Comparison of N-alkyl acridine orange dyes as fluorescence probes for the determination of cardiolipin.

The phospholipid (PL), cardiolipin (CL), is found almost exclusively in the inner membrane of mitochondria and loss of CL is considered as an important indication of cell apoptosis. Previously, 10-N-nonyl acridine orange (NAO) has been used as a fluorescent probe for the visualization of CL in mitochondrial cell membranes and in solution. In this work for the determination of CL, we have synthesized two new fluorescent probes, n-tetradecyl acridine orange (C14-AO), and n-octadecyl acridine orange (C18-AO) by reacting acridine orange with the corresponding n-alkyl bromide. Using excitation and emission wavelengths at about 500 and 525 nm and varying the percentage of methanol in water as the solvent, no interaction between CL and the fluorescent probes at 75% is noted but a proportional quenching of the fluorescence signal by CL is observed at 50% or less for C14-AO and 60% or less for C18-AO. Binding efficiency of these fluorescent probes to CL is compared using dye concentrations of 5, 10, and 20 muM. C18-AO shows a better sensitivity than C14-AO and NAO, respectively, but is less selective. For C14-AO, the detection limit and limit of quantitation are 0.07 and 0.21 muM, respectively, which are better than those previously reported for NAO. One anionic PL, phosphatidic acid, shows some quenching interference to both the C14 and C18 dyes but only at concentrations above the working range for sample analysis. The CL in mitochondrial membrane samples is determined by standard addition using C14-AO. The level of CL in the outer mitochondrial membrane compared to the inner membrane is significantly increased due to the addition of cadmium chloride into the cells causing cell apoptosis.

Polymer mediated capillary electrophoresis with attenuated total reflectance infrared microspectroscopy detection.

Polymer mediated capillary electrophoresis (CE) using poly(diallyldimethylammonium chloride) (PDDAC) in the electrolyte with end-column single reflection attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the capillary is placed approximately 1 microm from the internal reflectance element (IRE), at the focus of the ATR infrared microscope. Electrophoretic separations of benzenesulfonate, 1-naphthalenesulfonate and 1,5-naphthalenesulfonate in a NaCl and PDDAC electrolyte using either -11 or -15 kV are demonstrated with the CE-ATR FT-IR spectra providing identification of these compounds. Running electrolyte salt concentrations in the 0.85-1.7 M range are required due to the 50-80 mg mL(-1) sample concentrations. Increasing the NaCl salt concentration improves peak resolution but also increases analysis time. A PDDAC concentration as low as 0.0075% can facilitate the separation of the aromatic sulfonates while maintaining reasonable electroosmotic flow. Etching of the germanium IRE by the applied current, which can affect the intensity of the infrared beam, is corrected by fabrication of a plastic mounting post for the IRE to prevent current conduction through the IR instrument.

Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange.

Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 microM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4-20 microM cholesterol with the exception of PG at above 8 microM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO-CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 microM NAO provides an inverse linear fluorescence response at 0.2-10 microM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 microM and the limit of quantification is 0.6 microM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 microM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.

Capillary electrophoresis of phospholipids with indirect photometric detection.

The capillary electrophoresis (CE) separation of different anionic phospholipid classes including phosphatidic acids (PA), phosphatidylserine, phosphatidylinositol, phosphatidylglycerol (PG), and cardiolipin using indirect detection with adenosine monophosphate (AMP) is described. A standard mixture of PAs (C14, C16, and C18) can be separated in 10 min by CE using 5 mM AMP and 100 mM boric acid in 10% water--80% methanol--10% acetonitrile. Although nonionic surfactants such as Brij 35 can improve the CE resolution of PAs, the separation time and the baseline noise are both increased. Optimization of the organic solvent in the running electrolyte is important. Methanol provides faster electroosmotic flow than propanol, and 10% acetonitrile effectively reduces migration time further by a factor of 1.4-2.2, depending on the phospholipid. The concentration limit of detection ranges from approximately 2 to 6 mg/L, and the mass limit of detection is as low as 21 pg. Linearity from 19 to 100 mg/L is established for cardiolipin and C16-PG. Phospholipids in soybean and brain extract samples could be profiled.

