Current Analytical Chemistry - Volume 3, Issue 4, 2007

Triacylglycerols (TAGs) are widely distributed, if not universal, in edible plants and edible plant triacylglycerols are nutrients forming an important part of the human diet. TAGs of higher plants consist mainly of esters of glycerol with fatty acids, many of which are essential as human nutrients and are therefore in the center of increasing attention. Despite their importance, the molecular species of each TAG class have not yet been fully characterized. The use of state-ofthe- art techniques, especially in a combination such as that discussed in this review, permits the identification of not only TAG classes, but also their positional isomers and often also individual molecular species. Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in combination with high-performance liquid chromatography (HPLC) has proven to be a very valuable technique for analysis of different lipids from a variety of sources. This paper describes direct analyses of TAGs from natural oils, frequently very unusual, using LC-MS/APCI.

Transcription factors are a large and diverse group of proteins which bind to DNA response elements in the promoters of genes either activating or repressing gene expression. The purification and characterization of transcription factors has been improving at a rapid pace. New purification techniques, including oligonucleotide trapping, and improved methods of detection, including chromatin immunoprecipitation have been combined with improved characterization using mass spectrometry methods. Here, we review and give examples of many of these recent advances.

Phospholipids are the main constituents of biological membranes. Their amphiphilic character is responsible for the typical bilayer arrangement as the structural basis of biological membranes. In addition to their structural role, some phospholipids are of significant functional importance. They act as intercellular messengers and are involved in the pathogenesis of many diseases. Therefore, phospholipid research (“lipidomics”) has significantly advanced in the last decades, generating the need for fast, reliable, and informative analytical techniques. The aim of this review is to demonstrate the power of 31P nuclear magnetic resonance (NMR) spectroscopy for structural and analytical phospholipid research. High resolution 31P NMR provides information on the composition of phospholipid mixtures, whereas solid-state 31P NMR gives structural information about the sample phase and morphology. This review provides an introduction into the field and a short overview of currently used analytical and physicochemical methods to study these biomolecules. We will provide a theoretical description of 31P NMR spectroscopy and discuss methods for obtaining highly resolved phospholipid spectra. Selected applications of 31P NMR to aid phospholipid analysis and the investigation of phospholipid structures, membrane-peptide interactions, and enzyme activities are discussed. This review ends with an overview on 31P NMR applications to the analysis of body fluids, cells, and tissues.

Implementation of combinatorial chemistry and high throughput screening to biological targets has led to produce lipophilic and poorly water soluble lead compounds. Since these compounds show poor absorbability, high throughput methods for improving solubility and membrane permeability have been developed. Kinetic solubility of compounds after dilution of dimethyl sulfoxide solution into aqueous buffer is determined using high throughput methods with turbidimetry, nephelometry or UV assays. The kinetic solubility tends to be overestimated compared with the corresponding thermodynamic solubility obtained using a traditional flask shaking method. A new solid-dissolution method providing thermodynamic solubility similar to that in traditional method has been developed using a 96-well plate for equilibrium dialysis. In conventional Caco-2 assays, the membrane permeability (Papp) of certain lipophilic compounds was underestimated due to insolubility in the apical compartment and adhesion to the device, resulting in a poor relationship between the in vivo absorption fraction and the Papp values. The addition of a solubilizer into the apical compartment and bovine serum albumin in the basolateral compartment improved the relationship, enabling evaluation of widely diverse compounds. The usefulness of these newly-developed methods for compounds with poor physical properties will be discussed, with reviewing currently available high throughput methods for optimizing absorption.

Field-flow fractionation (FFF) is a family of separation techniques able to fractionate a broad range of macromolecules, nano- and micro-sized particles of different origin. Among FFF, flow FFF (FlFFF) shows the highest separation versatility, and today it is the only FFF technique with significant market relevance. Commercial FlFFF employs macro-scale, flat-type channels. The idea of hollow-fiber (HF) membranes as tubular, micro-column channels for FlFFF (HF FlFFF) dates back 1974, with fundamentals on HF FlFFF given only in the late 1980s, and outstanding applications reported only over the last ten years. Compared to standard FlFFF, the key advantage of HF FlFFF lies in the instrumental simplicity and low-cost of the fractionation device, and in the low volume able to favor on-line coupling with orthogonal methods. Last-generation HF FlFFF has been successfully applied to macromolecules and particles such as proteins and whole cells, and shown particularly suited to be coupled with time-of-flight (TOF) mass spectrometry (MS) for intact protein profiling and characterization. We review basics on HF FlFFF theory and instrumentation, the early investigations, and the most recent technical and methodological developments that have improved HF FlFFF to a performance normally achieved by conventional FlFFF.