a 50-µL dynamic mixer were used in the Eldex pump. Triplicate injections were made onto each column and peak areas were measured; the RSDs calculated for each set of triplicates were all e2%. Characterization of Chiron Compounds. To show the utility of using the CLND in conjunction with UV and MS detectors for simultaneous identification, quantitation, and purity assessment of small organic compounds, compounds produced at Chiron from a single synthesis and as a combinatorial mixture were analyzed. For chromatography of these Chiron compounds, a stock solution of each in DMSO was diluted either 1:50 in 10% MeOH, 0.2% TFA or 1:10 in 50% buffer B, and 50 or 10 µL, respectively, of each solution was injected onto the 1 × 150 mm column. Gradient elution conditions were modified for each sample, as listed in the figure legends. A dilution series of diphenhydramine in 10% MeOH, 0.2% TFA was prepared and used as the external standard to calibrate the CLND for quantitation. It was injected under conditions identical to those used for the test samples. The mass spectrometer was set to the positive ion detection mode using the default settings. A data-dependent fragmentation method involving two scan events was used throughout the chromatogram: the largest base peak ion with an intensity above a threshold of 5 × 105 counts in scan 1 was isolated at a width of 2 mass units and then energized and fragmented by collision with helium gas. The fragments of the base peak ion were subsequently identified in scan 2. This LC/MS method, in almost all cases, resulted in the fragmentation of the intended product and major impurities related to the intended product. The same peak integration parameters were used for all samples, and using peak areas, calibration curves were fit to the external standard by linear regression analysis with 1/x2 weighting. Quantitation of the Chiron compounds was achieved by interpolation on the calibration curve. Pharmacological Activity Measurements. As previously described,16 pharmacological activity was measured in a radioli(16) Stratton-Thomas, J. R.; Min, H. Y.; Kaufman, S. E.; Chiu, C. Y.; Mullenbach, G. T.; Rosenberg S. Protein Eng. 1995, 8, 463-70.
3342 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 2. Chemical structures of drugs used to calibrate the CLND.
gand competitive binding assay for the human urokinase plasminogen activator receptor using a peptide containing the first 48 residues of human urokinase plasminogen activator as the ligand. RESULTS AND DISCUSSION Flow Injection Analysis of Calibration Compounds. To validate the hypothesis that a nitrogen-specific detector would be useful as a universal quantitation tool for any compound that contains nitrogen, 10 diverse compounds were used to calibrate the CLND. The compounds chosen varied significantly in molecular scaffold, molecular weight, nitrogen content, and type of nitrogen-containing group, e.g., primary, secondary, and tertiary amines, aromatic and heterocyclic amines, and amides (see Figure 2 and Table 1). All were easily obtained at high purity. The response of the CLND to each compound and the variation in response between compounds was first measured by FIA. A dilution series of each compound was analyzed. The calibration curves fit by linear regression analysis to each dilution series are shown in Figure 3 and summarized in Table 1. Except for BSA, responses were linear from 6400 down to 50 pmol of nitrogen injected, indicating that the response of the CLND was directly proportional to the amount of analyte. Linearity for BSA did not extend below 200 pmol. The detection limit, defined as a signal-
Table 1. Linear Regression Analysis and Quantitation Errors for Calibration Compounds Analyzed by Flow Injection with Chemiluminescent Nitrogen Detection compound
Mr
diphenhydramine 291.8 doxepin 315.8 chlorpheniramine 390.9 diphenylalanine 312.4 triprolidine 332.9 dibucaine 379.9 6-nor-6-allyllysergic acid diethylamide (N-ALSD) 349.2 caffeine 194.2 angiotensin II 1046 bovine serum albumin 69 290
N atoms LOQa LODb per compd (pmol of N) (pmol of N) slopec y-intercept 1 1 2 2 2 3 3 4 13 816
50 50 50 50 50 50 50 50 50 200
25 25 25 25 25 25 25 25 25 100
1820 2051 1775 1823 1977 1685 1921 1928 2068 1931
-4461 -4472 3940 4001 26540 55580 -2461 -5701 -14550 -2951
R2 >0.9999 0.9999 >0.9999 >0.9999 >0.9999 0.9999 >0.9999 >0.9999 0.9999 0.9997
quantitation errord (%) 6.6 -5.2 6.8 3.6 -5.2 11 2.4 -3.1 12
a Limit of quantitation defined as a minimum signal-to-noise ratio of 5:1. b Limit of detection defined as a minimum signal-to-noise ratio of 3:1. Curves were fit using linear regression analysis with 1/x2 weighing. d Each drug was quantified using N-ALSD as the external standard at the 800 pmol of nitrogen injection.
