Structure-Dependent Response of a Chemiluminescence Nitrogen

Anal. Chem. 2007, 79, 718-726

Structure-Dependent Response of a Chemiluminescence Nitrogen Detector for Organic Compounds with Adjacent Nitrogen Atoms Connected by a Single Bond Bing Yan,*,| Jiang Zhao,‡ Kyle Leopold,§ Bin Zhang,| and Guibin Jiang†

School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, China; Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492; Codexis, Inc., Redwood City, California 94063; and Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

High-throughput screening (HTS) of chemical libraries is indispensable for drug discovery research. However, the HTS data quality for lead discovery, lead optimization, and quantitative structure activity relationship studies has been severely compromised due to the uncertain compound concentrations in screening plates. In order to address this issue, we compared various high-throughput technologies for quantification of compounds in microtiter plate format without the need for authentic compounds as standards and identified the chemiluminescence nitrogen detector (CLND) as the method of choice at the present time. However, the structure dependence of this detector has not been well studied. A proposed rule suggested that the only exception to equimolar response is for compounds that contain adjacent nitrogen atoms. The response should be zero when the adjacent nitrogen atoms are connected by a double bond and 0.5 when they are connected by a single bond. In this investigation, we studied a broad range of compounds with isolated and adjacent nitrogen atoms. We confirmed that compounds with isolated nitrogen atoms produce an equimolar response with a 15-20% variation depending on structures and compounds with adjacent nitrogen atoms connected by a double bond giving nearly zero response. We discovered that the CLND response for compounds containing adjacent nitrogen atoms that are connected with a single bond is highly structure dependent. Substitutions on the nitrogen atoms or nearby in the molecule can increase the CLND response to approach a value higher than the predicted value 0.5 (maximal value 0.82/nitrogen atom). Without substitution, much lower values than predicted (minimal value 0.0-0.08/nitrogen atom) are obtained. Therefore, the prediction of response of 0.5/nitrogen atom for compounds with adjacent nitrogen atoms connected by a single bond should be abandoned. Compounds with * Corresponding author: (e-mail) [email protected]. † Research Center for Eco-Environmental Sciences. ‡ Bristol-Myers Squibb Pharmaceutical Research Institute. § Codexis, Inc. | Shandong University.

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similar structures should be used to generate calibration curves for quantification of this class of compounds. High-throughput screening (HTS) is committed to identifying valid lead compounds for further optimization and drug development. The primary screening data are so crucial that thousands of compounds are denied further investigation solely based on these data. In quantitative structure-activity relationship (QSAR) studies, both positive and negative data points are crucial for establishing the model of drug interactions. However, it is a general mistake to believe that all compounds in the biological assay plates are present at the reported concentration. In fact, the quality of the screening data is questionable due to the fact that the concentration of compounds in biological assay plates is mostly uncertain. Such uncertainty results from several factors such as compound degradation in storage, unknown compound solubility in organic solvents during compound transfer and handling, and unknown compound aqueous solubility in assay plates. The impact of these flawed data to drug discovery efforts cannot be ignored. Three fundamental elements of compound quality are its identity, purity, and quantity. The compound identity can be sufficiently addressed by mass spectrometry. The relative purity of compounds can now be simultaneously obtained in LC/UV (or ELSD)/MS measurement. These relative purity data are obtained based on the assumption that all components in the solution can be detected by UV or evaporative light scattering detection (ELSD) with the same response factor. This assumption has been proven wrong. Therefore, the relative purity derived this way is not its absolute purity.1 The weights of compounds often include impurities that are not detectable by LC/UV/ELSD/MS. Therefore, concentrations calculated based on compound weights are not correct in most cases. This critical purity issue is now addressed by adopting high-throughput purification (HTP)2 for all compounds including those with high relative purity. Even when compounds are pure and their weights accurate at the time (1) Yan, B.; Fang, L.; Irving, M.; Zhang, S.; Boldi, A. M.; Woolard, F.; Johnson, C. R.; Kshirsagar, T.; Figliozzi, G. M.; Krueger, C. A.; Collins, N. J. Comb. Chem. 2003, 5, 547-559. (2) Yan, B.; Collins, N.; Wheatley, J.; Irving, M.; Leopold, K.; Chan, C.; Shornikov, A.; Fang, L.; Lee, A., Stock, M.; Zhao, J. J. Comb. Chem. 2004, 6, 255-261. 10.1021/ac061682x CCC: $37.00