Liquid chromatography of aromatic amines with photochemical derivatization and tris(bipyridine)ruthenium(III) chemiluminescence detection.

We have shown by flow injection that tris(bipyridyl)ruthenium(III) [Ru(bpy)3(3+)] chemiluminescence (CL) detection of some aromatic amines can be enhanced by on-line photochemical derivatization. Two of the aromatic amino acids, tryptophan, and tyrosine as well as the peptide phenylalanine-alanine and other primary aromatic amines such as L-dopa, phentermine, and tryptamine upon irradiation with UV light are found to give an increased CL signal on the order of 4-9 times that for nonirradiated compounds. For benzylamine, phenethylamine, and phenylalanine, the improved CL detectability upon photolysis is about 15-16 times better. Chemiluminescence detection limits of the photolyzed compounds are generally 2-20 pmol, significantly better than those by UV-Vis detection at 254 nm. GC-MS work has been done to identify the products of some of the photolysis reactions and explain the enhanced CL detectability. The fact that other aromatic amines without a one or two carbon spacer from the aromatic ring to the amine group such as aniline, m- and p-phenylenediamine, and N,N'-dimethylaniline did not show any CL signal improvement upon irradiation with UV light suggests that there is some selectivity in the reaction. CL detection of aromatic amino acids after on-line photochemical derivatization and HPLC has been shown.

Determination of minute enzymatic activities by means of capillary electrophoretic techniques.

Capillary electrophoretic analysis of enzymes, co-enzymes, substrates and other chemical species that can be linked to an enzymatic reaction is reviewed with 80 references. Both off-line and on-line assays of minute enzymatic activities are discussed. In addition to heterogeneous on-line enzyme assays, a special emphasis is given to a newly established on-line technique called electrophoretically mediated microanalysis (EMMA). The basic principle, procedure, and various detection modes of EMMA are discussed. The recent developments in on-line determination of various enzyme substrates as well as on-line enzyme kinetic studies are also summarized. Some potential future developments in the determination of enzymatic activities by means of CE are also presented.

On-line lactate dehydrogenase substrate and activity determinations by capillary electrophoresis.

Capillary electrophoresis is used to perform both substrate and enzyme assays using the lactate dehydrogenase (LDH) enzyme system. Polymer mediated stabilization with polyethylene glycol (PEG) can extend the lifetime of enzyme activity, increasing the number of assays that can be performed at room temperature. For the determination of lactate, the reduction of the cofactor nicotinamide adenine dinucleotide (NAD+) is monitored at 340 nm, while the determination of pyruvate is accomplished by following the oxidation of the cofactor nicotinamide adenine dinucleotide, reduced form (NADH) at 280 nm. Sample throughput is 1 min, excluding both the wash and equilibrium steps. Linearity is obtained in the clinical range for both lactate and pyruvate standards. In addition, bovine serum with spiked lactate is analyzed with an average recovery of above 90%. For the enzyme assay, the pyruvate reaction is chosen for its more favorable kinetics. Linearity is also established in the clinical range and the examination of serum spiked enzyme samples was possible.

Separation of phosphorylated sugars using capillary electrophoresis with indirect photometric detection.