c
Figure 3. Calibration curves for structurally diverse compounds analyzed by flow injection. Curves were fit by linear regression with 1/x2 weighting. The plot is displayed using log/log scales to equally space the points on the curves, which were generated by 2-fold serial dilutions of each compound. Plot symbols correspond to diphenhydramine (O), doxepin (b), chlorpheniramine (0), diphenylalanine (9), triprolidine (4), dibucaine (2), caffeine (3), 6-nor-6-allyllysergic acid diethylamide (1), angiotensin II (]), and bovine serum albumin ([).
to-noise ratio of 3, was 25 pmol of nitrogen for all compounds except BSA, which had a detection limit of 100 pmol of nitrogen. The reduced linearity and sensitivity observed with BSA was due to peak tailing during the infusion.17 The similarity of the slopes and y-intercepts in Figure 3 and Table 1 shows that the CLND response was equivalent for the calibration compounds when the amount of compound analyzed had been normalized for nitrogen content. Equivalence was further illustrated by calculating the quantitation error for each compound in the middle of the calibration range using 6-nor-6allyllysergic acid diethylamide (N-ALSD) as an external calibration standard. The quantitation errors ranged from -5.2 to +12% and exhibited a quantitation variance at the 95% confidence level of 12%. The equivalence of the linear responses of the CLND to the compounds tested indicates that response was independent of the chemical nature of the compounds and that any external standard could be used for accurate quantitation. FIA is a rapid method for quantitation based on total nitrogen response. However, its utility is constrained by the condition of (17) With ∼800 nitrogen atoms per molecule of BSA, 100 pmol of nitrogen is equivalent to ∼125 fmol (10 ng) of protein. This sensitivity with protein permits the quantitation of a protein at the femtomole or nanogram level without the need of an extinction coefficient.
Figure 4. Calibration curves for structurally diverse compounds analyzed by RP-HPLC with gradient elution. Curves were fit by linear regression with 1/x2 weighting. The plot is displayed using log/log scales to equally space the points on the curves, which were generated by 2-fold serial dilutions of each compound. Plot symbols correspond to diphenhydramine (O), doxepin (b), chlorpheniramine (0), diphenylalanine (9), triprolidine (4), dibucaine (2), caffeine (3), 6-nor-6-allyllysergic acid diethylamide (1), and angiotensin II (]).
the analyte. The presence of nitrogen impurities in the sample or solvent will distort results. For accurate measurements, the analyte must represent >90% of the nitrogen content of the sample and be dissolved in a diluent identical to the infusion buffer. RP-HPLC of Calibration Compounds. To be a widely useful quantitation tool in the pharmaceutical industry, the CLND should be compatible with gradient elution RP-HPLC, which separates the components of a sample so that they can be quantified individually. Accordingly, the calibration compounds tested by FIA (except BSA) were analyzed by RP-HPLC. A linear gradient elution in a mobile phase consisting of water/MeOH/IPA was used for the CLND analysis. Mobile-phase components used for RP-HPLC must be nitrogen-free, thereby requiring the use of an organic modifier other than acetonitrile, such as an alcohol. In addition, the highest grade solvents are recommended since most are not controlled for nitrogen content per se. The results of the linear regression analysis are shown in Figure 4 and summarized in Table 2. The calibration curves were linear from 6400 down to 25 pmol of nitrogen injected, with detection limits of e12.5 pmol. The similarity of the slopes and y-intercepts among the nine curves indicates that the response of the CLND under gradient elution conditions was similar for all nine compounds. Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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Table 2. Linear Regression Analysis and Quantitation Errors for Calibration Compounds Analyzed by RP-HPLC with Gradient Elution and Chemiluminescent Nitrogen Detection quantitation error (%)d 800 pmol 3200 pmol
compound
LOQa (pmol of N)
LODb (pmol of N)
slopec
y-intercept
R2
50 pmol
diphenhydramine doxepin chlorpheniramine diphenylalanine triprolidine dibucaine N-ALSD caffeine angiotensin II
25 25 25 25 25 25 25 25 25
12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5
2186 2647 2209 2176 2643 1933 2380 2040 2761
-9287 -891 11367 3330 -9670 -5434 -5721 -4025 1988
>0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
12 -13 12 -1.0 -11 24
6.9 -12 5.2 7.2 -12 15
6.5 -8.9 3.6 9.2 -12 17
14 -25
10 -21
13 -15
a Limit of quantitation defined as a minimum signal-to-noise ratio of 5:1. b Limit of detection defined as a minimum signal-to-noise ratio of 3:1. Curves were fit using linear regression analysis with 1/x2 weighing. d Each drug was quantified using N-ALSD as the external standard at the specified moles of nitrogen injection.