© 2007 American Chemical Society Published on Web 11/09/2006

they are archived, knowing the final compound concentration in the HTS plates is still problematic for at least three reasons. First, during compound dilution, distribution, and handling, a single solvent (normally DMSO) or a solvent combination is used for high-throughput automated operation. Unavoidable poor solubility of many compounds is responsible for the altered concentrations in the daughter plates. Second, pure compounds can decompose over time in storage and there is no routine method to quantify them. In our studies of 10 purified libraries with diverse structures stored in -70 °C as dry film over a period of 2 years, we found that half of them exhibited a different extent of degradation. Among them, ∼25% showed decomposition after only a few months. Third, solubility of compounds in aqueous solution is unknown. Many DMSO-soluble compounds are no longer soluble in aqueous medium.3 In our cell-based assays, we often observe the formation of compound solid in cell culture even though the involved compounds are perfectly soluble in DMSO. The adverse effect of quantification deficiency not only impacts the very foundation of the HTS, lead discovery, and QSAR, it also affects other drug discovery areas such as the optimization and quality control of parallel synthesis and HTP. In parallel synthesis, the product characterization heavily relies on LC/UV/MS. These data consist of information on the identity of products and their relative purity. They indicate nothing about the quantitative yield. With such data, it is very hard to improve synthesis yield. As a result, one does not know the amounts of synthetic products that are subjected to purification. Without knowing the amount of compound applied to the purification column, there is no way to know the purification efficiency or try to improve HTP protocol. Therefore, compound quantification at the synthesis, the compound storage, and the biological assay stages is critical to the success of the high-throughput drug lead discovery. Quantification of organic compounds using a standard calibration method is intrinsically time and labor intensive. For quantification of a large number of compounds, this method is inhibitory long and tedious. If the authentic compound is not available at the time of measurement, the quantification cannot be achieved. Therefore, there is a general need for a universal and structureindependent quantification method to rapidly determine the quantity of compounds without using authentic compounds and tedious calibration method. Such a high-throughput quantification method will make great impact in the area of lead discovery and, therefore, in the modern drug discovery research. In recent years, various techniques such as ELSD,4-7 CAD,8,9 and chemiluminescence nitrogen detector (CLND)10-28 have been (3) Popa-Burke, I., G.; Issakova, O.; Arroway, J. D.; Bernasconi, P.; Chen, M.; Coudurier, L.; Galasinski, S.; Jadhav, A. P.; Janzen, W. P.; Lagasca, D.; Liu, D.; Lewis, R. S.; Mohney, R. P.; Sepetov, N.; Sparkman, D. A.; Hodge, C. N. Anal. Chem. 2004, 76, 7278-7287. (4) Kibbey, C. E. Mol. Diversity 1996, 1, 247-258. (5) Hsu, B. H.; Orton, E., Tang, S. Y.; Carlton, R. A. J. Chromatogr., B 1999, 725, 103-112. (6) Fang, L.; Pan, J.; Yan, B. Bioeng. Biotechnol. (Comb. Chem.) 2001, 71, 162171. (7) Webster, G. K.; Jensen, J. S.; Diaz, A. R. J. Chromatogr. Sci. 2004, 42, 484490. (8) Teutenberg, T.; Tuerk, J.; Holzhauser, M.; Kiffmeyer, T. K. J. Chromatogr., A 2006, 1119, 197-201. (9) Go´recki, T;, Lynen, F.; Szucs, R.; Sandra, P. Anal. Chem. 2006, 78, 31863192. (10) Fujinari, E. M. In ; Developments in Food Science 39; Wetzel, D. L. B., Charalambous, G., Eds.; Elsevier Science B.V.: Amsterdam, 1998; p 431.

