Mitchell E. Johnson

Development of near-infrared fluorophoric labels for the determination of fatty acids separated by capillary electrophoresis with diode laser induced fluorescence detection†

Synthesis and characterization of a polymethine cyanine near-infrared (NIR) fluorophoric label for the derivatization and determination of fatty acids separated by capillary electrophoresis are described. The label contains an aromatic amine functionality, which is used to form a covalent linkage with the analyte. Various linking chemistries are explored, including direct amine–acid condensation using dicyclohexylcarbodiimide (DCC) as a carboxyl activating group. Spectrofluorimetry was used to probe the fluorescence efficiency of the label in order to assist in choosing a separation medium for capillary electrophoretic separation. A nonaqueous separation medium for capillary zone electrophoresis was used to provide high quantum efficiency for fluorescence and adequate solubility of fatty acid analytes. Diode laser-induced fluorescence detection following electrophoresis of a simple mixture of labeled fatty acids shows the applicability of this method to biologically relevant carboxylic acid analytes.

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the quantification of lipid-related extracellular metabolites in Saccharomyces cerevisiae.

A highly sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for the quantification of glycerophosphoinositol (GroPIns), glycerophosphocholine (GroPCho), glycerol 3-phosphate (GroP), inositol, and choline in the extracellular medium of Saccharomyces cerevisiae. The media samples were pretreated with a single two-phase liquid extraction. Chromatographic separation was achieved on a Waters Xbridge HILIC (150 mm × 4.6 mm, 5 μm) column under isocratic conditions using a mobile phase composed of acetonitrile/water, 70:30 (v/v) with 10mM ammonium acetate (pH adjusted to 4.5) at a flow-rate of 0.5 mL/min. Using a triple quadrupole tandem mass spectrometer, samples were detected in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curves were linear (r² ≥ 0.995) over the range of 0.5-150 nM, with the lower limit of quantitation validated at 0.5 nM for all analytes. The intra- and inter-day precision (calculated by coefficient of variation, CV%) ranged from 1.24 to 5.88% and 2.46 to 9.77%, respectively, and intra- and inter-day accuracy (calculated by relative error, RE%) was between -8.42 to 8.22% and -9.35 to 6.62%, respectively, at all quality control levels. The extracellular metabolites were stable throughout various storage stability studies. The fully validated method was successfully applied to determine the extracellular levels of phospholipid-related metabolites in S. cerevisiae.

Determination of fatty acid amides as trimethylsilyl derivatives by gas chromatography with mass spectrometric detection

Fatty acid amides are a newly emerging class of compounds with biological activity. The amides are formed enzymatically in vivo. Analysis of fatty acid amides has been accomplished by gas chromatography coupled with mass spectrometry. Fatty acid amides required derivatization prior to analysis at high temperatures due to thermal instability. Trimethylsilylation of fatty acid amides has been accomplished under optimum reaction conditions. The limit of detection for the silylated amides is approximately 1 pmol, with the lowest detected level being 700 fmol for the lauramide derivative. Quantitation of fatty acid amide derivatives can be accomplished by monitoring m/z 59 or m/z M-71, the only two major fragments formed in the ion trap mass spectrometer with electron impact ionization. The smaller fragment is the result of a newly reported, McLafferty-type rearrangement; M-71 resulted from loss of an n-pentyl fragment. Either peak gave four-five orders of magnitude linear dynamic range. Numerous trimethylsilylamides from C7 to C20 were separated under standard conditions. Elution was linear with the number of carbons and was systematically affected by the number and position of the double bonds.

Highly efficient microscale purification of glycerophospholipids by microfluidic cell lysis and lipid extraction for lipidomics profiling.

This article presents a novel method for small-scale lipidomics of bacterial cells by integrating extraction of glycerophospholipids on a microchip with a nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometer (nanoESI-Q-TOF MS/MS). The standard starting point for typical macroscale lipid analysis is a multiphase liquid-liquid extraction. Working with small populations of cells (1 to about 1000) requires a scaled down process in order to minimize dilution and facilitate the interface with microscale separation methods for sample cleanup and introduction to mass spectrometry. We have developed a microfluidic system that allows for lysis of bacterial cells, capture of lipids, and elution of captured lipids from a solid phase for microscale purification of lipids. The best on-chip extraction efficiency for glycerophospholipids was as high as 83.3% by integrating silica beads as the packing material with methanol as the eluent. A total of 10 successive measurements were evaluated indicating that the microchip packed with fresh silica beads is capable of being reused four times without any loss in the lipid extraction process. The initial screening based on high-resolution tandem mass spectrometry data along with a discovery profiling approach revealed the presence of 173 identified phospholipid species from microfluidic cell extracts. This work demonstrates the potential of incorporating microchip-based lipid extraction into cellular lipidomics research.

