High-performance liquid chromatography with spectrophotometric and electrochemical detection of a series of manganese(III) cationic porphyrins

Recent studies have revealed potent pharmacological activities of manganese-containing cationic porphyrins. An analytical method employing high-performance liquid chromatography with spectrophotometric and electrochemical detection (HPLC-UV/EC) suitable for in vivo applications is described for a series of manganese(III) cationic porphyrins with good separation and resolution. In particular, this method resolved the four atropisomers of manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+ or AEOL-10113), verified by mass spectrometry. Electrochemical and spectrophotometric methods of detection were compared using manganese(III) meso-tetrakis(1,3-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP5+ or AEOL-10150), the lead catalytic antioxidant of this series. Both methods of detection were quantitative, but electrochemical detection, although less specific for in vivo applications, appears to be considerably more sensitive than spectrophotometric detection.     

Detection of singlet oxygen and its role in dye-sensitized photooxidation in aqueous and micellar solutions

Indirect methods for the detection of singlet oxygen in dye-sensitized photooxidation based on its interception by some singlet oxygen acceptors in aqueous and micellar solutions are discussed. Mechanistic aspects and some applications of a very sensitive method using p-nitrosodimethylaniline in the presence of imidazole (RNO + imidazole method) are also treated. The technique of competition kinetics with a singlet oxygen quencher N-3 which can serve for the determination of the role of singlet oxygen is discussed as well. Such competition with tryptophan and guanosine shows that these substrates react exclusively or predominantly via the singlet oxygen mechanism in the presence of hematoporphyrin as sensitizing dye.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems. I. Characterization of subpopulations of cells.

Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.     

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective R_F values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.     

High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

Pulsed amperometric detection (PAD) is useful for detection of cephalosporins following separation on a C₁₈ column using an acetate buffer solvent with a small percentage of organic modifier. Under these conditions, the indirect PAD mode worked better than direct PAD, with IPAD outperforming both. A gradient program was demonstrated that allowed separation and sensitive electrochemical detection of eleven different cephalosporins with widely differing side chain structures. The cephalosporins could be detected to sub-micromolar levels with this separation. Applications of the method for quantitation of pharmaceutical formulations and for monitoring cephalexin in porcine serum were demonstrated. To improve the detectability of cephalexin, an on-column concentration scheme using separate concentration and elution solvents was applied to porcine serum.     

Capillary electrophoresis of chitooligosaccharides in acidic solution: Simple determination using a quaternary-ammonium-modified column and indirect photometric detection with Crystal Violet

Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 μM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC.     

Direct quantification of dimethylsulfoniopropionate (DMSP) with hydrophilic interaction liquid chromatography/mass spectrometry

A simple, derivatization free method for the direct determination of dimethylsulfoniopropionate (DMSP) using hydrophilic interaction liquid chromatography (HILIC)/mass spectrometry is introduced. DMSP is a zwitterionic osmolyte which is produced from marine plankton, macro algae and higher plants. Due to its central role in climate relevant geochemical processes as well as in plant physiology and chemical ecology there is a great interest in methods for its quantification. Since DMSP is labile and difficult to extract currently most protocols for quantification are based on indirect methods. Here we show that ultra performance liquid chromatography/mass spectrometry using a HILIC stationary phase is suitable for the direct quantification of DMSP from aqueous samples and microalgal extracts. The protocol requires minimal sample preparation and phytoplankton samples can be investigated after filtration of small volumes. The limit of detection is 20nM and the calibration curve is linear in the range of 60nM to 50μM. The use of [(2)H(6)]-DMSP as internal standard allows prolonged sample storage since it is transformed with the same kinetics as natural DMSP. This makes the method suitable for both laboratory and field studies.     

High-performance liquid chromatographic determination of mexiletine enantiomers in plasma using direct and indirect enantioselective separations

Two methods were developed for the determination of mexiletine enantiomers in plasma samples suitable for studies on the stereoselective disposition of this drug. Both methods used fluorescence detection to improve sensitivity and selectivity. The direct enantioselective separation was based on the chiral resolution of mexiletine-2-naphthamide derivatives on a Chiralcel OJ column. The calibration curves were linear over the concentration range 50–500 ng/ml for each enantiomer; therefore the method can be used only for therapeutic monitoring, drug interaction and multiple dose pharmacokinetic studies. The indirect method was based on the formation of diastereomers using o-phthaldialdehyde and N-acetyl-l-cysteine reagents. The diastereomers were resolved on a reversed-phase RP-18 column. The method proved to be suitable for single or multiple dose pharmakokinetic studies based on the loq quantification limit ng/ml) and the broader linear range (1–1000 ng/ml) obtained.     

Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals

We describe a protocol for the synthesis of mono-6(A)-(1-butyl-3-imidazolium)-6(A)-deoxy-beta-cyclodextrin chloride (BIMCD), a cationic, water-soluble cyclodextrin used in the chiral separation of amino acids and anionic pharmaceuticals by capillary electrophoresis. Starting from commercially available chemicals, BIMCD is synthesized in five steps. The first step involves a nucleophilic substitution between p-toluenesulfonyl chloride and imidazole to afford 1-(p-toluenesulfonyl)imidazole (A). In the second step, a nucleophilic substitution between beta-cyclodextrin and A affords mono-6(A)-(p-toluenesulfonyl)-6(A)-deoxy-beta-cyclodextrin (B). In the third step, a nucleophilic substitution between 1-bromobutane and imidazole affords 1-butylimidazole (C). In the fourth step, a nucleophilic addition between A and C affords BIMCD tosylate. In the final step, anion exchange using an ion-exchange resin yields BIMCD as a highly water-soluble solid. Each step takes up to 2 d, including the time required for product purification. The overall protocol requires approximately 6 d.     

Bioseparation and bioanalytical techniques in environmental monitoring

The growing use of antibody-based separation methods has paralleled the expansion of immunochemical detection methods in moving beyond the clinical diagnostic field to applications in environmental monitoring. In recent years high-performance immunoaffinity chromatography, which began as a separation technique in biochemical and clinical research, has been adapted for separating and quantifying environmental pollutants. Bioaffinity offers a selective biological basis for separation that can be incorporated into a modular analytical process for more efficient environmental analysis. The use of immunoaffinity chromatography for separation complements the use of immunoassay for detection. A widely used immunochemical detection method for environmental analyses is enzyme immunoassay. The objective of this paper is to review the status of bioaffinity-based analytical procedures for environmental applications and human exposure assessment studies. Environmental methods based on bioaffinity range from mature immunoassays to emerging techniques such as immunosensors and immunoaffinity chromatography procedures for small molecules.     

Some applications of reversed-phase high-performance liquid chromatography to oligosaccharide separations

Oligosaccharide separation on reversed-phase high-performance liquid chormatographic columns have been examined using a range of aqueous solvents. Addition of anionic, cationic and non-ionic surfactants, tetramethyl urea and organic solvents to the mobile phase cause faster elution of oligosaccharides, and allow the separation of the larger oligomers in an acceptable time. Addition of neutral, inorganic salts increase the retention factors considerably, and allows good resolution of some compounds poorly resolved in water alone.The mechanism operating in the separations approximates to that invoked in the solvophobic theory of reversed-phase chromatography. There is some evidence also of hydrogen bond effects. The improvements described should prove useful in the isolation and analysis of neutral oligosaccharides in general, and in structural analyses of polysaccharides in paritcular.     

Synthesis and characterization of methylated poly(L-histidine) to control the stability of its siRNA polyion complexes for RNAi

Poly(L-histidine) (PLH) with dimethylimidazole groups has been synthesized as a pH-sensitive polypeptide to control the stability of its small interfering RNA (siRNA) polyion complexes for RNA interference (RNAi). The resulting methylated PLH (PLH-Me) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Agarose gel retardation assay proved that the quaternary dimethylimidazole groups worked as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. PLH-Me exhibited no significant cytotoxicity despite the existence of cationic dimethylimidazole groups. By use of PLH-Me as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. These results suggest that PLH-Me controls the stability of siRNA polyion complexes by enhancing noncytotoxic siRNA delivery by optimizing the content balance of dimethyl, τ/π-methyl, and unmodified imidazole groups.     