tested as “universal detectors”, and they have shown different levels of promise in structural independent quantification. Using selective pure compound standards to make an average calibration curve, samples with the same core structure in a combinatorial library can be measured quantitatively by ELSD detectors with an error of 20-30%.6 However, when the core structure varied in cases of many diverse libraries, the quantification results showed significant structure dependence even using an average calibration curve made from many diverse compounds. Another evaporationbased technique, CAD, has shown ELSD-like quantification features.8,9 Recently, a series of papers10-28 have been published reporting the application of a chemiluminescence nitrogen detector. It was reported that, over a linear range of 2 orders of magnitude, the detector exhibited an equimolar response with (10% average error for compounds studied.20 In a CLND, organic molecules are fully oxidized in a 1050 °C furnace where oxygen is introduced. The nitrogen atoms in a sample are quantitatively converted to nitric oxide. Then NO reacts with ozone to form nitrogen dioxide in the excited state (NO2*), which subsequently relaxes to its ground state and emits chemiluminescent light. The light amplified by the photomultiplier tube is proportional to the nitrogen content in the analyte. Speculatively, the sample combustion and the follow-up reaction chain are potential processes to introduce structure dependence if any in the quantification. The obvious limitation of CLND is that it can only be applied to compounds containing nitrogen. However, an estimate of 90% of the ∼65 000 developmental and marketed drugs in the commercial database MDL Drug Data Report (MDDR) contain nitrogen, indicating that the CLND can be widely used in pharmaceutical analysis. Among nitrogen-containing compounds, those with adjacent nitrogen atoms are frequently synthesized for drug screening because they are more likely to possess druglike properties in terms of log P, solubility, and bioavailability. They (11) Fitch, W. L.; Szardenings, A. K.; Fujinari, E. M. Tetrahedron Lett. 1997, 38, 1689-1692. (12) Borny, J. F. A.; Homan, M. E. Curr. Trends. Dev. Drug Discovery 2000, 18, 514-519. (13) Brannegan, D.; Ashraf-Khorassani, M.; Taylor, L. T. J. Chromatogr. Sci. 2001, 39, 217-221. (14) Yurek, D. A.; Branch, D. L.; Kuo, M. J. Comb. Chem. 2002, 4, 138-148. (15) Petritis, K.; Elfakir, C.; Dreux, M. J. Chromatogr., A 2002, 961, 9-21. (16) Bizanek, R.; Manes, J. D.; Fujinari, E. M. Pept. Res. 1996, 9, 40-44. (17) Shi, H.; Strode, J. T. B., III; Taylor, L. T.; Fujinari, E, M. J. Chromatogr., A. 1996, 734, 303-310. (18) Shi, H.; Taylor, L. T.; Fujinari, E. M. J. Chromatogr., A 1997, 757, 183191. (19) Shi, H.; Taylor, L. T.; Fujinari, E. M. J. High Resolut. Chromatogr. 1996, 19, 213-216. (20) Taylor, E. W.; Qian, M. G.; Dollinger, G. D. Anal. Chem. 1998, 70, 33393347. (21) Harrison, C. R.; Lucy, C. A. J. Chromatogr., A 2002, 956, 237-244. (22) Taylor, E. W.; Jia, W. P.; Bush, M.; Dollinger, G. D. Anal. Chem. 2002, 74, 3232-3238. (23) Nussbaum, M. A.; Baertschi, S. W.; Jansen, P. J. J. Pharm. Biomed. Anal. 2002, 27, 983-993. (24) Bhattachar, S. N.; Wesley, J. A.; Seadeek, C. J. Pharm. Biomed. Anal. 2006, 41, 152-157. (25) Shah, N.; Gao, M.; Tsutsui, K.; Lu, A.; Davis, J.; Scheuerman, R.; Fitch, W. L.; Wilgus, R. L. J. Comb. Chem. 2000, 2, 453-460. (26) Lane, S.; Boughtflower, B.; Mutton, I.; Paterson, C.; Farrant, D.; Taylor, N.; Blaxill, Z.; Carmody, C.; Borman, P. Anal. Chem. 2005, 77, 4354-4365. (27) Letot, E.; Koch, G.; Falchetto, R.; Bovermann, G.; Oberer, L.; Roth, HansJorg. J. Comb. Chem. 2005, 7, 364-371. (28) Corens, D.; Carpentier, M.; Schroven, M.; Meerpoel, L. J. Chromatogr., A 2004, 1056, 67-75.

