Separation of the glucuronides of entacapone and its (Z)-isomer in urine by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method was developed for the separation of the 3-O-glucuronides of entacapone and its (Z)-isomer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intended for use as an adjunct in the treatment of Parkinson’s disease. Urine samples spiked with synthetic 3-O-glucuronides were used to study the effects of running buffer pH, composition and applied voltage on separation of the closely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved by increasing the running buffer ionic concentration. Changes in pH had little effect on the separation, whereas increase in sodium dodecyl sulfate (SDS) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphate (25 mM)–borate (50 mM)–SDS (20 mM) running buffer, pH 7.0, in a 75 μm×60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in the UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 μg/ml (5.2·10 ⁻⁷M) (injection 0.5 p.s.i./8 s). The linear detection range was 2–100 μg/ml (r²>0.999). Good repeatability of injection and relative migration times were obtained.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Determination of aloenin,barbaloin and isobarbaloin in Aloe species by micellar electrokinetic chromatography

Aloenin, barbaloin and isobarbaloin in JP Aloe¹, Aloe barbadensis (Aloe vera) and Aloe arborescens Miller var. natalensis Berger (Aloe arborescens Miller) were determined by micellar electrokinetic chromatography (MEKC) with 50 mM sodium dodecyl sulfate. Aloenin, barbaloin and isobarbaloin were well separated by MEKC and as little as 5.5 pg/11 nl of the three compounds could be detected. The determination took around 14 min.     

An alkali tolerant α-l-rhamnosidase from Fusarium moniliforme MTCC-2088 used in de-rhamnosylation of natural glycosides

Analkali tolerant α-l-rhamnosidase has been purified to homogeneity from the culture filtrate of a new fungal strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chromatography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa using SDS-PAGE analysis. The K_m value using p-nitrophenyl-α-l-rhamnopyranoside as the variable substrate in 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s⁻¹giving the values of k_cat/K_m is 3.12 × 10⁴M⁻¹ s⁻¹. The pH and temperature optima of the enzyme were 10.5 and 50 °C, respectively. The purified enzyme had better stability at 10 °C in basic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-l-rhamnosidase has potential for enhancement of wine aroma.     

Exploitation of micellar medium for photochemical-spectrofluorimetric flow-injection determination of fenvalerate

A photochemical flow-injection methodology for the fluorimetric determination of fenvalerate, a nonfluorescent pyrethroid pesticide, is proposed. The sample was irradiated by ultraviolet light inside a reaction coil in a nonstop mode and the photodegradation products were monitored by spectrofluorimetry at λ_exc=277 nm, λ_em=329 nm. The exploitation of a micellar medium resulted in a pronounced fluorescence enhancement with a concomitant tenfold sensitivity improvement. Several surfactants were evaluated, and best results were obtained with sodium dodecylsulphate (SDS). The calibration plot was linear within 2.0×10⁻⁶ and 1.0×10⁻⁵ mol l⁻¹ fenvalerate. The proposed system is very stable, handles 30 samples h⁻¹ and requires only 60 μmol SDS per determination. The detection limit is 5×10⁻⁷ mol l⁻¹. The method was evaluated in the determination of fenvalerate in tap water.     

Retention model for the resolved enantiomers of felodipine on chiral-AGP using micellar mobile phases

A retention model for the chiral separation of an uncharged solute, felodipine, on CHIRAL-AGP, using a micellar mobile phase is proposed. The model assumes the presence of two stereoselective sites and each enantiomer was found to interact with different sites. Addition of a chiral aliphatic alcohol, (+)-(S)-2-octanol, preferentially interacted with the binding site for (?)-(S)-felodipine. The monomeric form of the micellar agent (Tween® 20) competed with the enantiomers for the adsorption sites, and the formation of a 1:1 complex between the enantiomers and the micelles was assumed. The retention of the solutes was effectively controlled by adding small quantities (<1.63 × 10^?3 M) of the nonionic detergent Tween 20 to the mobile phase. Baseline separation was achieved by addition of 1.0 mM n-octylamine to the mobile phase; 8.14 × 10^?4 M Tween 20 in phosphate buffer pH 7.0. The separation factor (α = 1.74) was unaffected by the detergent concentration in the presence of 1.0 mM n-octylamine. © 1995 Wiley-Liss, Inc.     

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids

Laser-induced native fluorescence detection with a KrF excimer laser (λ=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-μm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3σ) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; α-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7–170 nM.     