Adenosine monophosphate (AMP) and naphthalene disulfonate (NDS) have been characterized as electrolytes for the indirect photometric detection of phosphorylated sugars and other organophosphorus compounds of biochemical interest. This work has resulted in the CE separation on an uncoated capillary using 5 mM AMP and 100 mM boric acid at pH 7.2 of six metabolites (glucose-6-phosphate [G6P], fructose-6-phosphate [F6P]), fructose-1,6-bisphosphate [F-1,6-P], dihydroxyacetone phosphate [DHAP], glyceraldehyde-3-phosphate [G3P], and 2-phosphoglycerate [2-PG] or 3-phosphoglycerate [3-PG]) found in the glycolytic pathway. The detection limits using a 5-sec injection time were between 0.5 and 1 mg/L for these compounds, with the exception of G3P. Resolution between 3-PG and 2-PG is possible by the addition of magnesium ion, although the separation time is longer. A successful separation of five monophosphorylated sugars (G6P, F6P, ribose-5-phosphate [R5P], sucrose-6-phosphate [S6P], and 2-PG) has been performed using the same conditions as for the glycolytic pathway separation. A separation of bisphosphorylated sugars (glucose-1,6-bisphosphate [G-1,6-P],F-1,6-P, ribulose-1,5-bisphosphate [Ru-1, 5P], and sedoheptulose-1,7-bisphosphate [S-1, 7P]) could not be performed with AMP unless magnesium chloride was added. With NDS, a separation of these bisphosphorylated sugars can be obtained without the addition of magnesium chloride.

Colorimetric determination of macrolide antibiotics using ferric ion.

Macrolide antibiotics such as erythromycin, oleandomycin, spiromycin, and tylosin are found to react with Fe(3+) in the presence of an acetic acid-sulfuric acid mixture to form a colored product having a useful absorption band at 592 nm. Troleandomycin forms only a weakly colored product upon reaction. The molar absorptivity is about 2900 1 mol(-1) cm(-1) for erythromycin and the detection limit is 5 mug ml(-1). This colorimetric method permits the analysis of fermentation broths containing either erythromycin or tylosin without a separation step.

Microbore liquid chromatography of tertiary amine anticholinergic pharmaceuticals with tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection.

The post-column chemiluminescent reaction of six anticholinergic alkaloid compounds with tris(2,2'-bipyridine)ruthenium(III) (Ru(bpy)3(3+)) is applied to microbore high-performance liquid chromatography (HPLC). At flow rates less than 200 microL/min, the capillary mixing cell in which Ru(bpy)3(3+) and the analyte are mixed directly allows for good light detection. In contrast, a diminished signal occurs at these low flow rates with conventional post-column mixing in a tee. Optimal chemiluminescent pH conditions for atropine, scopolamine, dicyclomine, cyclopentolate, cyclobenzaprine, and procyclidine are determined at moderately basic conditions (pH 7 to 9). 2-Butanone is found to be compatible with the chemiluminescent reaction, whereas tetrahydrofuran and propionitrile cause an increase in background noise and a chemiluminescent signal loss. As 2-butanone is more nonpolar than acetonitrile, it assists in the elution of these hydrophobic anticholinergic compounds. Five anticholinergic compounds are resolved successfully with a PRP-1 polymeric column and a slightly basic mobile phase, but a C8 silica column is better suited for the more hydrophobic compounds (cyclobenzaprine, procyclidine, and dicyclomine).

Determination of substrates using poly(ethylene glycol)-stabilized dehydrogenase enzymes by microlitre per minute flow injection.

Flow injection (FI), at a flow rate of microliter min-1, is an effective method for enzymic substrate determination using low concentrations of poly(ethylene glycol) (PEG)-stabilized soluble enzymes. PEG stabilizes dehydrogenase enzymes for at least several days by promoting sub-unit association. Band broadening of knitted open tubular reactors is reduced as flow rate decreases below 300 microliter min-1 and a small tubing diameter is important for a faster rate of absorbance signal increase with residence time. Small (0.5 microliter) sample injections also ensure narrow FI peaks. The determination of several substrates such as pyruvate, lactate, and cortisone using appropriate PEG-stabilized enzymes is demonstrated with this FI instrument at 25 or 50 microliters min-1 with sample throughputs of the order of 2-3 min per sample. The determination of lactate in serum samples is also possible. The advantage of this method, sample throughput, is not sacrificed but enzyme consumption is considerably less, compared to standard ml min-1 FI.

