Abstract: Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2′-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a “routine” marker of oxidative DNA damage in vivo.

Abstract: Detection was by UV absorbance (after pre-column derivatization of dehydroascorbic acid with 1,2-phenylenediamine). When absent, isoascorbic acid could be used as internal standard. Isocratic separation was accomplished in 11 min using the eluent, methanol-water (5:95, v/v) containing potassium dihydrogen phosphate (50 mM) and the counterion hexadecyltrimethylammonium bromide (5 mM). Sample preparation steps using Sep-pak C18 cartridge were minimal. Ten ppm could be detected for each compound with good reproducibility (c.v. < 2%). The method was used to determine vitamin C content in selected foods and beverages.

Abstract: Particle beam liquid chromatography/mass spectrometry (PB/LC/MS) was used to analyze finished drinking water for non-volatile organic compounds. 500 liters of finished water were extracted with an on-line continuous liquid/liquid extractor with dichloromethane at pH 7.4. PB/LC/MS was an excellent tool to detect and identify ng/L concentrations of alkylphenol polyethoxylates (n = 3−8), materials which went undetected by on-column gas chromatography/mass spectrometry. In addition, alkylphenol polyethoxylate carboxylates with 2–7 degrees of ethoxylation could be detected without chemical derivatization.

Abstract: Analytical-scale supercritical fluid extraction (SFE) of polar organics from solid samples was enhanced by derivatizing the analytes to less polar under static SFE conditions using reagents including trimethylphenylammonium hydroxide and boron trifluoride in methanol. After derivatization, the analytes were extracted with CO 2 using standard SFE techniques and analyzed without additional treatment using conventional capillary GC

Abstract: We report a novel aqueous derivatization of methylmercury chloride by NaBH 4 to mehtymercury hydride (CH 3 HgH), a volatile and unexpectedly stable species which we determined has a half-life of approximately 2 h. The analytical apparatus consisted of a purge and trap (PT) unit linked to a gas chromatograph (GC), in line with a Fourier transform infrared spectrometer (FTIR). The sample was purged with nitrogen, and volatile compounds were concentrated in a cold trap

Abstract: The analysis of carbohydrates has been always hampered by their lack of UV absorbance above 200 nm, which is an especially challenging problem in capillary electrophoresis due to the very small (nl) sample volumes injected. The introduction of 2-aminopyridine as derivatizing agent allows sensitive direct UV detection of saccharides in the fmol range. However, due to the requirement of the presence of a free aldehyde group only aldoses and uronic acids can be determined. This limitation was recently overcome by means of precolumn derivatization withp-aminobenzoic acid or ethylp-aminobenzoate, which permits the analysis of fructose with a lower mass detection limit of 0.3 and 0.14 pmol, respectively. The detection limits for aldoses were even as low as 15 and 7 fmol. A more universal approach is the use of indirect UV detection, which permits the analysis of carbohydrates, including (1–2)-linked disaccharides and aldonic acids, at the lower pmol level without the need for derivatization.

Abstract: A variety of different peptide-mapping schemes are presented, with emphasis on the development of procedures which can be done with limited quantities (i.e. 5 pmol) of protein. Results are obtained from model proteins which contain disulfide bonds, which must be broken prior to fragmentation of the protein. A reaction involving the simultaneous use of tributylphosphine and 2-methylaziridine to reduce and alkylate the disulfide bonds is employed, due to favorable attributes of these reagents for the scaled-down procedure. The traditional performic acid oxidation reaction to cleave cystine groups is also successfully used with low-picomole quantities of protein. Three different protein digestion reagents are used: trypsin, chymotrypsin, and cyanogen bromide. Each reagent produces a unique mixture of peptides. Capillary electrophoresis is used to separate the peptides, offering high separation efficiencies, short analysis times, and compatibility with small sample sizes. In addition to the conventional use of UV detection for underivatized peptides, laser-induced fluorescence detection is employed in conjunction with an arginine-selective derivatization reaction. This latter procedure for derivatization and detection offers an alternative peptide-mapping mode, in which only the arginine-containing peptides are detected, and is useful in simplifying the peptide maps of large proteins.

Abstract: Mixtures of several basic proteins have been used to test CZE capillaries with surfaces modified by new pretreatment procedures; the performance obtained has been compared with that achieved using capillaries treated by procedures described in the literature. It has been shown that addition of non-ionic polyvinylalcohols (PVA) to CZE buffer solutions deactivates even bare, i.e. untreated, fused silica surfaces and renders them suitable for separations of basic proteins. The performance obtained from such surfaces was comparable with that of capillaries modified by the more elaborate procedures of etching, silanol derivatization, and/or adsorptive coating (again with polymers). A home-made device is described which enables derivatization and coating reactions to be performed on fused silica capillaries under an inert atmosphere, i.e. one free from oxygen and water.

