Design, construction and application of a simple packed capillary high performance liquid chromatography/electrospray mass spectrometry system

Abstract

A simple packed-capillary high performance liquid chromatography (HPLC) system for the analysis of trace (picogram) mixtures by electrospray mass spectrometry is described in detail. This simple and cost-effective design is constructed from conventional hardware generally available in the modern LC/MS laboratory. Short slurry-packed vitreous silica capillary columns (5 cm ؋ 200 µm i.d.) are prepared in minutes from a variety of commercial bulk packing materials by fast, medium pressure bomb packing. Precise submicroliter per minute gradient elution of the capillary column is achieved by precolumn flow splitting of a conventional, analytical-scale, gradient HPLC pump operating at 0.5 to 3.0 mL/min. flow rates. Chromatographic system dead volume is reduced to a minimum via unique integration of the splitter, injector, capillary column and electrospray emitter into a single assembly, thereby markedly improving chromatographic performance and ease of use. System performance is demonstrated through several applications, including the determination of caffeine in untreated urine, the analysis of tryptic peptide mixtures harvested from electrophoretic gels and the determination of trace impurities in pharmaceutical bulk drugs. © 1997 by John Wiley & Sons, Ltd. This laboratory provides structural characterization support for the research and development of a wide range of pharmaceutical agents including both conventional 'small molecule' synthetics and biopolymers derived from recombinant technology. As such, we are routinely challenged by the demand for rapid and cost effective analyses of complex mixtures spanning diverse chemical classification. Such analyte diversity has required the implementation of nearly all sample ionization and 'hyphenated' introduction methods, the number of which precludes the luxury of maintaining costly, functionally dedicated mass spectrometry (MS) systems.This challenge has intensified due to acceleration of the pharmaceutical development process, prolific drug template expansion through combinatorial chemistry, and the need for decisive metabolite and degradant characterization in early high volume testing models and preclinical formulation design studies. Such early investigations are further hindered by severe sample size limitations rendered tractable only through marked chromatographic scale reduction.While several 'nano-scale' liquid separation methods have been successfully interfaced with mass spectrometry for such purposes including capillary electrophoresis (CE), supercritical fluid chromatography (SFC), and capillary electrochromatography (CEC), packed capillary HPLC methods with electrospray ionization (ESI) mass spectrometric detection are the most versatile and easiest to implement.1-11 HPLC and ESI-MS are well suited to each other and to the analysis of the widest range of highly functionalized, thermally labile, and reactive biologically active species. Adsorption mode HPLC, arguably the dominant modern chromatographic separation method, has the further advantage of being ...

Determination of in Vitro- and in Vivo-Formed DNA Adducts of 2-Amino-3-methylimidazo[4,5-f]quinoline by Capillary Liquid Chromatography/Microelectrospray Mass Spectrometry

“…Although these techniques never flourished, they do have niche applications. With compatible flow rates, the coupling of capillary LC to nano-or microelectrospray is a good marriage of sensitivity and specificity that has begun to receive attention (28)(29)(30)(31). Several groups have recently illustrated the usefulness of these capillary LC/ MS/MS on-line techniques with proteins and other biologically related analytes (29,32,33).…”

Quantification of the Heterocyclic Aromatic Amine DNA Adduct N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline in Livers of Rats Using Capillary Liquid Chromatography/Microelectrospray Mass Spectrometry: A Dose−Response Study

“…The recent emergence of micro- and nanoflow electrospray ionization ( μ ESI and n ESI) sources has resulted in profound decreases in the limits of detection attainable using mass spectrometry . Furthermore, operating at microliter and nanoliter flow rates has enabled the coupling of packed capillary liquid chromatography systems to mass spectrometry thereby allowing the detection of analyte compounds in biological samples. − Specifically, a capillary LC/μESI-MS method was recently developed to detect IQ−DNA adducts in the kidney of nonhuman primates undergoing carcinogen bioassay at levels approaching 1 adduct in 10 7 bases . However, with current studies aimed at exposing animals to carcinogen levels close to what would be expected in humans, it is essential to develop a capillary LC/ μ ESI-MS method capable of detecting less than 1 adduct in 10 8 bases.…”

Proteomics and proteoforms: Bottom-up or top-down, how to use high-resolution mass spectrometry to reach the Grail

“…This coupling also allowed to free itself from 2D GE as a prerequisite. The sensitivity of the whole process has been improved by miniaturization (from the proteolytic sample to the gas-phase ions, with the nanoLC-ESI coupling [27][28][29][30]) and by technological optimization (from the ion production to signal-to-noise ratios), with new ionic optics [31,32] and the use of electronic ion-trapping systems instead of scanning ion detection [33], such as linear ion traps, orbitraps or ICR cells. In parallel, the link between mass spectrometry data (peptide mass and sequence elements) and the genome and proteome databases of a given organism has been automated by developing specific bioinformatics tools that are continually evolving to follow the technological evolution [34].…”