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Figure 1. Previously proposed exceptions to CLND equimolar response rule.

are increasingly made for lead screening studies. The CLND technique offers a unique opportunity in drug discovery research as a quantitative detector that does not require pure, authentic standards. Several papers discussed the potential application of CLND in the quantitative analysis of compound archive and screening libraries.24-28 In our previous1,6 and current research, we compared various detectors and identified CLND as the method of choice to analyze the reaction yield of high-throughput parallel synthesis, to evaluate and optimize the purification recovery, and to quantify compounds after a long storage. CLND can be used as a flow injection analysis device or an on-line detector for separation instruments such as HPLC. Unlike other HPLC detectors, CLND presents more instrumental complexity and requires more optimization to reach the optimal operational conditions. We carried out significant optimization before the application of HPLC/UV/CLND, and the instrument was maintained and operated by a dedicated operator. Although previous reports have reported equimolar response with a 10% error,20 it was also found that the sole exception to equimolar response of the CLND arises from chemical structures containing adjacent nitrogen atoms (Figure 1), such as azo or azide groups, presumably because the adjacent nitrogen atoms have a tendency to be released as N2 gas upon incineration. It was speculated that this deviation was predictable and can be corrected. A proposed rule of thumb is that the response should be 0 when adjacent nitrogen atoms are connected by a double bond and 0.5 when adjacent nitrogen atoms are connected by a single bond (Figure 1). To the best of our knowledge, this rule has been used widely without solid validation. For CLND to play a more significant role in critical tasks such as combinatorial and parallel synthesis, high-throughput purification, quantification in compound archives, and quantification of compounds in biological assay plate, it is urgent to further validate the CLND method to keep pace with the drug discovery needs. During our preliminary investigation, we noticed that compounds with adjacent nitrogen showed inconsistent and often unpredictable results. We therefore carried out a series of investigations on the structure dependence of compounds containing adjacent nitrogen atoms and report our findings below. EXPERIMENTAL SECTION Chemicals. Methanol, water, and DMSO were purchased from Burdick & Jackson (Muskegon, MI). Argon and oxygen came from Airgas (Radnor, PA). Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) without further purification. Their purities were analyzed using HPLC/UV/CLND and these purities were considered in concentration calculation. Library compounds were made and purified in-house. Instruments. An HP1100 HPLC instrument controlled by ChemStation software was used by running a linear gradient (0720

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100% methanol in water with 0.05% TFA) over 4 min, with a 0.5min wash at the end followed by 0.5-min re-equilibration. The flow rate is 1.7 mL/min, and the sample injection volume is 10 µL. An in-line diode array UV detector was used. Chromatographic separation was performed on a MetaChem Polaris C18 column (5 µm, 5 cm × 2.0 mm i.d.) held at a constant temperature of 40 °C. A postcolumn splitter diverted most of the postseparation flow to waste, while sending ∼300 µL/min to the nitrogen detector. An Antek 8060 CLND detector was used for nitrogen detection, with the pyrotube heated to 1050 °C and gas flows of 50 mL/min argon and 252 mL/min oxygen. The following modifications were made to the 8060 module to improve system stability and reproducibility: (1) The original stainless steel nebulizer was replaced with one sapphire-tipped assembly kit. This new kit made significant improvement to the reproducibility because its ability to deliver more stable spray. (2) The nebulizer was adjusted monthly to deliver a symmetrical spray, and this practice facilitated a stable performance from the CLND detector. (3) The original quartz pyrotube was replaced with a ceramic pyrotube. This upgrade was originally designed to reduce etching effect from fluoride, but it also improved the system stability. Calibration. Caffeine standard was made fresh weekly in volumetric flask at a concentration of 4.1 mM (N) in methanol. This solution was subjected to serial dilution to make solutions with the following concentrations: 2.05, 1.03, 0.51, 0.25 mM (N). These five solutions were later used as calibration standards. Another caffeine solution (1.03 mM (N)) was made separately in a volumetric flask and was used as an external standard, during calibration and subsequent sample analysis. The calibration solutions were analyzed by CLND in triplicate from low to high concentration. Injections of methanol blank and external standard, both in triplicates, were made at the beginning, between each change of concentration, and at the end. As a result, the methanol blank and the external standard were each analyzed 18 times. Prior to calibration curve determination, the following steps were taken to validate system performance. All chromatographs of methanol blank were visually inspected for any detectable signals. Should there be any signals in the blank, the injector module was rebuilt and the experiment repeated. Peak areas of all 18 external standard samples were averaged, and any sample with a deviation of more than 10% from average was defined as an outlier. Existence of any outlier often indicates an unstable spray at the nebulizer, a condition that can be fixed by adjusting the relative position between the capillary and the sapphire orifice. Once the calibration set passed validation, peak areas were averaged for samples of each concentration and divided by the average peak areas of external standard to generate a unitless ratio. A calibration plot was then made. A R2 value of 0.98 or higher was obtained. The external standard concentration was also confirmed at this point. Sample Preparation and Analysis. About 12.5 mg of each testing compound was weighed and dissolved in methanol. All the expected compound concentration calculations were corrected for their purity. The solution was transferred into a 250-mL volumetric flask. For less soluble samples, up to 10 mL of DMSO was used to dissolve the sample. The flask was filled with methanol to the calibrated mark to make 50 µg/mL stock solution,

