Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from uterine myoma and cervical cancer patients

Capillary electrophoretic (CE) profiling analysis combined with pattern recognition methods is described for the correlation between urinary nucleoside profiles and uterine cervical cancer. Nucleosides were extracted from urine specimens by solid-phase extraction in affinity mode using phenylboronic acid gel. CE separation was carried out with an uncoated fused-silica capillary (570 mm×50 μm I.D.) maintained at 20°C, using 25 mM borate–42.5 mM phosphate buffer (pH 6.7) containing 200 mM sodium dodecyl sulfate as the run buffer under the applied voltage of 20 kV. A total of 15 nucleosides were positively identified in urine samples (2 ml) from eight uterine myoma (benign tumor group), 10 uterine cervical cancer (malignant tumor group) patients and 10 healthy females (normal group) studied. The star symbol plots drawn based on each mean concentration of nucleosides normalized to that in normal group enabled one to discriminate malignant and benign groups from normal group. In addition, canonical discriminant analysis performed on the nucleoside data of 28 individual urine specimens correctly classified into three separate clusters according to groups in the canonical plot.     

Enantiomeric separation of tramadol hydrochloride and its metabolites by cyclodextrin-mediated capillary zone electrophoresis

The enantiomeric separation of tramadol hydrochloride and its major metabolites, O-demethyltramadol (M1) and N-demethyltramadol (M2) was studied using cyclodextrin (CD)-mediated capillary zone electrophoresis (CZE). Influence of the choice of type and concentration of CD, capillary temperature, length of capillaries, buffer pH and the addition of polymer modifier on the chiral separation of tramadol and its metabolites was evaluated. It was found that the drug and the metabolites can be baseline-separated simultaneously by using 50 mM phosphate buffer (pH 2.5) containing 75 mM methyl-β-CD, 220 mM urea and 0.05% (w/v) hydroxypropylmethyl cellulose.     

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.     

Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.     

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm×75 μm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

Determination of bromhexine and ambroxol in pharmaceutical dosage forms,urine and blood serum

Data presented in this paper show that bromhexine and its pharmacologically active metabolite can easily be determined by capillary zone electrophoresis. The composition of the running buffer had a significant effect on the reproducibility of the migration time for which a carrier solution containing 30 mM phosphate buffer (pH 3.0), 5 M urea and 10% (v/v) acetonitrile was used. The method was validated with respect to its response linearity and reproducibility. The method is suitable for the determination of bromhexine and ambroxol in several samples such as pharmaceuticals, urine and serum. Photodiode-array detection permitted the rapid identification of both drugs in the sample analyzed.     

Determination of cocaine and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Brownlee C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.2, containing 1.29·10⁻⁴M tetrabutylammonium phosphate (12.5:10:77.5, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 235 nm. The method was used to study the pharmacokinetics of cocaine after an intravenous (i.v.) bolus dose (4 mg/kg).     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

Stereoselective determination of R-(−)- and S-(+)-prilocaine in human serum by capillary electrophoresis using a derivatized cyclodextrin and ultraviolet detection

A capillary electrophoresis (CE) method for the quantification of R-(−)- and S-(+)-prilocaine in human serum was developed and validated. Stereoselective resolution was accomplished using 15 mM heptakis(2,6-di-methyl)-β-cyclodextrin and 0.03 mM hexadecyltrimethylammonium bromide (HTAB) contained in 100 mM phosphate buffer, pH 2.5. Solid-phase extraction was used as a sample preparation technique to remove endogenous interferences. A 72-cm uncoated fused-silica capillary at a voltage of 25 kV and 30°C was used for the analysis. The detection limits for R-(−)- and S-(+)-prilocaine were 38 ng/ml using 1 ml of human serum and the limits of quantitation were 45 ng/ml. The calibration curve was linear over the range of 45–750 ng/ml with procainamide as the internal standard. Precision and accuracy of the method were 2.86–8.50% and 3.29–7.40%, respectively, for R-(−)-prilocaine, and 3.94–9.17% and 2.0–6.73%, respectively, for S-(+)-prilocaine. The CE method was compared to an existing chiral HPLC method in terms of sensitivity and selectivity for the routine analysis of the drug.     

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.     

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxyholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) × 50 μm I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 mM) in 25 mM borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 mM phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 × 10⁻⁴ − 3 × 10⁻⁵ mol l⁻¹ with a detection limit of 1 pmol. The method was validated and applied to an 11β-hydroxysteroid dehydrogenase assay in tissues.     

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.     

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm×75 μm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Development of a capillary electrophoretic separation of an N-(substituted)-glycine-peptoid combinatorial mixture

Capillary electrophoresis was used for the separation of a combinatorially synthesized N-(substituted)-glycine (NSG) peptoid mixture. This mixture consisted of 24 trimeric compounds sharing a common backbone structure but differing in the side chain attached at the N-terminal residue. Standards of the individual components were unavailable so that development of the separation was based on the mixture. A variety of buffer additives were investigated to enhance the CE resolution of this diverse mixture. Ion-pairing agents, cyclodextrins and organic modifiers were all evaluated as buffer additives. The best separations were achieved using a combination of buffer additives, each serving a different purpose in the separation. Heptane sulphonic acid (HSA) was used to reduce hydrophobic intramolecular interactions. Methyl-β-cyclodextrin was used to provide host–guest interactions in order to resolve the very hydrophobic components of the NSG-peptoid mixture. The optimized run buffer consisted of 250 mM sodium phosphate buffer, pH 2.0, with 25 mM HSA and 40 mg/ml BCD and resulted in the resolution of 21 peaks for the 24 peptoids in the combinatorial mixture.     

Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin

We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semi-preparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60–360 mM phosphate buffer gradient was found to give better separation than elution with a 60–180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

High-performance liquid chromatographic determination of cocaine and its metabolites in serum microsamples with fluorimetric detection and its application to pharmacokinetics in rats

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 μl). Chromatographic separation is achieved on a Hypersil BDS C₁₈ column (100×2.1 mm, 5 μm) with an isocratic mobile phase consisting of methanol–acetonitrile–25.8 mM sodium acetate buffer, pH 2.6, containing 1.0·10⁻⁴ M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.