Analytical and biological evaluation of high throughput screen actives using evaporative light scattering, chemiluminescent nitrogen detection, and accurate mass LC-MS-MS.

In this investigation the utility of evaporative light scattering detection (ELSD) combined with HPLC-MS was demonstrated as a key component of a bioassay-guided fractionation, or "biofractionation" technique, for the evaluation of high throughput screen actives. ELSD provided on-line analyte mass information that was critical for the classification of the samples. Chemiluminescent nitrogen detection (CLND) was also evaluated for sample concentration estimation for nitrogen-containing compounds, and accurate mass LC-MS-MS analysis was employed for rapid structural confirmation and elucidation of components previously identified as active via biofractionation.

Chiral separations of polar compounds by hydrophilic interaction chromatography with evaporative light scattering detection.

The chiral separations of drug substances and underivatized amino acids were demonstrated in this study through the use of hydrophilic interaction chromatography (HILIC). The polar character of the model compounds presented challenges for their analysis by traditional modes of chromatography, but through the employment of multimodal chromatography utilizing the HILIC mechanism and cyclodextrin- or teicoplanin-derivatized stationary phases, effective resolution was achieved. The analytes lacked sufficient ultraviolet chromophores, requiring their determination by evaporative light scattering detection. HILIC was demonstrated to represent a novel technique for the facilitation of chiral chromatography by providing an environment of solubility and retention that could not be achieved through the use of the traditional methods of reversed-phase, normal-phase, or polar organic mode.

High-performance liquid chromatographic-electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening.

Within the pharmaceutical industry, significant resources have been applied to the identification of new drug compound leads through the use of high-throughput screening (HTS). To meet the demand for rapid analytical characterization of biologically active samples identified by HTS, the technique of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) has been utilized, and the application of this technique specifically for the integration of natural product sample mixtures into modern HTS is reviewed. The high resolution provided by reversed-phase HPLC coupled with the gentle and relatively universal ionization facilitated by the electrospray process has had significant impact upon a variety of procedures associated with the HTS of natural products, including extract sample diversity evaluation, dereplication, structure elucidation, preparative isolation, and affinity-based biological activity evaluation.

Hydrophilic interaction chromatography-electrospray mass spectrometry analysis of polar compounds for natural product drug discovery.

For the drug discovery efforts currently taking place within the pharmaceutical industry, natural product extracts have been found to provide a valuable source of molecular diversity which is complementary to that provided by traditional synthetic organic methods or combinatorial chemistry. However, there exists a need for analytical tools that can facilitate the separation and characterization of components from these sources in a rapid manner. Specifically, the evaluation of highly polar compounds (i.e., compounds that cannot be retained on traditional reversed-phase stationary phases) has been challenging, and a hydrophilic interaction chromatography-electrospray ionization mass spectrometry (HILIC-ESI-MS) method was developed to meet this need. In this investigation, amide-, Polyhydroxyethyl Aspartamide-, and cyclodextrin-based packings provided superior performance for the analysis of a set of polar natural product compounds. The properties of the mobile-phase buffers also greatly impacted the separations, and relative to other volatile buffering agents, ammonium acetate at a concentration of approximately 6.5 mM was determined to facilitate optimal HILIC retention, reproducibility, and durability. An optimized HILIC-ESI-MS system was successfully applied for the analysis of complex natural product mixtures. The techniques described in this report should also prove useful for the analysis of polar compounds from synthetic sources of molecular diversity such as combinatorial chemistry.

Analysis of trifluoroacetic acid in lyophilized natural products by capillary electrophoresis.

A rapid and simple method for the capillary electrophoretic determination of residual trifluoroacetic acid in lyophilized natural products is described. The technique utilizes 2,6-naphthalenedicarboxylic acid as a separation buffer additive providing indirect ultraviolet absorption detection. Using this method, acceptable precision, accuracy, selectivity, range and linearity were achieved.

Capillary electrophoresis of biotechnology-derived proteins.