and 1.5 mL of the solutions was transferred into a 2-mL capped vial for analysis. The analysis was performed by triplicate injections of each testing sample. Control samples, a methanol blank, and an external standard were again inserted at the beginning, between every nine injections, and at the end of the sequence. Once the sequence was completed, a validation process similar to the one during calibration was also performed. The peak areas of each sample were averaged and then divided by the average of peak areas of the external standards immediately preceeding and following the sample injections. This ratio was then applied to the calibration curve for quantitation. Data Analysis. A Visual Basic-based program was written to extract data from ChemStation. During each data acquisition sequence, an autoreport scheme was set up within ChemStation to generate a generic report for each injection. A new folder was created for the whole sequence with a subfolder for each injection, and a file named “Report.txt” in each subfolder. The software, when browsed to the sequence folder, generated a list of the subfolders. It would then open the “Report.txt” file and extract the following information from the peak list within: sample name, time of analysis, retention time, peak width, peak area, peak height, and chromatographic purity. A summary of all peak tables was written to a tab-delimited text file that can be transferred to Excel for further analysis. RESULTS AND DISCUSSION LC/CLND/UV System Optimization. We used caffeine as one of the standards for calibration and an external standard incorporated into the sample analysis routine. With our instrument configuration, a linear response was observed reproducibly at a nitrogen concentration below 16.5 mM (N) with 10-µL injection volume (data not shown). Our working concentration range was set below 8 mM (N), which is well within the linear range. Caffeine was used to generate a calibration curve over the range of sample concentration from 0.5 (N) to 8 mM (N). The regression analysis gave an equation of y ) 1.0022x - 0.012 with a R2 ) 1.0. When the concentration was higher than 16.5 mM (N), a nonlinear response was observed. When the concentration was less than 0.06 mM (N), there was a greater influence from integration parameter setting during our experiment. This range was selected based on two factors: it was well within the linear range and it also provided enough UV response on the in-line DAD detector. In the course of our study, we first noticed that there is a month-to-month variation of the CLND response. As much as a 13% variation for the caffeine standard was observed in a 4-month study. To correct this fluctuation, we incorporated caffeine standard measurements. An external standard measurement of 50 µg/mL caffeine (or 1 mM nitrogen) in methanol was incorporated at the beginning and the end of each queue, as well as between every nine injections. Peak area of sample detected was divided by the average peak area of the two adjacent external standards, and the ratio is fitted to the calibration curve to determine the nitrogen concentration. To further validate the suitability of using caffeine as a standard, we tested 15 compounds 1-15 (Figure 2) to compare their response relative to caffeine. The purity of 1-15 was analyzed using HPLC/UV/CLND, and CLND responses were corrected for the compound purities. The response per nitrogen

Figure 2. Chemical structures of 15 compounds used in correcting caffeine calibration curve.

Figure 3. Plot of per nitrogen CLND response of 15 commercial samples relative to that of caffeine (assigned to 1.0). The chart shows that an average of 95% of response is obtained for these compounds compared to that of caffeine.

of each compound was plotted in Figure 3. Twelve of the 15 samples exhibited lower response than that of caffeine, and 3 samples exhibited a response comparable to that of caffeine. The average response is 95% relative to caffeine. We use this value as a correction factor for caffeine calibration, and all compound analyses were adjusted accordingly. When samples elute into the CLND, the solvent composition or the content of organic solvent may affect nitrogen response of Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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Figure 4. Relative CLND response at different solvent compositions. With HPLC pump running isocratic at each solvent composition, the peak areas of three 10-µL injections of 50 µg/mL caffeine (1 mM nitrogen) solution were averaged.

