Quality Assessment of Combinatorial Pooled Libraries Using Mass Spectrometry

Lambert, Pierre‐Herve; Bertin, Sophie; Fauchère, Jean‐Luc; Volland, Jean‐Paul

doi:10.2174/1386207013331057

Cited by 9 publications

(5 citation statements)

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“…Our efforts toward obtaining a universal quantitative detection regime have been focused upon the evaporative light-scattering detector (ELSD) and the chemiluminescent nitrogen detector (CLND). Both of these detectors have been used for a number of years to aid quantification and purity assessment in conjunction with high-throughput chemistry programs. − …”

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Toward Single-Calibrant Quantification in HPLC. A Comparison of Three Detection Strategies: Evaporative Light Scattering, Chemiluminescent Nitrogen, and Proton NMR

et al. 2005

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There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.

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“…Taylor et al found the absolute error for a set of chemically and structurally diverse compounds averaged approximately ±10%. Shah et al used flow injection analysis (FIA) in conjunction with CLND and MS to analyze up to 1000 compounds per day.…”

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Toward Single-Calibrant Quantification in HPLC. A Comparison of Three Detection Strategies: Evaporative Light Scattering, Chemiluminescent Nitrogen, and Proton NMR

et al. 2005

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“…Mass analyzers of the single and triple-stage types have played roles in the quantitative study of drug metabolism 69 and endogenous biologically active substances [72][73][74] and in the qualitative study of combinatorial chemistry. [146][147][148][149] These mass analyzers coupled with ESI, APCI and TSP are favorable for combinations with liquid separation techniques, such as HPLC and capillary electrophoresis (CE).…”

Section: ·1 Quadrupole Mass Spectrometermentioning

confidence: 99%

Biomedical and Biological Mass Spectrometry

Mano

Goto

2003

ANAL. SCI.

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By the end of the 20th century, mass spectrometry (MS) had evolved drastically as a major analytical tool in biomedical and biological research. Recent mass spectrometers, designed for ease of handling, came into wide use in the 1990s. Soft ionization methods, which were made practical in the second half of the 1980s, allowed for the sensitive analysis of nonvolatile and thermolabile compounds by MS. Electrospray ionization (ESI) 1,2 and matrix-assisted laser desorption ionization (MALDI) 3 methods, made it possible to ionize large biomolecules and to transfer them to the gas phase, thereby becoming a valuable tool in both protein and peptide analysis [4][5][6] and in the study of drugs and their metabolites [7][8][9][10][11][12] in the human body. Many reviews of MS have recently been published, [13][14][15][16][17][18][19] and in particular, the number of reviews describing biochemical, biological, and biomedical MS have increased dramatically over the last ten years. Initially, there were many commentary reviews on new techniques, such as soft ionization methods and mass analyzers, 13-15 and more recently, review articles have focused on the biological and biomedical applications of MS. 18-21 Above all, two major fields, drug discovery and proteomics, have made marked advances based on MS.Articles in the MEDLINE database were searched, and the results are illustrated in Fig. 1 and Table 1. Over the last 15 years, a total of 79220 MS articles were placed in the database, with a 2.5-fold increase from 1987 to 1999. The number of articles using fast atom bombardment (FAB) as an ionization method remained almost constant during this period. This ionization method is a practical soft ionization procedure, which was applied to the analysis of peptides [22][23][24][25][26][27][28][29][30][31] and endogenous biological substances [32][33][34][35] in the 1980s. Atmospheric pressure chemical ionization (APCI) is a suitable method 36 for the ionization of middle or weakly polar low-molecular-weight molecules, and can be easily coupled to conventional highperformance liquid chromatography (HPLC), used at a flow rate of around 1 mL/min. [37][38][39][40][41][42][43][44][45][46][47][48] However, the number of articles concerning APCI have increased slowly, because most drugs developed recently contain polar groups which can be ionized without difficulty by the ESI technique. Japan This review focuses on biological and biomedical mass spectrometry, and covers a selection of publications in this area included in the MEDLINE database for the period 1987 -2001. Over the last 15 years, biological and biomedical mass spectrometry has progressed out of all recognition. The development of soft ionization methods, such as electrospray ionization and matrix-assisted laser desorption ionization, has mainly contributed to the remarkable progress, because they can easily produce gas-phase ions of large, polar, and thermally labile biomolecules, such as proteins, peptides, nucleic acids and others. The innovations of ionization meth...

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“…Another important factor which impacts the quality of the results from any HTS assay is the collection or library of compounds. The quality of the compound collection screened is paramount and has been discussed in many published reports. − From an analytical perspective, the purity of a compound collection is now being addressed by many laboratories, typically by MS-based methods either for the characterization or purification of compounds. − In our laboratories, all of the library compounds are purified using LC/MS, where fractionation is based on the molecular ion of the target compound. , Compounds are then controlled for identity and quantitated in their respective DMSO stock solutions by flow injection analysis (FIA) MS/UV/chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD).…”

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Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results

Popa‐Burke¹,

Issakova²,

Arroway³

et al. 2004

Anal. Chem.

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As part of an overall systems approach to generating highly accurate screening data across large numbers of compounds and biological targets, we have developed and implemented streamlined methods for purifying and quantitating compounds at various stages of the screening process, coupled with automated "traditional" storage methods (DMSO, -20 degrees C). Specifically, all of the compounds in our druglike library are purified by LC/MS/UV and are then controlled for identity and concentration in their respective DMSO stock solutions by chemiluminescent nitrogen detection (CLND)/evaporative light scattering detection (ELSD) and MS/UV. In addition, the compound-buffer solutions used in the various biological assays are quantitated by LC/UV/CLND to determine the concentration of compound actually present during screening. Our results show that LC/UV/CLND/ELSD/MS is a widely applicable method that can be used to purify, quantitate, and identify most small organic molecules from compound libraries. The LC/UV/CLND technique is a simple and sensitive method that can be easily and cost-effectively employed to rapidly determine the concentrations of even small amounts of any N-containing compound in aqueous solution. We present data to establish error limits for concentration determination that are well within the overall variability of the screening process. This study demonstrates that there is a significant difference between the predicted amount of soluble compound from stock DMSO solutions following dilution into assay buffer and the actual amount present in assay buffer solutions, even at the low concentrations employed for the assays. We also demonstrate that knowledge of the concentrations of compounds to which the biological target is exposed is critical for accurate potency determinations. Accurate potency values are in turn particularly important for drug discovery, for understanding structure-activity relationships, and for building useful empirical models of protein-ligand interactions. Our new understanding of relative solubility demonstrates that most, if not all, decisions that are made in early discovery are based upon missing or inaccurate information. Finally, we demonstrate that careful control of compound handling and concentration, coupled with accurate assay methods, allows the use of both positive and negative data in analyzing screening data sets for structure-activity relationships that determine potency and selectivity.

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Quality Assessment of Combinatorial Pooled Libraries Using Mass Spectrometry

Cited by 9 publications

References 0 publications

Toward Single-Calibrant Quantification in HPLC. A Comparison of Three Detection Strategies: Evaporative Light Scattering, Chemiluminescent Nitrogen, and Proton NMR

Toward Single-Calibrant Quantification in HPLC. A Comparison of Three Detection Strategies: Evaporative Light Scattering, Chemiluminescent Nitrogen, and Proton NMR

Biomedical and Biological Mass Spectrometry

Streamlined System for Purifying and Quantifying a Diverse Library of Compounds and the Effect of Compound Concentration Measurements on the Accurate Interpretation of Biological Assay Results

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