Abstract: The cross-link was liberated by exhaustive proteolytic digestion and derivatized with PITC. The derivatives were resolved by HPLC. The present method enables one to determine simultaneously protein-constituting amino acids and ∈-(γ-glucyl)lysine and thereby facilitates the monitoring of progress of proteolytic digestion. The PTC-∈-(γ-glutamyl)lysine peak was collected and confirmed by conversion to a stable PTH form followed by HCl hydrolysis

Abstract: A simple and highly sensitive method for the determination of histamine (HA) was developed using ion-pair, reversed-phase HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation-exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation-exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing (R)alpha-methylhistamine, a potent and specific H3-receptor agonist, which could not be separated from HA by cation-exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypothalamus that (R)alpha-methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30-60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.

Abstract: Uric acid is generated by the oxidation of purines, but no enzyme is present to oxidize it further. It is present in plasma at a concentration approaching 500 J.lIIlollL and has been proposed as an important antioxidant. 1 In agreement with this proposal experiments in vitro have shown the protective effect of uric acid against oxidative damage. The first and major product of uric acid oxidation is allantoin, and it has been suggested that allantoin may be useful as a 'marker' of free radical reactions in vivo.2,3 However, methods currently used for the analysis of allantoin in body fluids are tedious and complicated, and since allantoin is very polar' it is difficult to obtain resolution from other serum constituents that have a similar polarity on a reverse-phase HPLC column. In the method of Grootveld and Halliwelluric acid is first analysed by HPLC, and allantoin is then measured in a separate procedure which involves initial separation and fraction collection by HPLC, hydrolysis and derivatization of allantoin, and finally separation of the derivatives by HPLC. We have developed a unique chromatographic assay to determine uric acid and allantoin simultaneously in plasma ultrafiltrate which is a significant improvement on previous HPLC methods.

Abstract: Two novel and convenient methods for obtaining volatile mercury and methylmercury species for analysis that allows non-volatile analyte solutions to be identified by element-specific and species-selective techniques are described. Non-volatile aqueous solutions of mercury(II) and/or methylmercury(II) compounds are converted into volatile forms (including hydrides) using NaBH4 or LiB(C2H5)3H, followed by chromatographic separation. The volatile derivatives separated on a column are detected by atomic absorption spectrometry or mass spectrometry.

Abstract: A stereospecific HPLC assay has been developed for the determination of the enantiomers of fluoxetine and an active metabolite, norfluoxetine, in plasma and tissue. Following derivatization with R-1-(1-napthyl)ethyl isocyanate, the diasteriomeric derivatives are resolved on a 5 μ.m silica column using a iso-octane/tetrahydrofuran mobile phase with fluorescence detection. The assay has a sensitivity of 5 ng/ml in plasma and 25 ng/gm in tissue. The calibration curves are linear over the range of 5–1000 ng/ml. The assay appears to be readily applicable to the study of enantioselective fluoxetine pharmacokinetics in animals and humans.

Abstract: The substitution patterns of methyl cellulose as well as of a thexyldimethylsilyl cellulose after permethylation were determined by hydrolysis and separation of the resulting partially methylated glucoses without further derivatization by h.p.l.c.. On an amine-modified silica column the solutes get separated into glucose, 2,3,6-tri-O-methyl glucose and the groups of mono-O-methyl-and di-O-methyl glucoses. A chromatographic run on a reversed-phase column enables the identification of the single mono-O-methyl- and di-O-methyl glucoses. In this way, a determination of both the average degree of substitution and the substitution pattern of cellulose derivatives is possible. Comparison of the results with those obtained by standard methylation analysis including g.l.c.-m.s. proves the correctnes of the method employed.

Abstract: Six chiral derivatization reagents, viz., (+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-DBD-APy), (–)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-DBD-APy), (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-NBD-APy), (–)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-NBD-APy), (+)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-ABD-APy) and (–)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-ABD-APy), have been synthesized to permit separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC). The reagents react with carboxylic acids at room temperature in the presence of activation agents (2,2′-dipyridyl disulfide and triphenylphosphine). The maximum emission wavelengths of the diastereomeric amide derivatives formed from D-naproxen (Nap) with D-DBD-APy, D-ABD-APy and D-NBD-APy were approximately 580 nm (excitation at 470 nm), 585 nm (excitation at 470 nm) and 540 nm (excitation at 470 nm), respectively. The emission wavelengths of the derivatives shifted towards the blue, and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The fluorescence intensities of three derivatives at pH 2–11 were higher in neutral than in acidic and alkaline solutions. The diastereomers derived from anti-inflammatory drugs and N-acetylamino acids were efficiently resolved by a reversed-phase column (5 µ Inertsil ODS-2) with a water-acetonitrile mixture as mobile phase. The detection limits (S/N = 2) of DBD-APy-Nap, NBD-APy-Nap and ABD-APy-Nap on HPLC chromatograms were 10, 15 and 30 fmol, respectively.