Dual Detection Approach to a More Accurate Measure of Relative

Aug 13, 2008 - The accurate determination of compound purity is crucial for characterizing library purity, monitoring the stability of storage compoun...
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J. Comb. Chem. 2008, 10, 746–751

Dual Detection Approach to a More Accurate Measure of Relative Purity in High-Throughput Characterization of Compound Collections Andrew Lemoff† and Bing Yan*,†,‡ St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and Shandong UniVersity, Jinan, China 250100 ReceiVed June 17, 2008 The accurate determination of compound purity is crucial for characterizing library purity, monitoring the stability of storage compounds, and obtaining meaningful high-throughput screening results. However, current high-throughput techniques for the determination of compound purity are inadequate. We evaluated on-line chromatography detectors, including UVTWC, UV214, and ELSD detectors, in a series of studies of 233 compound mixtures prepared with known compositions. Results indicate that both UVTWC and UV214 overestimate the minor component in a mixture whereas ELSD underestimates the minor component. An average of UVTWC and ELSD purities gives a more accurate measure of the relative purity for a wide range of compounds in various purity ranges. This technique was applied to 959 compounds from our compound collection to more accurately determine their relative purity. The study of interactions between small molecules and proteins or cells is a critical undertaking in drug discovery and chemical biology research. These molecular probes, coming from parallel synthesis, natural products isolation, or one-at-a-time synthesis, are stored in compound repositories. Due to the postpurification processing and the continuous degradation in storage, the purity of compounds needs to be repeatedly surveyed using high-throughput analytical techniques during their screening and storage life cycle. On the other hand, the determination of compound purity is also essential for quality control of compound library synthesis and production. However, the reported relative purity of a compound is often an uncertain number without knowing the details of how it was measured. For example, the purity of the same sample can be reported to have a higher than its true purity by UV254 or evaporative light scattering detector (ELSD) detection methods or a lower value by UV214 or total wavelength content, 210–400 nm (UVTWC), methods. Several techniques that are commonly used to determine the quantitative purity of a compound of interest are not well suited to a high-throughput environment. The use of standards to generate calibration curves for analysis of each compound in a compound library is not feasible due to the lack of authentic compound standards. Flow-injection NMR analysis1 has been shown to quantify samples accurately and recover most of samples. However, the time required for spectral interpretation is still a bottleneck. Elemental analysis is another accurate technique.2 However, it is time-consuming and requires too much sample; therefore, it is not suitable for compound collections that contain compounds with * To whom correspondence should be addressed. Phone: +9014952797. Fax: +9014955715. E-mail:[email protected]. † St. Jude Children’s Research Hospital. ‡ Shandong University.

microgram quantity. Chemiluminescent nitrogen detection (CLND)3-9 is compatible with a medium-throughput operation, but it is not widely used. Due to difficulties in obtaining quantitative purity of compounds in repositories or libraries, relative purity is often an acceptable measure for compound quality considering the fact that the relative purity is very close to the true quantitative purity if compounds are chromatographically purified.9,10 One commonly used technique for determining compound relative purity is LC/UV/ELSD/MS.10-19 This method is fast and requires little sample. Compounds and their impurities are separated on a reverse-phase HPLC column, which can take only a couple of minutes in the case of ultraperformance liquid chromatography (UPLC). The compounds are identified by their mass-to-charge ratio using mass spectrometry and the purity of the compound of interest can then be determined by UV at a single wavelength or over a range of wavelengths with a photodiode array detector, or by ELSD. Although these detection methods are widely used, each has its own unique drawbacks that make the use of a single detector problematic. Although mass spectrometry is an excellent technique for determining the identity of a compound, the ion current is highly dependent on the ionization efficiency of the compound and therefore mass spectrometry does not yield accurate purity information. UV absorbance at a single wavelength does not always yield accurate purity either, as the absorbance of compounds is wavelength dependent. For example, purity measured by UV absorbance at 254 nm is often overestimated because many impurities may have no absorption at this wavelength.10 UV detection using a PDA allows for analysis over a wide range of wavelengths and is better than a single wavelength UV detector. However, some compounds do not have a UV chromophore and therefore are invisible to the PDA. ELSD

10.1021/cc800100g CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

Lemhoff and Yan

Journal of Combinatorial Chemistry, 2008 Vol. 10, No. 5 747

Table 1. Commercial Compounds Used in the Study number

compound name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

hydrocortisone acetate hydrocortisone cortisone acetate cortisone progesterone cyproterone acetate mifepristone dexamethasone prednisone Fmoc-ornithine(Alloc) Fmoc-glutamic acid(OcHx) Fmoc-phenylglycine Fmoc-O-tBu-serine Fmoc-isoleucine Fmoc-lysine(Alloc) Fmoc-phenylalanine 2′,3′-O-isopropylideneadenosine N-(R)-tosyl-L-arginine methyl ester phthalylsulfacetamide warfarin perphenazine

does not require that the compound contain a UV chromophore, but detection does depend on the volatility of the compound. Highly volatile compounds are not detected, and low molecular-weight compounds (