c
The accuracy of the response over the entire calibration range was determined by calculating quantitation errors for each compound at low, middle, and high points in the curve. The quantitation variances at the 95% confidence level were 31, 25, and 23% for injections of 50, 800, and 3200 pmol of nitrogen, respectively, with an overall average variance of 27%. The overall average quantitation error was 11%. This is a higher variance than that observed by FIA. The possible effect of gradient elution conditions was examined in an effort to account for this higher variance. Diphenylalanine was diluted in different percentages of reversed-phase buffer B. For each dilution sample, triplicate injections of 1 nmol were analyzed by flow injection in an infusion buffer consisting of the same percentage of buffer B. The RSD of the average peak areas at the percentages tested was 3.6%, indicating that the response of the CLND was essentially identical across the entire range of buffer B compositions (data not shown). Thus, it is likely that the increased variability observed with quantitation via RP-HPLC was due to differences in peak shape and, therefore, peak integration between compounds, and/or small differences in sample recovery off the column. Taken together, these data indicate that, in conjunction with RP-HPLC, the linear response of the CLND was equivalent for the compounds tested and response was independent of mobile-phase composition. Thus, the CLND can be used with gradient elution for accurate quantitation with an absolute average quantitation error of ∼(10%. The precision of the CLND response when used with gradient elution RP-HPLC was measured by quadruplicate injections of a mixture containing the nine calibration compounds, with all nine at the same nitrogen concentration. Under the chromatography conditions used, the compounds were all baseline-resolved and exhibited acceptable peak shape in the water/MeOH/IPA mobile phase (Figure 5). Peak widths in the CLND trace were nearly identical to those in the UV trace. The baseline remained flat over the entire gradient, making quantitation relatively simple. For each compound, peak areas were measured and averaged. All compounds exhibited RSDs of e2% for the quadruplicate injections. In addition, peak areas in the CLND trace showed less variability than those in the UV trace. In the CLND trace, peak areas exhibited an average deviation of ∼9% around the mean peak area, whereas, after adjusting for mass of each compound injected, UV peaks areas deviated ∼150% at 214 nm and ∼120% at 270 nm. 3344 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 5. Chromatogram of a mixture of the calibration compounds separated by RP-HPLC with gradient elution and detected by UV absorption and chemiluminescent nitrogen. The injected sample contained 2 nmol each of caffeine (1), triprolidine (2), chlorpheniramine (3), diphenylalanine (4), 6-nor-6-allyllysergic acid diethylamide (5), angiotensin II (6), diphenhydramine (7), doxepin (8), and dibucaine (9).