Simple Determination of Trace Amounts of Anionic Surfactants in River Water by Spectrophotometry Combined with Solid-phase Extraction

A simple and sensitive specrophotometric method combined with solid-phase extraction (SPE) for the simultaneous determination of sodium linear-dodecylbenzenesulfonate (DBS) and sodium dodecyl sulfate (SDS) is described. The C2 (ethyl group bonded silicagel) cartridge could be repeatedly used more than 500 times for SPE, and it enabled the anionic surfactants to be concentrated by 50-fold. The calibration graph for DBS was linear in the range from 1.6×10^?8 M to 5.0×10^?7 M and for SDS from 2.0×10^?9 M to 3.0×10^?7 M. The relative standard deviation (n=5) for 5.0×10^?7 M DBS was 3.1% and for 2.5×10^?7 M SDS was 1.7%. The proposed method was applied to the simultaneous determination of DBS and SDS in river-water samples.     

Capillary zone electrophoretic determination of the four vitamin C esters l-ascorbyl-2-phosphate,l-ascorbyl-2-sulfate,l-ascorbyl-2-diphosphate and l-ascorbyl-2-triphosphate in fish feed,plasma and tissue

A new and fast method for the determination of l-ascorbyl-2-phosphate, the 2-diphosphate, 2-triphosphate and the 2-sulfate ester in feed, fish plasma and tissue samples by capillary zone electrophoresis is described. The substances were extracted with a 200 mM potassium phosphate buffer pH 4.0. Separation was achieved using a 50 μm uncoated fused-silica bubble capillary of 40 cm length to the detector, which was set at 254 nm, and a 80 mM tricine buffer at pH 9.2. Repeatability checked on a trout feed sample revealed a migration time variation of 1.2% (n=24) and an area calculation variation of 2.8% (n=24). Detector response for l-ascorbyl-2-phosphate was linear up to at least 100 μg ml⁻¹. After administration of a high dose of l-ascorbyl-2-phosphate to rainbow trout, the compound was found in the stomach and intestine but not in plasma, proving a fast conversion to vitamin C.     

Micellar electrokinetic chromatography for the determination of urinary desmosine and isodesmosine in patients affected by chronic obstructive pulmonary disease

The presence in urine of desmosine (DES) and isodesmosine (IDES), two crosslinked amino acids unique to the elastic fiber network, can be used as a specific indicator of degradation of mature elastin. Compared to methodologies so far available, the capillary electrophoretic technique reported here seems to be suitable and convenient for determining desmosines in urine of patients affected by chronic obstructive pulmonary disease (COPD). By using 35 mM sodium tetraborate pH 9.3 containing 65 mM SDS as the background electrolyte, the peaks of DES and IDES could be detected in hydrolyzed urine samples from controls and patients. Owing to the simultaneous determination of endogenous urinary creatinine used as appropriate internal standard, the amount of these amino acids could be accurately quantified. The results obtained were of the same order of magnitude as the data already reported in the literature for COPD patients. Thus micellar electrokinetic chromatography (MEKC) may be considered as a reliable technique for studying the turnover of the elastic fiber in clinical conditions.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Simultaneous determination of nicotinic acid and its metabolites in rat urine by micellar electrokinetic chromatography with photodiode array detection

Nicotinic acid, nicotinamide and their possible metabolites were successfully separated within 17 min by micellar electrokinetic chromatography using 50 mM borate buffer (pH 9.0) containing 150 mM sodium dodecyl sulfate as the running buffer. Calibration curves for all compounds showed good linearity in a range of 5 μg/ml and 250 μg/ml with good correlation. The present method did not require any clean-up procedures and made it possible to determine all metabolites without interference on a photodiode array detector. Urine samples collected from Wistar male rats were analyzed after high-dose oral or intravenous administration of nicotinic acid or nicotinamide. Metabolic pathways of nicotinic acid in male Wistar rats are also discussed.     

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k_cat significantly increased while K_m was little changed after correction to free substrate concentration. The ratio of k_cat to K_m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10⁻² M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.     