the sample by disturbing the splitting ratio and the nebulization process. If this is true, compounds eluting at various times in a gradient elution may be subjected to different conditions and result in variable responses. We therefore carried out experiments to test the solvent effect on CLND response. The splitting ratio was tested by comparing solvent volumes collected at different isocratic solvent compositions. Since most compounds elute to detector between 30 and 90% methanol in a gradient separation, caffeine samples was analyzed using various isocratic HPLC conditions from 30 to 90% methanol at 10% intervals. This experiment was done in triplicate, and the result is summarized in Figure 4. There is about 5-7% variation in CLND response going from more aqueous to more organic solvent. Therefore, the variation in CLND response at static conditions is minimal. It should be noted that some solvent effect can be observed when comparing data obtained at two extremes (nearly pure organic or pure water). Under the dynamic situation, we compared CLND responses from 15 compounds at the gradient elution (0-100% methanol in water in 7 min) where compounds would elute at various retention times and at a step gradient (step to 100% methanol after a 30-s hold at 10% methanol). The variations in response were well within experimental error. After a series of optimization and validation of the system, we obtained a set of experimental conditions to carry out investigations to be described below. Further Validation and Application of CLND in HighThroughput Chemistry. It is now a common practice to purify compounds in a high-throughput format. However, the quantification of these compounds is not straightforward when the amount is too low to be accurately weighed and when the authentic standard compounds are not available. It is under such a circumstance that we have used high-throughput quantification with HPLC/UV/CLND in our routine compound analysis. We present here some data to demonstrate the application of this method in high-throughput chemistry and also to further validate this quantification method. The first study was performed by comparing purified compound quantity determined by HPLC/UV/CLND with that from weighing. This study involved several libraries containing 30-55 compounds. One such example is shown in Figure 5. Results suggested that HPLC/UV/CLND data are comparable to real 722 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

Figure 5. Comparison of compound weights measured by weighing and CLND determination. Fifty-two compounds from a small library were purified by HPLC, dried, and weighed. These samples were also subjected to HPLC/UV/CLND quantification and the weights, in micromoles, based on CLND quantification were compared with their weights. The relative standard deviation (RSD) was determined to be 9 ( 8%.

Figure 6. Application of CLND in determining the synthesis yield and the quantitative recovery of high-throughput purification. A small library containing 39 compounds was first analyzed by HPLC/UV/ CLND to quantify the amounts of products. The library was then purified by high-throughput purification, and the purified compounds were dried and weighed. The final weights are compared with the weights determined by CLND before the purification. The average weight of this library from weighing was 127 ( 55 µmol after purification and that from CLND was 191 ( 65 µmol before purification. The average purification recovery was 66.5%. The purification recovery can also be calculated for each compound. These data were used to guide the optimization of the high-throughput purification protocol.

weight with a variation of 10%. This analysis provided important synthesis yield data that can guide synthesis optimization. The HPLC/UV/CLND method was further used in the purification recovery evaluation studies. One example is shown in Figure 6. The reaction yield of this library was deduced from compound quantity determined by HPLC/UV/CLND following a separate HPLC/UV/MS analysis. The weight of purified compounds was obtained after high-throughput purification. A purification recovery of 66.5% was obtained by comparing the amount of compounds before (CLND) and after (weighing) purification. The lower recovery was expected in our high-speed purification with a one-compound-one-fraction collection scheme. In separate experiments, recoveries close to 90% were achieved when multifractions were collected for each injection in a slower purification process. These applications of CLND were crucial for control of the proper operation of library chemistry and purification. Another area of CLND application is the quantitative analysis of the amount of compounds in archived plates and in assay plates.

Table 1. Some Simple Nitrogen-Containing Compoundsa

Table 2. Substituted Indolesa

a

Note: All values are from the average of triplicate injections.

Table 3. Substituted Nitro-Containing Compoundsa

a


Popa-Burke et al. reported the use of CLND in examination of compound quantity in HTS plates and its impact on biological data.3 Therefore, all three areas lacking quantitative assessment in drug discovery can now be potentially addressed by the CLND method. From our evaluations, CLND is a general quantification method without using authentic standard compounds. However, we and others28 often observed the quantification inconsistencies when compounds with adjacent nitrogen atoms were analyzed. The previously proposed rule for predicting quantification for compounds with adjacent nitrogen atoms does not always offer correct results. To better understand the CLND process and validate CLND as a quantitative tool in combinatorial chemistry and drug discovery, we carried out a series of investigations to determine the structure dependence of CLND response for compounds containing adjacent nitrogen atoms. CLND Studies of Compounds with Isolated Nitrogen Atoms. The compounds studied were substituted indoles, substituted imidazoles, amines, and nitro-substituted compounds. In order to first evaluate CLND effectiveness in analyzing compounds without adjacent nitrogen atoms, compounds as shown in Tables 1-3 were studied by HPLC/UV/CLND. Compounds were weighed to the nearest 0.01 mg, and then their solutions were made and analyzed quantitatively by HPLC/UV/ CLND. Compound per nitrogen response relative to caffeine was calculated, and the values are shown in Tables 1-3. These compounds are often considered as the class of “wellbehaved” compounds. According to previous reports, they should all give 1.0 ( 0.1 response per nitrogen. Actually, we found that most amines, imidazoles, and urea compounds (Table 1) and