These results all demonstrate good reproducibility of the CLND response for replicate injections and between different compounds. The CLND responds to changes in nitric oxide gas levels produced from the incineration of nitrogen-containing analytes (see Figure 1). Therefore, the CLND response should not be affected by changes in sample concentration and mobile-phase flow rate, in contrast to a UV detector which is concentrationdependent. In a test of this hypothesis, 1-nmol aliquots of diphenylalanine were injected onto three different RP-HPLC columns of 0.5-, 1-, and 2-mm i.d. and eluted at appropriately scaled flow rates, 12.5, 50, and 100 µL/min, respectively, to generate varying concentrations of the compound at elution. Compared to the 1-mm-i.d. column, the UV detector response from the 0.5mm-i.d. column eluate was ∼4-fold greater, and from the 2-mmi.d. column ∼2-fold lower (data not shown), indicating that the concentration of the compound at elution varied as a function of the difference in column diameter and flow rate. On the other hand, the responses from the CLND were identical for these columns. This observation demonstrated that the CLND response was dependent only on total mass injected. Characterization of Chiron Compounds. For simultaneous compound identification and quantitation, the RP-HPLC eluate was split between the CLND and a mass spectrometer. The MS method utilized a data-dependent fragmentation method where
Figure 6. Simultaneous on-line characterization for identity, quantity, and purity of an impure, pharmacologically active compound derived from a combinatorial library. (A) Sample was chromatographed by RP-HPLC using a linear gradient from 5 to 95% B in 30 min. (B) Mass spectrum of the major peak of RP-HPLC chromatogram. (C) Mass spectrum of the fragments of the predominant ion in (B). The concentration of the intended product in the stock solution was measured by CLND. The calculated potency of the intended product in the impure sample was based on the concentration of the intended product as measured by CLND and the pharmacological activity exhibited by the sample. The actual potency of the intended product was measured using the same compound purified to >95%.
the predominant ion in a base peak scan was isolated and fragmented, and the fragment ions were identified by a second scan event. This LC/MS technique allowed for the simultaneous confirmation of identity from a single RP-HPLC injection by allowing one to check the consistency of the fragmentation pattern with the proposed structure of the compound. An example of the utility of simultaneous UV, chemiluminescent nitrogen, and MS detection during gradient elution RP-HPLC (LC/UV/N/MS) is the following characterization of an impure small organic molecule that, in crude form following a synthesis, exhibited pharmacological activity in vitro (Figure 6). This compound was an analogue of a lead compound derived from a combinatorial library, identified by its competitive binding to human urokinase plasminogen activator receptor. A sample of this crude was characterized for identity, purity, and quantity of the intended product from a single HPLC injection. The chromatogram in panel A shows the presence of one major component in both the UV and CLND traces. A scan of the spectral base peak intensity across the chromatogram revealed that the major peak contained an ion with a mass-to-charge ratio corresponding to the MH+ ion of the intended chemical product (panel B). The structure of this compound was verified by the fragment ions identified in the second scan event (panel C). Based on absorbance at 214 nm, the intended product was 68% pure. Its concentration, as measured by CLND, was 0.81 mM in the stock solution. Using this concentration measurement and the competitive binding results, an in vitro potency value of 360 nM was calculated for the compound. This calculation assumed that the intended product exhibited the predominant activity in the sample. To verify both that the intended product was the primary source of activity and that the calculated potency value was accurate, ∼10 mg of the intended product was subsequently purified to >95% by preparative RP-HPLC (data not shown). This material was dissolved at a concentration of 10 mM (w/v), corroborated by CLND (data not shown). The in vitro potency of the pure compound was determined to be 320 nM. The measured in vitro potency for the purified product was indistin-
guishable from the calculated potency of the compound in the crude sample. These data demonstrate that the potency value of a compound can be determined before purification. Thus, this method can expedite structure-activity relationship studies by obviating the need for purification, since the analogues are expected to exhibit the pharmacological activity in the crude samples. Another example of the utility of LC/UV/N/MS is the characterization of multiple products resulting from a single synthesis on a solid-phase support (Figure 7). RP-HPLC analysis of a sample of the crude products from a synthesis to generate an isoxazole analogue revealed the presence of four major components, two of which did not contain nitrogen. Again using the data-dependent fragmentation method, the last three peaks were determined to be the intended product (compound 3) and two intermediates from incomplete reactions (compounds 1 and 2). The first peak, labeled with an asterisk in the UV trace, was an unidentified impurity. The structure determined for compound 1 by MS was supported by the CLND response because it did not contain any nitrogen atoms and, therefore, did not generate a signal in the CLND. Compounds 2 and 3, both of which contained one nitrogen atom, were quantified. The results showed that compound 2 was present in the sample at ∼1.5-fold greater concentration than compound 3. The yields per milligram of resin were calculated by multiplying the measured concentrations by the total volume of sample and then dividing that value by the total amount of solid-phase support used for the synthesis. The yield of compound 2 was 45 nmol/mg of resin, and that of compound 3 was 30 nmol/mg of resin. To date, there has not been a method described to rapidly and accurately perform a quantitative characterization of “equimolar” mixtures of compounds produced by combinatorial synthesis methods. With MS, it has been possible to identify the components of a mixture and determine the percentages of intended products synthesized. With the use of the CLND in conjunction with RP-HPLC and MS, it is possible to measure the concentration of each component in a mixture, and that, together with identity, Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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Figure 7. Characterization of multiple products resulting from a single synthesis. The sample was chromatographed by RP-HPLC using a linear gradient from 5 to 95% B in 20 min. The structure of each compound is shown next to its corresponding UV peak. The asterisk indicates an unidentified impurity. Purities were determined using the UV trace at 214 nm. Concentrations refer to the stock solution. Yields are per milligram of solid-phase support (resin).
