Compound libraries purity assessment

Yurek, D. A., Branch, D. L., and Kuo, M. S. (2002) Development of a system to evaluate compound identity, purity, and concentration in a single experiment and its application in quality assessment of combinatorial libraries and screening hits. 

After the library development process passes the stage of development and optimization of chemical reactions, the requirement of high throughput becomes more and more important. Usually, hundreds of samples need to be analyzed for evaluation of building blocks and the characterization of the full production library requires analysis of thousands of samples. At this moment detailed structural analysis of synthesized compounds is substituted by confirmation of the fact that intended compound has been synthesized. Purity assessment and quantification of intended compounds are the other two goals of analysis. 

Independence of the detector s response from a compound chromophore or ionizability makes it more universal for quantification applications than UV or MS. Recently, the evaporative light-scattering detector has been introduced as a useful tool for quantification and purity assessment of compounds in combinatorial libraries [36, 41]. 

Figure 11-4. Purity assessment is a critical component in the decision process by the chemist as to whether their isolated compound is of sufficient quality to be submitted for compound registration and biological testing. To facilitate automated and rapid purity assessment of compound libraries, applescripts and visual basic scripts are used. (A) Total ion current chromatogram shows two components. (B) Extracted ion chromatogram for the expected product identifies its retention time. (C) Mass spectrum observed for the expected product. (D) UV 220-nm chromatogram indicates the expected product is approximately 75% pure. (E) UV 254-nm chromatogram indicates the expected product is approximately 66% pure.

Purity assessment of combinatorial hbraries is an important issne. It has led to the nse of alternative detectors next to MS, snch as UV-DAD and ELSD. Stractural characterization and pnrity assessment of compound libraries obtained by combinatorial parallel synthesis using LC-APCI-MS and MS-MS, UV-VIS DAD, and NMR has been reported by Dulery et al. [18]. 

Synthesis control has two tasks directly associated with it. These are to identify or verify the identity of a combinatorial component and to determine the purity of the synthetic product. When characterizing a parallel library it is a relatively easy task to obtain a molecular weight from a small amount (femto-mole) of compound and thereby obtain a crude identification of the product. This circumvents the need to perform more difficult NMR or IR spectral interpretation and sample introduction maybe performed by a simple flow-injection atmospheric pressure ionization (API)/MS system. Purity assessment is typically based on area percentage normalization of the total ion chromatogram, assuming equivalent ionization of impurities and parent compounds, or a secondary detector, such as UY... 

Yurek DA, Branch DL, and Kuo M-S. Development of a System To Evaluate Compound Identity, Purity, and Concentration in a Single Experiment and Its Application in Quality Assessment of Combinatorial Libraries and Screening Hits. J Comb Chem 2002 4 138-148. 

Fast LC-MS methods have been used to assess library quantity and purity, as well as to triage purification of compounds. Zeng et al. [51] developed one of the first fully automated analytical/preparative LC-MS systems for the characterization and purification of compound libraries derived by parallel synthesis. The system incorporated fast, reverse-phase LC/ESI-MS analysis (5-10 minutes). Post-data-acquisition purity assessment of compound ti-braries was performed automatically with software control. Compounds that were below a threshold level of purity were automatically purified with HPLC. The real-time purity assessment eliminated the need for postpurification analysis or pooling of fractions collected. 

Further improvement of throughput in LC-MS analysis may be achieved by step-gradient elution. This elution format is essentially an on-line solid-phase extraction (SPE) process, where the samples are loaded onto the column, washed with aqueous mobile phase to remove water soluble impurities, and compounds are eluted with a mobile phase of high organic content. The technique combines the simplicity of FIA with the benefit of the removal of impurities and buffer components before mass spectrometry detection. In this case, selectivity is achieved by mass spectrometry alone without chromatographic separation. The technique has been used for compound purity assessment and quantitation. An on-line back-flush SPE-MS technique has been used by Marshall for quality assessment of the combinatorial libraries [112]. This back-flush elution procedure provides a very effective in-line removal method... 

Thus, in the result of automated data processing one can confirm the identity of library components, perform purity assessment based on several independent techniques (UV, MS, ELSD), estimate quantities of compounds, obtain information about side products, etc. The results may be presented using tabular output, which has certain strengths and weaknesses. On one side, a lot of different information about compounds can be captured in one table, such as sample ID, purity, estimated amount of material, etc. the data can be easily sorted and transferred to various databases. An example of tabular output where compounds were sorted based on their average purity is presented in [34], On the other side, the amount of information usually captured in a table is much more than is nec-... 

LC-MS is usually used for rapid purity assessment of the compound libraries. Because LC-MS has advantages over FIA-MS in its ability to analyze mixtures and to provide more complete characterization of compound libraries, it has replaced FIA-MS as the standard analysis tool in... 

A recent report (41) has highlighted the usefulness of CE to determine the enantiomeric purity of individuals coming from parallel synthesis, either during chemical assessment to help revising the experimental protocols and as a library QC tool to determine the stereochemical quality and purity of chiral libraries. The authors studied the Pictet-Spengler reaction of chiral tryptophans with carbonyl compounds on SP to provide tetrahydro-P-carbolines, and clearly highlighted the reaction conditions-dependent racemization of the reaction products. 

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