Liquid chromatography of antihistamines using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection.

The separation and detection of five antihistamine drugs commonly found within over-the-counter allergy and cold pharmaceutical products was performed by HPLC with chemiluminescence (CL) detection. Comparable detection limits at 5-10 pmol were found for the antihistamines by both UV at 214 nm and tris(2,2'-bipyridine) ruthenium(III) CL. However, urine samples were found not to generate as large an unretained peak by CL detection as compared to those peaks by UV detection at 214 and 254 nm. For example, the pheniramine peak representing 0.15 microgram/ml was almost totally obscured at 214 nm. Quantitative results received for three antihistamine commercial samples ranged from 4 to 8% error in accuracy when an internal standard was used to compensate for short term detector drift.

Simple methods for the qualitative identification and quantitative determination of macrolide antibiotics.

Pyrolysis-gas chromatography is shown to be a rapid straightforward method for the qualitative differentiation of the macrolide antibiotics erythromycin, oleandomycin, troleandomycin, spiramycin and tylosin. Organic salts do not interfere and identification of erythromycin and troleandomycin in commercial products is viable. Spectrophotometric quantitation of these same five antibiotics after reaction with concentrated sulphuric acid is studied at about 470 nm. Reaction conditions such as acid concentration, time and temperature are provided. The sugar moieties of the antibiotics are proposed as the reactive sites. Detection limits are about 0.2-1.0 microg ml-1 [corrected] and analysis of pharmaceutical products should be possible.

Indirect electrochemical detection of cations with cerium(III) in the mobile phase.

Cerium(III) has been used in the mobile phase for indirect electrochemical chromatography of cations. Lithium, sodium, ammonium, and potassium ions have been separated within 4 min and detected with both glassy carbon and Kel-F wax carbon paste electrodes. With a Kel-F wax/graphite electrode at 0.7 V, the detection limit for sodium, ammonium, and potassium ions are 1.5, 1.1, and 2.0 ppm, respectively.

Pre- and postcolumn derivatization chemistry in conjunction with HPLC for pharmaceutical analysis.

Descriptions of derivatization reactions applied to high-performance liquid chromatography for pharmaceuticals classified as alkaloids, amines, antibiotics, barbiturates, carbonyl/carboxylic compounds, catecholamines, hydroxy compounds, steroids, sulfur compounds, and miscellaneous are summarized.

Enzymatic determination of phosphate in conjunction with high-performance liquid chromatography.

A selective assay for orthophosphate in complex matrices was developed based on the nucleoside phosphorylase catalyzed conversion of inosine and orthophosphate of hypoxanthine. The enzyme reaction was using only 0.28 units/assay was allowed to proceed for 30 min before quenching. Separation of inosine and hypoxanthine was performed by reversed-phase high-performance liquid chromatography. Quantitation of the hypoxanthine peak was found to be linear with orthophosphate up to 30 micrograms/g. A detection limit of 0.75 ppm could be obtained after dialysis of the commercial enzyme. Interference studies showed that the enzymatic assay unlike the colorimetric molybdate-blue technique was essentially unaffected by complex matrices such as serum, urine, polyphosphates, and phosphoesters.

Fluorometric determination of hydrazine and ammonia separately or in mixtures.

Hydrazine and ammonia are often added to boiler water to inhibit corrosion. The reagents o-phthalaldehyde and mercaptoethanol have been found to form derivatives with hydrazine and ammonia which can be determined by fluorimetry. Because the optimum pH values for formation of the hydrazine and ammonia derivatives were different, analysis of mixtures of the two components without prior separation was possible. Simulated wet-lay-up boiler water samples containing 5-200-mug/ml levels of hydrazine and ammonia have been analysed with an average relative error of about 10%.