Biological, Medical, and Clinical Analysis

An increasingly available and attractive clinical analysis method, LC?MS nevertheless fails to directly benefit from the knowledge and experience of clinicians steeped in the immunoassay tradition.

This installment of "Milestones in Chromatography" discusses the events leading to the development of the amino acid analyzer near the end of the 1950s at the Rockefeller Institute of Medical Research by Moore, Stein, and Spackman.

Biologists entering liquid chromatography?mass spectrometry (LC?MS) from a background of general LC rarely use their mass spectrometer to its full potential. Isotope peaks offer huge possibilities both in semi-quantitative interpretation of structure and in quantitative labelling studies. This article examines charge state, "easy" isotopes, such as chlorine, the slightly harder problem of sulphur compounds, and finally looks at a method for improved measurement of heavy labels in a metabolic study.

The two most popular mechanical techniques for sample preparation for large-scale protein production are concussion and liquid shear.

Protein unfolding and aggregation can be serious considerations when designing laboratory and preparative chromatographic purification steps. This problem has been studied most thoroughly within the contexts of reversed-phase chromatography and hydrophobic interaction chromatography. However, there are currently no robust methods for resin selection capable of predicting adsorbed-phase protein stability as a function of amino acid sequence, secondary or tertiary structure, or resin characteristics.

The authors discuss a solid-phase extraction (SPE) clean-up procedure for drugs present in biofluids.

Hyaluronic acid (HA) is a naturally occurring, unbranched polysaccharide that consists of alternately repeating D-glucuronic acid and N-acetylglucosamine units. This biopolymer is present throughout all mammalian systems but occurs primarily in synovial (joint) fluid, vitreous humor, and various loose connective tissues (such as rooster comb) (1). HA is of enormous commercial interest for ophthalmic, medical, pharmacological, and cosmetic applications.

A simple HPLC procedure is described for the determination of bendroflumethiazide (BMFT) in pharmaceutical formulations and urine samples. No interferences from common additives or other drugs frequently administered with BMFT or from endogenous compounds in urine samples were found. The lack of an organic solvent in the mobile phase reduces the risk of environmental contamination and human toxicity.

A new detection method based upon aerosol charging was examined for its applicability and performance with high performance liquid chromatography (HPLC). Our results demonstrate universal detection of nonvolatile analytes with response magnitude that is independent of analyte chemical properties, four orders of magnitude dynamic range, low nanogram, lower limits of detection, and < 2% relative standard deviation response variability. Broad applicability was demonstrated for a range of methods including those using gradient elution, reversed phase, hydrophilic interaction, and ion chromatography; normal and narrow bore column formats; and in combination with other detectors (for example, UV detectors, evaporative light-scattering detectors, and mass spectrometers).

Formic acid often is used for the analysis of peptides in proteomic studies by HPLC-MS, due to its volatility and reduced signal suppression. However, poorer chromatographic performance can be obtained in comparison with trifluoroacetic acid or nonvolatile phosphate buffers due to increased overloading, which can occur even for extremely small sample masses. Comparison of a highly inert silica-ODS and a wholly polymeric phase indicated that overloading effects on both are very similar and caused by the mutual repulsion of solute ions on the hydrophobic column surface.

A new detection method based upon aerosol charging was examined for its applicability and performance with high performance liquid chromatography (HPLC). Our results demonstrate universal detection of nonvolatile analytes with response magnitude that is independent of analyte chemical properties, four orders of magnitude dynamic range, low nanogram, lower limits of detection, and < 2% relative standard deviation response variability. Broad applicability was demonstrated for a range of methods including those using gradient elution, reversed phase, hydrophilic interaction, and ion chromatography; normal and narrow bore column formats; and in combination with other detectors (for example, UV detectors, evaporative light-scattering detectors, and mass spectrometers).

The author focuses on some key considerations in the manufacture and use of microplates, particularly the 96-well plate.

A simple, rapid, and sensitive method is presented for clinical measurements of ascorbic acid and malondialdehyde in human serum.

This article establishes the estimation of the uncertainty associated with the chromatographic determination of biogenic amines.

New miniaturized liquid-membrane extraction designs are especially suited for speciation studies of metal ions in small amounts of biological fluids.

The authors evaluate the performance of multiplexed 96-lane CE with laser-induced fluorescence detection for high-throughput separation&#150;based assays using fluorescein isothiocyanate as a model compound and fluorescein as an internal standard.

SEC analysis of proteins. (Tosoh Biosep LLC)

The Death Cap mushroom is the cause of most mushroom-related poisonings in the world. The author has developed a highly efficient, sensitive CE technique that toxicologists and forensic analysts can use to determine the poisonous peptides in body fluids of affected patients.

In this article, the authors review recent developments in the research of whole-column-imaging detection for capillary electrophoresis (CE). Whole-column-imaging detection was developed for capillary isoelectric focusing, for which it proved to be an ideal detector. Several whole-column-imaging detectors -- including refractive index gradient imaging, UV-absorption imaging, and fluorescence imaging detectors -- have been studied. The capillary isoelectric focusing UV-absorption imaging technique even has been commercialized. The development of whole-column-imaging detection itself facilitates CE studies in many directions such as in electrophoretic dynamics within narrow channels, new separation modes, and two-dimensional separations. Whole-column-imaging detection also finds application in capillary zone electrophoresis.

This month's "Directions in Discovery" gives a brief overview of turbulent- flow liquid chromatography, a technique for fast separations. Two examples demonstrate the use of the technology for high-throughput bioanalytical separations in drug discovery.