Applications of capillary electrophoretic techniques for the analysis of biotechnology-derived proteins continue at a steady pace. This review summarizes the analyses of these proteins achieved using capillary isoelectric focusing, micellar electrokinetic capillary chromatography, capillary electrophoresis/mass spectrometry and capillary gel electrophoresis. A very brief summary of each technique is included. Application of capillary isoelectric focusing to the analysis of recombinant tissue plasminogen activator in quality control type applications is examined in some detail. Analyses of dosage forms of biotechnology derived proteins of pharmaceutical importance as well as the identification and examination of the peptides, obtained after enzymatic cleavage of proteins, by capillary electrophoresis have been included. Examples of capillary gel electrophoresis as a substitute sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of biotechnology-derived proteins are also reviewed.

Analysis of recombinant human growth hormone in Escherichia coli fermentation broth by micellar high-performance liquid chromatography.

A method for the reversed-phase high-performance liquid chromatographic (HPLC) determination of recombinant methionylaspartyl-human growth hormone (MD-HGH) in Escherichia coli fermentation broth is described. The technique utilizes mobile phases containing n-propanol and the anionic surfactant sodium dodecyl sulfate (SDS) under micellar conditions at pH 6.4. The methodology is directly applicable to the analysis of samples solubilized via sulfitolysis in the presence of SDS, and offers superior resolution in comparison with chromatography in the absence of the surfactant. Using this method, acceptable precision (day-to-day R.S.D. = 4.9%), accuracy, selectivity, range, linearity and ruggedness were achieved.

Capillary electrophoretic separations of biotechnology-derived proteins in E. coli fermentation broth.

A capillary electrophoresis (CE) method was developed for the analysis of a recombinant DNA protein in a fermentation broth matrix. A polyacrylamide-coated capillary was employed to eliminate electroosmotic flow, facilitating relatively rapid run times. Selectivity and efficiency were enhanced significantly through the incorporation of magnesium sulfate and acetonitrile, an organic modifier, into the separation buffer. Both of these additives appeared to influence the separation by diminishing the interaction of the protein analyte with the charged micelles present in the buffer. The quantitation capabilities of the CE method were validated and found to be comparable to the standards set by chromatographic techniques previously developed for similar applications.

Capillary electrophoretic analysis of serine hydroxymethyltransferase in Escherichia coli fermentation broth.

Serine hydroxymethyltransferase (SHMT) expressed in Escherichia coli was analyzed in fermentation broth through the use of capillary electrophoresis (CE), a method which provided advantages over the traditional techniques of slab gel electrophoresis and chromatography. In addition, via CE the difficult resolution and quantitation of SHMT holoenzyme and apoenzyme were achieved. Using this method, a pyridoxal-5'-phosphate (PLP) cofactor/SHMT dimer molar ratio of 0.65 was estimated to be present in holoenzyme in the absence of excess PLP. This determination correlated well with results obtained by other techniques, including electrospray ionization mass spectrometry (ESI-MS). CE and ESI-MS analyses both provided evidence for significant differences between the folded conformations of SHMT holoenzyme and apoenzyme.

Micellar electrokinetic capillary chromatography of proteins.

Separations of model proteins obtained under denaturing conditions in the presence of micellar concentrations of ionic surfactants displayed high resolution and efficiency using either bare silica or C18-derivatized silica capillaries. Superior migration time reproducibility was achieved through the use of the C18-derivatized capillaries (run-to-run migration time % RSD = 0.2), relative to that obtained in bare silica capillaries (run-to-run migration time % RSD = 2.2), in the absence of buffer replenishment. The effects of surfactant concentration and pH upon the separation of a mixture of five model proteins of varying ionic and hydrophobic character were investigated, and the application of this technique to the analysis of a recombinant DNA-derived protein in fermentation broth was demonstrated.

Anion-exchange chromatography of DNA restriction fragments.

The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied. The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1. The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range. Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest. The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved. Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing. Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.