a


substituted indoles (Table 2) all gave per nitrogen responses within 10% of the predicted values. We also noticed that they all have a value smaller than 1.0. This suggests that CLND hardly produces artifactual higher signals, and the major issue for this technique may be to deal with various factors such as compound solubility, combustion efficiency, oxidation rate, and other unknown factors that cause signal loss in the detection process. Although most compounds gave responses within the expected range, it should be mentioned that a subtle structure dependence of response was noted. All six indole derivatives 25-30 gave a higher per nitrogen response (0.94 ( 0.02) than that of five nitro compounds 30-34 (0.87 ( 0.09). We speculate that, due to structural variations, some unavoidable signal loss may be structure dependent and an error range of 10-20% is possible. Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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Table 4. Molecules with Adjacent Nitrogen Atoms: Class 1a


a Note: All values are from the average of triplicate injections. *, Values are obtained by assigning response from NdN as 0.0. **, See text for explanation.

We also noticed that, in previous studies, the 10% error range was concluded on the basis of the study of a limited number of compounds. CLND Studies of Compound with Adjacent Nitrogen Atoms: Classes 1-5. Compounds with adjacent nitrogen atoms always gave lower responses, but to our knowledge, the relationship of their responses and their structures is not questioned before. When the adjacent nitrogen atoms have a double bond connection (class 1), a nitrogen molecule will form and the response is near zero (Figure 1). This rule is confirmed by data in Table 4. In 35 and 39, only a signal from one nitrogen atom is observed. In 38, between the two adjacent nitrogen atoms, there is significant double bond character due to two conjugated carbonyl groups at both ends and the response of 38 is close to zero. In 36, after assigning the response of sNdNs as 0.0, the per nitrogen response for the remaining four nitrogen atoms is 0.86. When the adjacent nitrogen atoms are connected with a single bond, the CLND per nitrogen response varies widely ranging from 0.0 to 0.82 (Tables 5-7). It is important to reveal the structural dependence, and the underlining mechanism is important for the application of CLND in the quantification of organic compounds. First, an array of substituted pyrazole derivatives (Table 5, class 2) was analyzed. The CLND responses from this class of compounds with adjacent nitrogen atoms are shown in Table 5. Compare 40, 42 to 41, 43; there is a trend that nitrogen or ring substitution will cause an increase in CLND response of the compound. From the responses of 43 and 44, it seems that the electronegativity of substations also plays a role in controlling the response. It seemed that this substitution effect might be one of the key factors that caused structure dependence in CLND response of compounds with adjacent nitrogen atoms. We therefore carried out more experiments to test the generality of the substitution effect. We next tested an array of hydrazine derivatives (class 3). The CLND responses of this class of compounds with adjacent nitrogen 724 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

a Note: All values are from the average of triplicate injections. *, Values are obtained by assigning response from isolated nitrogen atom as 1.0.


a


atoms are shown in Table 6. The substitution effect was again confirmed by nitrogen quantification results. The unsubstituted hydrazine 48 only gave a response of 0.31 while the substitution increased the responses in 49, 50, and 54 (0.48, 0.71, and 0.67). All other compounds except 52 also show such an effect. The lower response of 52 indicates that the extended conjugation in the compounds may cause an increased double bond character


a

Note: All values are from the average of triplicate injections. *, Values are obtained by assigning response of isolated nitrogen atom as 1.0.

between two adjacent nitrogen atoms and, therefore, reduce its response. On the basis of studies of pyrazole and hydrazine derivatives, we carried out investigation of more pyrazole/hydrazine compounds. The CLND responses of the fourth class of compounds with adjacent nitrogen atoms consistently show the effect of nitrogen substitution (Table 7). Those compounds share the same core structure and only their substitutions are varied. There are three structural groups: 55-63 have in average 0.60 and 0.82 per nitrogen response. These compounds all have nitrogen substitutions and their responses are higher than 0.5, the rule of thumb previously proposed (Figure 1). The other two groups, 64-66 and 67-71 gave much lower response between 0.0 and