Robust Utilization of Phospholipase-Generated Metabolites, Glycerophosphodiesters, by Candida albicans: Role of the CaGit1 Permease

ABSTRACT Glycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. In Saccharomyces cerevisiae, a single gene, GIT1, encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, the Candida albicans genome contains four open reading frames (ORFs) with a high degree of similarity to S. cerevisiae GIT1 (ScGIT1) Here, we report that C. albicans utilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis of C. albicans GIT1 (CaGIT1) (orf19.34), the ORF most similar to ScGit1, abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [3H]GroPIns at acidic and physiological pHs, while reintegration of a GIT1 allele into the genome restored those functions. Several lines of evidence, including the detection of internal [3H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparent Km of 28 ± 6 μM. Notably, uptake of label from [3H]GroPCho was found to be roughly 50-fold greater than uptake of label from [3H]GroPIns and roughly 500-fold greater than the equivalent activity in S. cerevisiae. Insertional mutagenesis of CaGIT1 had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [3H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [3H]GroPIns and [3H]GroPCho. Screening of a transcription factor deletion set identified CaPHO4 as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.

Selective fluorescence derivatization and capillary electrophoretic separation of amidated amino acids

Abstract Selectively derivatized amide-terminated amino acids were separated by micellar electrokinetic capillary chromatography (MECC). The amides were selectively derivatized by deactivating all primary amines in the sample mixture by acetylation, converting the amides to primary amines by Hofmann rearrangement, and tagging the resultant amines with fluorescein isothiocyanate (FITC). The fluorescent amide derivatives were detected by confocal laser-induced fluorescence. The use of the MECC mode was mandated by very similar charge and size characteristics of the derivatized amides. Separation of 11 amino acid amides was carried out in bare fused silica capillaries in 10 m M borate and 90 m M sodium dodecyl sulfate buffer in under 15 min. Analysis of dispersion and mobility behavior suggests that hydrophobicity is the primary determinant of micelle/buffer partitioning, and, therefore, mobility; relative hydrophobicity based on values for amino acids is an adequate predictor of elution order for derivatives without charged side-chains. Selectivity of the reaction was estimated to be as great as 100, perhaps greater; the estimation was limited by ability of FITC to derivatize at low concentrations. Rearrangement reaction yields and kinetics were found by NMR spectroscopy to depend on side-chain identity. Yields were 80–100% for the rearrangement, and half-lives were 10–30 min for 100 m M solutions of several representative amino acid amides. Reaction products were not purified, for the sake of simplicity, and a number of impurity peaks appeared in the electropherograms. Because of the high efficiency of the separation (optimal plate heights below 2 μm), resolution was usually adequate to mitigate this potential problem.

The Use of Solid‐Phase Supports for Derivatization in Chromatography and Spectroscopy

Abstract Derivatization, or chemical modification of analytes, is often required for species that are only weakly detectable by common spectroscopic methods. Derivatization is most commonly performed in homogeneous solution or using phase‐transfer catalyzed reactions. However, the use of solid phase supports for performing the same reactions has a number of advantages. The sample can be “cleaned up” on the same phase, eliminating interfering matrix components or excess reagent. The process naturally concentrates the analyte, providing higher sensitivity, but also, under favorable circumstances, provides for more efficient reactions relative to solutions of the same original concentration. This review explores the uses to which such supports have been put, primarily in fluorescence derivatization for chromatographic applications. Some of the considerations in applying these techniques are described, and they are shown to be an extremely useful format for derivatization.

Ion trap mass spectrometry of trimethylsilylamides following gas chromatography

Fatty acid amides are a class of compounds with newly discovered biological activity. The ion trap mass spectrometric characteristics of silylated fatty acid amides were examined. Silylation of primary fatty acid amides is required prior to gas chromatography owing to thermal instability of the underivatized compound. The trimethylsilylated amides do not yield a molecular ion under normal electron ionization conditions (70 eV). With methane as a chemical ionization gas, the [M+H]+ ion appears. The [M+H]+ ion also appears when the helium buffer gas pressure is increased in the ion trap. There are three fragments other than the [M+H]+ peak that are predominant in the ion trap mass spectra of these compounds. Two of the fragments have been reported previously, namely the m/z 59 and the [M-71]+ fragments. The fragment of m/z 72 was identified and is the result of a rearrangement. Isotopic labeling was used to confirm fragment identity and the composition of the rearrangement products. Fragmentation patterns were affected by the amide chain length and concentration.

Characterization of a Rugged, Open-Gap Flow Cell for Confocal Laser-Induced Fluorescence Detection in Capillary Electrophoresis

Construction details and performance characteristics of an open-gap flow cell for fluorescence detection in capillary electrophoresis are described. The flow cell is created by separating two pieces of capillary by a small (90 μm) gap. The gap is surrounded with buffer and grounded, and the application of electric fields to both inlet and outlet capillaries causes the material in the inlet capillary to flow across the gap. The use of a simple confocal optical arrangement for laser-induced fluorescence detection allows straightforward application of the gap flow cell to detection in capillary electrophoresis. The signal-to-noise ratio is measured to be about a factor of 2 better than that for on-column confocal detection at nanomolar concentrations over a wide range of pinhole diameters. The detection limit for fluorescein isothiocyanate is in the low picomolar range. Detection of a simple mixture of amino acids that have been derivatized with fluorescein isothiocyan ate demonstrates the stability and utility of the gap. Increased tailing is observed with the gap cell, with average asymmetry of about 1.4 near the center of the gap. Dispersion characteristics as a function of position in the gap are interpreted as dilution of the analyte as it flows across the gap. Fortunately, resolution and theoretical plates, measured by using least-squares fitting, are not significantly different from on-column separations, in spite of the tailing.