Monitoring the formation of Maillard reaction products of glucosamine with fibrinogen and human serum albumin using capillary electrophoresis.

Formation of Maillard reaction products (MRP) of glucosamine (GlcN) with fibrinogen and human serum albumin (HSA), under simulated physiological conditions, was detected by fluorescence (excitation/emission: 340/420 nm) and UV/Vis (max. 275 nm) spectroscopy. The application of polyacrylamide gel electrophoresis demonstrated the generation of high-molecular-weight fibrinogen and HSA MRP by GlcN. A simple and rapid capillary electrophoresis method was developed to separate MRP formed by the reaction of GlcN with proteins from GlcN autocondensation products.

Capillary electrophoresis separation of autocondensation glycation products of glucosamine.

Glucosamine nonenzymatically forms autocondensation glycation products under physiological conditions. Many studies have reported the effectiveness of oral doses of glucosamine alone or in combination with the galactosamine containing chondroitin in treating osteoarthritis. However, none of these studies has considered whether it is the glucosamine itself and/or one or more of its autocondensation products that exert this effect. A capillary electrophoresis method was developed to monitor the nonenzymatic formation of autocondensation glycation products of glucosamine, galactosamine, and mannosamine under physiological conditions. Major components were detected and separated by CE with a UV detector. The effects of concentration and incubation time on product species were determined. The method described is simple, rapid, and effective.

The optimization of HPLC-UV conditions for use with FTIR detection in the analysis of B vitamins.

The water-soluble vitamins thiamine (B(1)), riboflavin (B(2)), pantothenic acid (B(5)), and pyridoxine (B(6)) are separated by high-performance liquid chromatography. The mobile phase, column temperature, and flow rate are optimized so that the chromatograph can be used with a Fourier-transform infrared (FTIR) detector. Reproducibility, linearity, and detection limits are evaluated for method validation. Finally, this method is successfully transferred to liquid chromatography-FTIR with a standard mixture.

Perspectives on analyses of nucleic acid constituents: the basis of genomics.

The recent mapping of the human genome was a tremendous achievement made possible to a large degree by the development of analytical methods for sequencing purine and pyrimidine bases in nucleic acids. In the last 3 decades, the number of analyses of nucleic acids and their constituents by HPLC and capillary electrophoresis (CE) has exploded. These techniques have been used not only for genomics, but also for the determination of free nucleotides, nucleosides and their bases in body fluids and tissues. Although a large number of HPLC and CE papers have been published on nucleic acid constituent applications, relatively little has been written on the mechanisms of the separations. However, to optimize analytical conditions knowledgeably and rapidly, it is important to know why and how these separations occur and the factors that affect them. The HPLC methods for the analysis of nucleic acid constituents and the information available on some of the mechanisms of separation of nucleotides, nucleosides and their bases, as well as the analysis of these compounds by CE and the factors that affect these separations are discussed.

Capillary electrophoretic separation by double-strand polyaniline-coate capillaries of the advanced glycation endproducts formed from N-alpha-acetyl-L-lysine with reducing sugars.

Capillary electrophoresis using a capillary coated with a double-strand coating of polyaniline:poly(methyacrylate-co-acrylic acid) (PAN:P[MA-AAI) was used to separate advanced glycation endproducts (AGEs) formed at 37 degrees C from model systems containing either glucose (Glc), fructose (Fru), or glyceraldehyde (GA) and N-alpha-acetyl-L-lysine (NALys). The presence of the P(MA-AA) as a second strand in the polymer allows the maintenance of the conductive state of the PAN at a wide pH range. Effects of buffer pH and coating concentration on the electroosmotic flow (EOF) were investigated. More AGE species can be detected for the GA/NALys mixtures using this coated capillary than upon an uncoated capillary. The coating procedure is simple and the stability of the coated capillary is good.

Analysis of C60 and C70 fullerenes using high-performance liquid chromatography-fourier transform infrared spectroscopy.

The performance of Fourier transform infrared spectroscopy (FT-IR) detection coupled to high-performance liquid chromatography for the analysis of C60 and C70 fullerenes was investigated. The isocratic separation method involved an octadecylsilane (ODS) column and an acetonitrile-toluene (1:1) mobile phase. The hyphenated system was designed with a split valve to control eluent volume leading to the FT-IR detector; this allowed for additional coupling of the liquid chromatograph to ultraviolet-visible detection. On-line FT-IR spectra of C60 and C70 were matched with standard off-line FT-IR spectra from the literature. In addition, with band chromatograms individual fullerenes can be identified using FT-IR active modes known specifically for each fullerene. Few changes to a pre-existing HPLC-UV method were necessary for the HPLC-FT-IR method, and there was no need for fraction collection to identify the fullerenes C60 and C70.

Enhanced analysis of purine and pyrimidine bases by the use of double-strand polyaniline coatings in micellar electrokinetic capillary chromatography.

A double-strand polymeric complex, which suppresses electroosmotic flow relative to fused-silica, is described. The polymeric complex contains a strand polyaniline (PAN) with the second strand containing polyacrylic acid (PAA) and methacrylate (MA) groups. The complex is referred to as PAN:P(AAMA). This polymeric complex has pH-controlled electroactive and hydrophobic characteristics and can be easily coated onto fused-silica. Enhanced separations of theophylline, theobromine, caffeine and adenine, thymine, uracil and cytosine were obtained by the use of the coated capillary in the micellar electrokinetic capillary electrophoresis (MEKC) system. The purine and pyrimdine bases were separated on the coated capillary with a 20 mM, pH 7 phosphate buffer which contains 0.05 M sodium dodecyl sulfate (SDS) as an additive.

Mass spectral characterization of tetracyclines by electrospray ionization, H/D exchange, and multiple stage mass spectrometry.

Electrospray ionization (ESI) and collisionally induced dissociation (CID) mass spectra were obtained for five tetracyclines and the corresponding compounds in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The number of labile hydrogens, x, could easily be determined from a comparison of ESI spectra obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M + H]+ and [M - H]- ions and the exchanged analogs, [M(Dx) + D]+ and [M(Dx) - D]- , and produced by ESI using a Sciex API-III(plus) and a Finnigan LCQ ion trap mass spectrometer. Compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(N) spectra of the un-deuterated and deuterated species. Protonated tetracyclines dissociate initially by loss of H2O (D2O) and NH3 (ND3) if there is a tertiary OH at C-6. The loss of H2O (D2O) is the lower energy process. Tetracyclines without the tertiary OH at C-6 lose only NH3 (ND3) initially. MSN experiments showed easily understandable losses of HDO, HN(CH3)2, CH3 - N=CH2, and CO from fragment ions. The major fragment ions do not come from cleavage reactions of the species protonated at the most basic site. Deprotonated tetracyclines had similar CID spectra, with less fragmentation than those observed for the protonated tetracyclines. The lowest energy decomposition paths for the deprotonated tetracyclines are the competitive loss of NH3 (ND3) or HNCO (DNCO). Product ions appear to be formed by charge remote decompositions of species de-protonated at the C-10 phenol.

The Separation and the Characterization of Long Chain Fatty Acids and Their Derivatives by Reversed Phase High Performance Liquid Chromatography.

The potential for the application of chromatography to the analysis of fatty acids was first realized by A. J. James and A. J. P. Martin in 19521. These two noted scientists successfully separated the iso- and ante-isomers of short chain free fatty acids by gas liquid chromatography (GLC). Even today, 35 years later, the method of choice for characterization of fatty acids is capillary gas chromatography with a mass spectrometer as a detector. However, high performance liquid chromatography (HPLC) is now becoming competitive in the separation of fatty acids, especially on the preparative scale.