0.28. The common feature of these compounds is that they do not have any substitutions on at least one set of adjacent nitrogen atoms. Their responses are much lower than 0.5, the value previously proposed for such compounds. Compounds 67-71 are pyrazole compounds that do not have nitrogen substitutions. To consider these data more quantitatively, we attribute 1.0 per nitrogen response to the amino group in the molecule and leave only 0.0-0.13 per nitrogen response to the pyrazole core. In contrast, 55-57, after subtraction of 1.0 for the same amino group, give a per nitrogen response of 0.60-0.71 for the pyrazole core demonstrating the substitution effect. Compounds 58-63 also have one nitrogen atom substituted and, in addition, have a methyl group and a carboxyl group on Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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the pyrazole ring. These compounds gave responses ranging from 0.68 to 0.82. Comparing structures and responses, we are convinced that a substitution on the nitrogen atom correlates with a high response close to the expected 1.0 per nitrogen response while the nature of substitution groups on the pyrazole ring is also very important in regulating how a compound will respond in CLND detector. Compounds 64-66 both contain a pyrazole core and hydrazine group along with other substitutions. They gave per nitrogen response ranging from 0.18 to 0.28. This series of responses are lower than pyrazole core responses in 55-57, suggesting the importance of the nature of substitutions themselves. This no doubt adds more complexity in structure dependence of CLND detection. The substitution effect can be understood in term of how surrounding chemical bonding energy affect the N2 formation inside CLND. If more chemical bonds or higher bonding energy surround the nitrogen atoms, they tend to be pulled toward the mother molecule rather than form N2 and leave the molecule. We rationalize that this is likely the root cause for the structure dependence we observed for compounds containing adjacent nitrogen atoms connected by a single bond. Triazoles and other related compounds are the fifth class of compounds with adjacent nitrogen atoms, and their results are shown in Table 8. General lower values for triazoles 72-76 suggest an increased tendency for these compounds to form nitrogen gas under CLND conditions and become undetectable. The adjacent nitrogen atoms in 77 and 78 can be considered substituted and their per nitrogen responses are in 0.72-0.85 range. This is similar to other nitrogen-substituted compounds shown in Tables 5-7. However, if we take the response of two adjacent nitrogen atoms with a double bond in between as zero in 72-76, the per nitrogen responses of this class of compounds are from 0.90 to 1.03, that is right on the predicted value. This class of compounds can be classified as “well behaved” after applying the rule that azo compounds produce zero response in CLND process. CONCLUSION Compared with other HPLC detectors, CLND presents more instrumental complexity and requires more optimization. After instrument and process optimization, a large number of compounds can be analyzed quantitatively and efficiently without the need for authentic compounds as primary standards. CLND data are valuable when combined with the relative UV purity data in the quality control of parallel synthesis, the optimization of highthroughput purification, the quality check of compounds in storage, and eventually the quantification of compounds in biological assay plates. In our investigation of the structure dependence of CLND response, we confirmed that, when compounds contain isolated nitrogen atoms, the response is close to quantitative with a variation about 10-20% depending on structures. When adjacent nitrogen atoms are connected by a double bond, the response is

726 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007


a Note: All values are from the average of triplicate injections. *, Values are obtained by assigning response of isolated nitrogen atom as 1.0.

nearly zero, indicating the strong tendency for these compounds to form N2 molecule and become undetectable. When nitrogen atoms are connected by a single bond, it was predicted previously that a response of 0.5/nitrogen atom should be observed. In this work, we presented compelling evidence that this rule is wrong and should be abandoned. Our data demonstrated that, in compounds with adjacent nitrogen atoms connected by a single bond, the CLND response is highly structure dependent. Substitutions on the nitrogen atoms or nearby in the molecule can increase the CLND response to approach a value higher than the predicted value 0.5 (maximal value 0.82/nitrogen atom). Without substitution on nitrogen or in the molecule, much lower values than predicted (minimal value 0.0-0.08/nitrogen atom) are obtained. Based on our studies, a structurally similar calibration compound should be used for this class of compounds in the quantitative analysis using CLND. ACKNOWLEDGMENT We acknowledge the financial support from National Science Foundation of China (20577029). Received for review September 27, 2006. AC061682X

September

6,

2006.

Accepted

Structure-Dependent Response of a Chemiluminescence Nitrogen

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