Frequently one or more auxiliary detection methods such as UV, nuclear magnetic resonance (NMR), ELSD, and more recently, CLND, are combined with LC-MS to provide a better overall assessment of library purity. These different detection methods often provide complementary capabilities for detection selectivity, range of sensitivity, and linearity of response.
Kibbey  compared HPLC quantitation of combinatorial libraries with ELSD and UV detection methods. The ELSD detector response is independent of chemical structural and requires no chromophore, which makes it well suited to HPLC analyses of mixtures of dissimilar compounds. Furthermore, the ELSD exhibits a nearly equivalent response to compounds within a structural class. Hence, rapid quantitation of compound libraries may be carried out with the use of a single external standard. Hsu et al.  reviewed the theory of ELSD and the design of commercial instruments. The application of ELSD to library analysis was illustrated with examples from the authors' library synthesis program. Complemented by UV detection for purity assessment and mass spectrometry for product identification, ELSD was the only technique that afforded sufficient accuracy and sensitivity for high throughput library analysis. Fang el al.  examined 42 compounds from seven different combinatorial libraries with a high throughput LC-MS/UV/ELSD method and 33 commercial and standard compounds with both high throughput and standard quantitation methods. It was demonstrated that compounds with low molecular weight (<300 Da) are generally less responsive to ELSD and can result in a purity measurement by ELSD that appears higher than when measured by UV. Quantitation with a general UV calibration curve generally gives higher precision than with a general ELSD calibration curve. A calibration curve from structurally related compounds is needed for better quantitation. Fitch et al.  made one of the earliest descriptions on the use of LC/CLND for the assessment of solid-phase synthesis of combinatorial libraries that contain nitrogen in the structure. Taylor et al.  evaluated a CLND detector as a nearly universal quantitation tool for nitrogen-containing compounds. CLND produced a linear response from 25 pmol to 6400 pmol of nitrogen in the molecule for a set of chemically and structurally diverse compounds with FIA and gradient HPLC. 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 HPLC for rapid and accurate quantitation down to low-picomole levels with only a single external standard. The authors also demonstrated the combination of LC/UV/CLND/MS as a generic method for rapid identification, quantitation, and purity assessment of small organic compounds. Shah et al.  developed a method for high throughput quality control of combinatorial library compounds from parallel synthesis with a combination of FIA-MS and flow-injection CLND. Compounds were characterized by mass spectrometry and concentration was determined by CLND with a throughput of 60 compounds/h. Dulery et al.  developed a strategy of a generic fast HPLC method with diode-array detection (DAD) and MS to provide structure and purity information. In addition, complementary NMR analyses were performed on selected compounds to provide a better structural characterization of the expected compounds and their potential side products. In a recent report, Yurek et al.  described the development and use of a new system for the simultaneous determination of identity, purity, and concentration of library components produced by parallel synthesis. The system makes use of HPLC with DAD, ELSD, CLND, and TOF-MS detectors (HPLC/DAD/ELSD/CLND/TOF-MS). The use of exact mass capability of TOF-MS along with CLND provides a synergistic combination that enables identification of target and side-product structures and determination of concentrations and purities in a single analysis.
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