One-Minute Full-Gradient HPLC/UV/ELSD/MS Analysis to Support High-Throughput Parallel Synthesis

Abstract

High-throughput parallel synthesis of library compounds for early drug discovery requires high-throughput analytical methods to confirm synthesis, identify reaction products, and determine purity. An ultrafast 1.0-min HPLC/UV/ELSD/MS method was developed and compared to our standard 2.5- and 5.0-min methods in order to determine if the faster method was appropriate to evaluate compound synthesis and determine purity. In addition to using standard test mixtures, a 400-member library produced by high-throughput parallel synthesis was used for comparing the various methods. Mass spectrometric detection was used for compound identification, while UV and ELSD data offered purity assessment. Compared to our longer separations, chromatographic separation achieved using the 1.0-min method was sufficient for compound evaluation and purity assessment. This ultrafast 1.0-min HPLC/UV/ELSD/MS method is expected to increase analytical throughput tremendously, provide important information faster, and reduce the overall cycle time from synthesis to screening.

Development and Application of an Automated Solution Stability Assay for Drug Discovery

“…However, many labs do have automated HPLC and LC-MS systems, such as those used for drug development studies, combinatorial synthesis monitoring, and bioanalysis. [22][23][24][25][26][27][28][29] So far, there are no reports of using an HPLC autosampler for online sample addition, buffer addition, incubation, and injection or to quantitate the stability of drug candidates.…”

Integration of a rapid automated solubility classification into early validation of hits obtained by high throughput screening

“…With cycle times of only 3-5 min and the capability of multiplexing it is possible to achieve throughputs of more than 1000 samples per day with only g amount of sample [4][5][6][7].…”

High Throughput Chemistry in Drug Discovery

“…The use of scavenger reagents and supported sequestration approaches alongside catch and release methodologies certainly improved the purity and quality of combinatorial chemistry reactions. However, it has been the development of fast, automated LC-MS analysis systems 124 ''purify all'' to take over from previous triage processes, 126 where moderate to good purity compounds were typically processed into screening without additional purification, and only the less successful reactions were purified. The ability to estimate concentration using LC methods 127,128 has added a further level of quality into library compound in screening, as assay level concentrations can now also be determined with greater confidence rather than assuming only a single concentration across an entire collection.…”