Figure 8. Characterization of an “equimolar” mixture of diketopiperazines synthesized by combinatorial methods. The sample was chromatographed by RP-HPLC using a linear gradient from 5 to 65% B in 30 min. Each number above a peak in the UV trace corresponds to the mass of an intended product identified in that peak. Each number in the CLND trace corresponds to the concentration of that product in the stock solution. In cases where a peak represented two compounds, the concentration shown is the sum of the two compounds.
provides an assessment of synthesis efficiency and quality. An example of the characterization of a mixture of 24 diketopiperazines synthesized by combinatorial methods is shown in Figure 8. The components of this mixture were well separated by RPHPLC, and 21 of the 24 components were identified on the basis of parent ion masses. The separation efficiency for this mixture in a water/MeOH/IPA mobile phase was equivalent to that observed for the same mixture in a water/acetonitrile mobile phase (data not shown). Quantitation of each peak corresponding to an intended product showed that the mean concentration in the mixture was 1.01 mM per compound with a standard deviation of 0.34 mM. Thus, for this mixture, 88% of the compounds intended to be synthesized were present in the mixture, and of 3346 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
those compounds present, the measured concentration was generally within 35% of the predicted concentration. CONCLUSIONS The CLND has been demonstrated to be a universal quantitative detector for compounds that contain nitrogen, down to lowpicomole levels. The combination of CLND with UV and MS detection during RP-HPLC provides a generic method for rapid identification, quantitation, and purity determination of small organic compounds, particularly in cases where the compounds to be analyzed are either impure and/or only available in limited amounts. In addition, this technique has utility for identification and quantitation of impurities and metabolites related to the
intended product. We have routinely used this methodology to evaluate individual compounds derived from the deconvolution of mixtures that were produced in the low-microgram range and exhibited pharmacological activity in vitro. By combining the concentration data with the biological activity data of these samples, compounds were accurately rank-ordered for potency (unpublished data). This rank ordering helped prioritize which compounds to further develop. The CLND can be directly applied to both chemical synthesis and pharmacological screening processes and include applications such as determining yield of intended synthetic product, generating accurate pharmacological potency values, and identifying quantitative structure-activity relationships. We believe that the ability to perform these types of analytical characterizations will accelerate drug discovery and make it more efficient by driving more quantitative analyses into the current drug discovery paradigm.
ACKNOWLEDGMENT The authors gratefully acknowledge Antek Instruments, Inc., in particular Jean-Francois Borny, for their commitment to turn the CLND into an everyday workhorse, the Small Molecule Drug Discovery group at Chiron for support of this work, especially Paul Renhowe and Steve Rosenberg for pharmacologically active samples, Michelle Stempien for assistance with the in vitro pharmacological assay, Judit Csejtey for supplying compounds, Heatherbell Fong for her skilled editing, and, finally, the reviewers for their helpful comments.
Received for review April 13, 1998. Accepted June 23, 1998. AC980402D
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