Susceptibility ofParacoccidioides brasiliensis conidia to products of oxidative metabolism

The toxic effect of components of the peroxide-peroxidase-halide system onParacoccidioides brasiliensis conidia was investigated. By itself, hydrogen peroxide was lethal at a concentration of 0.5M. The addition of peroxidase (14 U/ml) and KI (5 × 10⁻⁴M) markedly reduced the amount of hydrogen peroxide (from 5 × 10⁻¹ to 5 × 10⁻⁶M) required to kill 99% of the conidia. The lethal effect of the system suggested that it may play a role in host defense againstP. brasiliensis.     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Analysis of lignocelluloses and lignins from Arundo donax L. and Miscanthus sinensis Anderss., and hydroliquefaction of Miscanthus

In a comparative investigation of the chemical composition of Arundo donax L. and Miscanthus sinensis Anderss. the following experiments were performed: ash determination and ash characterization by energy dispersive X-ray analysis; determination of solubility in cyclohexane/ ethanol, hot water, 1% hydrochloric acid and sodium hydroxide; C, H and N determination; determination of Klason lignin and acid soluble lignin content; sulfuric acid hydrolysis followed by borate complex ion exchange chromatography of the monomeric sugars; isolation of milled wood lignins (MWL) and dioxane lignins (DIL) and their analysis by C, H, and OMe determination; quantitative FTIR spectroscopy of MWL; recording the molecular weight distribution curves using high performance size exclusion chromatography (HPSEC); calculation of average molecular weights, such as M_w and M_n; calculation of the heating values of the lignocellulosics and their components. The quantitative composition of the lignin from the three basic phenylpropane units is presented.M. sinensis was submitted to hydroliquefaction. The conversion process yielded 35% of a net product oil (NPO) with low oxygen content (11%), low viscosity (10⁻² NS m⁻²), low asphaltene content (3·5%) low molecular weight (M_w 200) and with a specific gravity of c. 0·93 g cm⁻³. The NPO has a heating value of 39·4 MJ kg⁻¹ and contains 55% of the carbon of the starting material and 59% of the combined heating value from the biomass and the hydrogen used for hydroliquefaction. The process yields 28% water which contains 58% of the original oxygen of the biomass. The process gives rise to 9 g solids and 32–35 g gases, whose energy content can easily be recovered.     

Determination of monohydroxyethylrutoside in heart tissue by high-performance liquid chromatography with electrochemical detection

7-Monohydroxyethylrutoside (monoHER) is one of the components of the registered drug Venoruton. It showed a good protection against the cardiotoxic effects of doxorubicin. The analysis of monoHER was developed to study the pharmacokinetic profile of the drug in heart tissue. MonoHER was extracted from heart tissue homogenate with methanol. The supernatant was diluted 1:1 (v/v) with 25 mM phosphate buffer and injected onto a reversed-phase ODS column. The mobile phase consisted of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogenphosphate (pH 3.4), 10 mM acetic acid and 36 μM EDTA. The retention time of monoHER was about 5.2 min and no endogenous peaks were interfering. The lower limit of quantification was 0.072 nmol g⁻¹ wet heart tissue. The calibration line was linear up to 24 nmol g⁻¹. The within-day accuracy and precision of the quality controls (0.12, 1.2 and 12.0 nmol g⁻¹) were smaller than 17 and 19%, respectively. The between-day accuracy and precision were better than 6 and 11%, respectively. The recovery of monoHER from heart tissue ranged from 104.1 to 114.3% and was concentration independent. MonoHER was stable in heart tissue when stored at −80°C for 6 months. Repeated injection of monoHER from aliquots of 7.2 nmol g⁻¹ placed on the sample tray at 4°C for 24 h showed a decrease in the concentration of 30.3%. Analyzing sample duplicates in a mirror image sequence could compensate for the influence of this gradual decrease. The small sample volume allowed one to measure monoHER in the hearts of mice.     

Optimization of separation of a complex mixture of natural and synthetic corticoids by micellar liquid chromatography using sodium dodecyl sulphate: Application to urine samples

A systematic optimization of the separation of a mixture of corticoids by micellar liquid chromatography, using sodium dodecyl sulphate as surfactant, a Hypersil (250 mm×3.2 mm I.D.) C₁₈ column, a flow-rate of 0.5 ml min⁻¹, and UV absorbance detection at 245 nm has been carried out. Several mobile phases consisting of sodium dodecyl sulphate and different organic modifiers were tested of which tetrahydrofuran, PrOH and BuOH were finally selected. On the basis of analysis time, resolution and number of compounds separated, a mobile phase containing 36 mM sodium dodecyl sulphate and 1.91% butanol allowed the separation of thirteen corticoids out of sixteen in about 27 min. Under these conditions the optimal concentration of sodium dodecyl sulphate was found to be 36 mM. A bivariant optimization method for the mobile phase BuOH–sodium dodecyl sulphate corrobored these results. The effects of temperature, ionic strength and flow-rate effect have also been studied. The most important analytical figures of merit were assessed and compared with those obtained using conventional mobile phases. The optimized method was applied to human urine samples of subjects administered with Dezacor® (tablets containing 30 mg of the active ingredient deflazacort) with and without sample preparation.