Microemulsion electrokinetic chromatographic analysis of some polar compounds

This study presents the optimization of a microemulsion electrokinetic chromatographic (MEEKC) electrolyte solution by using UV detection and with the method, simultaneous separations of chemically, biochemically and pharmaceutically related anionic and cationic compounds. Representatives of the compound groups were from isoflavonoids, benzodiazepines, metanephrines, diuretics and peptide hormones. The MEEKC separations under basic conditions were first optimized using a two-component isoflavonoid mixture as the sample and an electrolyte containing 10 mM tetraborate as the main buffer (pH 9.5). The stable microemulsion phase was adjusted with various amounts of octane, 1-butanol and sodium dodecyl sulfate (SDS). An only acidified electrolyte solution used in the study was made of phosphoric acid (pH 1.8) containing octane, SDS and ethyl acetate. The analyses with isoflavonoids showed that electrophoretic mobilities of the investigated compounds were highly related to the concentrations of SDS and 1-butanol with linear and parabolic correlation, respectively. However, addition of octane gave linear correlation only at low concentrations. In most cases four to six structurally related compounds and even 13 diuretics with various polar properties were separated from each other in basic microemulsion medium. The acidified MEEKC electrolyte gave good resolution for anionic metanephrines.     

Analysis of trifluoroacetic acid in lyophilized natural products by capillary electrophoresis

A rapid and simple method for the capillary electrophoretic determination of residual trifluoroacetic acid in lyophilized natural products is described. The technique utilizes 2,6-naphthalenedicarboxylic acid as a separation buffer additive providing indirect ultraviolet absorption detection. Using this method, acceptable precision, accuracy, selectivity, range and linearity were achieved.     

Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes

A label-free electrochemical method for the detection of DNA-PNA hybridization using a water-soluble, ferrocene-functionalized polythiophene transducer and single-stranded PNA probes on the nanogold modified electrode is investigated. Nanogold modified electrodes can largely increase the immobilization amount of ss-PNA capture probe and lead to an increase of the electrical signal. The ferrocene-containing cationic polythiophene do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after DNA-PNA hybridization, cationic polythiophene is adsorbed on the DNA backbone, giving a clear hybridization detection signal in differential pulse voltammetry (DPV). Very good discrimination against non-complementary DNA and four-base mismatch DNA is observed. These studies show that the proposed method can provide an alternative for expanding the range of detection methods available for DNA hybridization.     

Rhodium(I) hydrogenation in water: Kinetic studies and the detection of an intermediate using C{H} PHIP NMR spectroscopy

The mechanism for hydrogenation of dimethylmaleate in water using cationic rhodium complexes with water-soluble bi-dentate phosphines has been investigated using kinetics and a novel method for the indirect detection of intermediates in catalytic hydrogenation reactions, whereby a late intermediate was detected. A mechanism is proposed involving fast, irreversible substrate binding followed by a rate-determining reaction with dihydrogen.     

Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.     

Acidified acetonitrile and methanol extractions for quantitative analysis of acylcarnitines in plasma by stable isotope dilution tandem mass spectrometry

We have compared two sample preparation methods for the analysis of plasma acylcarnitines by tandem mass spectrometry. Extraction from liquid plasma using acetonitrile was compared with the widely used methanol extraction from plasma spotted on filter paper. The recovery and reproducibility of the acetonitrile extraction were improved by acidification with 0.3% formic acid. The acidified acetonitrile and methanol extractions have the same limit of detection and upper linearity limit for all acylcarnitine species studied. The correlation coefficients between the two methods were greater than 0.988 and the slopes of the linear regressions ranged from 0.901 to 1.070. The extraction of acylcarnitines by acidified acetonitrile from liquid plasma yielded results comparable to those obtained by methanol extraction from plasma spotted on filter paper.     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

Simultaneous quantification of the enantiomers of verapamil and its N-demethylated metabolite in human plasma using liquid chromatography-tandem mass spectrometry

A stereoselective bioanalytical method for the simultaneous quantification of the enantiomers of verapamil and its active main metabolite norverapamil in human plasma has been developed and validated. The samples were analysed by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) in the Selected Reaction Monitoring (SRM) mode using a deuterated internal standard. The stationary phase used for the chiral separation was a Chiral-AGP. The enantiomers of verapamil were selectively detected from those of norverapamil by the mass spectrometer due to different molecular masses, although there was a chromatographic co-elution. Thus, time-consuming procedures like achiral preseparation or chemical derivatisation could be avoided. Higher detection sensitivity than earlier published methods based on fluorescence detection was obtained, although a mobile phase of high water-content and high flow-rate was introduced into the electrospray interface (85% aqueous ammonium acetate pH 7.4 +15% acetonitrile at 0.6 ml/min). The enantiomers of verapamil and norverapamil could be quantified at levels down to 50 pg and 60 pg/500 microl plasma sample, respectively, with R.S.D. in the range of 3.6-7.8%. The presented method was successfully applied to an in vivo intestinal absorption and bioavailability study in humans, using the Loc-I-Gut method.