Distinguishing Healthy and Carcinoma Cell Cultures Using Fluorescence Spectra Decomposition with a Genetic-Algorithm-Based Code.

In this paper, we analysed the steady state fluorescence spectra of cell suspensions containing healthy and carcinoma fibroblast mouse cells, using a genetic-algorithm-spectra-decomposition software (GASpeD). In contrast to other deconvolution algorithms, such as polynomial or linear unmixing software, GASpeD takes into account light scatter. In cell suspensions, light scatter plays an important role as it depends on the number of cells, their size, shape, and coagulation. The measured fluorescence spectra were normalized, smoothed and deconvoluted into four peaks and background. The wavelengths of intensities' maxima of lipopigments (LR), FAD, and free/bound NAD(P)H (AF/AB) of the deconvoluted spectra matched published data. In deconvoluted spectra at pH = 7, the fluorescence intensities of the AF/AB ratio in healthy cells was always higher in comparison to carcinoma cells. In addition, the AF/AB ratio in healthy and carcinoma cells were influenced differently by changes in pH. In mixtures of healthy and carcinoma cells, AF/AB decreases when more than 13% of carcinoma cells are present. Expensive instrumentation is not required, and the software is user friendly. Due to these attributes, we hope that this study will be a first step in the development of new cancer biosensors and treatments with the use of optical fibers.

Repetitive Detection of Aromatic Hydrocarbon Contaminants with Bioluminescent Bioreporters Attached on Tapered Optical Fiber Elements.

In this study, we show the repetitive detection of toluene on a tapered optical fiber element (OFE) with an attached layer of Pseudomonas putida TVA8 bioluminescent bioreporters. The bioluminescent cell layer was attached on polished quartz modified with (3-aminopropyl)triethoxysilane (APTES). The repeatability of the preparation of the optical probe and its use was demonstrated with five differently shaped OFEs. The intensity of measured bioluminescence was minimally influenced by the OFE shape, possessing transmittances between 1.41% and 5.00%. OFE probes layered with P. putida TVA8 were used to monitor liquid toluene over a two-week period. It was demonstrated that OFE probes layered with positively induced P. putida TVA8 bioreporters were reliable detectors of toluene. A toluene concentration of 26.5 mg/L was detected after <30 min after immersion of the probe in the toluene solution. Additional experiments also immobilized constitutively bioluminescent cells of E. coli 652T7, on OFEs with polyethyleneimine (PEI). These OFEs were repetitively induced with Lauria-Bertani (LB) nutrient medium. Bioluminescence appeared 15 minutes after immersion of the OFE in LB. A change in pH from 7 to 6 resulted in a decrease in bioluminescence that was not restored following additional nutrient inductions at pH 7. The E. coli 652T7 OFE probe was therefore sensitive to negative influences but could not be repetitively used.

Fiber-Optic Chemical Sensors and Fiber-Optic Bio-Sensors.

This review summarizes principles and current stage of development of fiber-optic chemical sensors (FOCS) and biosensors (FOBS). Fiber optic sensor (FOS) systems use the ability of optical fibers (OF) to guide the light in the spectral range from ultraviolet (UV) (180 nm) up to middle infrared (IR) (10 µm) and modulation of guided light by the parameters of the surrounding environment of the OF core. The introduction of OF in the sensor systems has brought advantages such as measurement in flammable and explosive environments, immunity to electrical noises, miniaturization, geometrical flexibility, measurement of small sample volumes, remote sensing in inaccessible sites or harsh environments and multi-sensing. The review comprises briefly the theory of OF elaborated for sensors, techniques of fabrications and analytical results reached with fiber-optic chemical and biological sensors.

Micellar electrokinetic chromatography with short-end injection for rapid separation and simultaneous determination of aesculin, aesculetin, and phenylephrine in pharmaceutical preparations.

A fast micellar electrokinetic chromatography (MEKC) method for simultaneous assay of aesculin, aesculetin, and phenylephrine was developed and validated. The separation was carried out in a fused-silica capillary (50 µm id, total length 64.5 cm, effective length 8.5 cm) with UV detection at 210 nm, temperature 25°C and separation voltage -25 kV. The samples were loaded hydrodynamically at a pressure of -50 mbar for 6 s. The background electrolyte of pH 8.6 contained 20 mM boric acid, 60 mM SDS, and 5% (v/v) of methanol. The calibration curves were linear in the range 10-500 µg/mL for aesculin and aesculetin and 12.5-625 µg/mL for phenylephrine. The RSD values of corrected peak areas were 0.6-1.2% (n = 6) when determining 0.2 mg/mL of aesculin and aesculetin and 0.25 mg/mL of phenylephrine in prepared standard mixtures. The method was successfully applied to the assay of aesculin and phenylephrine in a pharmaceutical preparation (RSD  =  1.9-2.0%; n  =  3) and the robustness of the method for both, the determination of analytes and the system suitability test parameter values, was evaluated with the use of Plackett-Burman design.

Determination of phenolic acids in plant extracts using CZE with on-line transient isotachophoretic preconcentration.

A novel transient ITP-CZE for preconcentration and determination of seven phenolic acids (caffeic acid, cinnamic acid, p-coumaric acid, ferulic acid, protocatechuic acid, syringic acid, and vanilic acid) was developed and validated. Effects of several factors such as control of EOF, pH and buffer concentration, addition of organic solvents and CDs, and conditions for sample injection were investigated. Sample self-stacking was applied by means of induction of transient ITP, which was realized by adding sodium chloride into the sample. The CZE was realized in 200 mM borate buffer ((w)(s)pH 9.2) containing 37.5% methanol, 0.001% hexadimethrine bromide, and 15 mM 2-hydroxypropyl-ß-CD. Under the optimal conditions for analysis, analytes were separated within 20 min. Linearity was tested for each compound in the concentration range of 0.1-10 µg/mL (R = 0.9906-0.9968) and the detection limits (S/N = 3) ranged from 11 ng/mL (protocatechuic acid) to 31 µg/mL (syringic acid). The validated method was applied to the ethanolic extract of Epilobium parviflorum, Onagraceae. The method of SPE was used for the precleaning of the sample.

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection.

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100-300 microg/mL; r(2)=0.997) and K(+) (15-75 microg/mL; r(2)=0.997) when using ethanolamine (100 microg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 microg/mL for GlAm and 2.9 microg/mL for K(+). The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6-103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.

Separation and determination of ketoprofen, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography.

Simple micellar electrokinetic chromatographic (MEKC) method was developed for the determination of ketoprofen as the active substance and methylparaben and propylparaben as preservatives in a semisolid pharmaceutical preparation. Separation was carried out with a fused silica capillary and UV detection at 200nm. Optimized background electrolyte was 50mM tricine buffer containing 30mM sodium dodecyl sulfate as surfactant and 15% (v/v) of methanol. Single separation took about 13min. No statistically significant differences were found when comparing the results with those of RP-HPLC method reported in literature.

Determination of selected antioxidants in Melissae herba by isotachophoresis and capillary zone electrophoresis in the column-coupling configuration.

A method utilising isotachophoresis and capillary zone electrophoresis in the column coupling configuration with UV detection at 320 nm was developed for separation and determination of five phenolic acids (rosmarinic, p-coumaric, ferulic, caffeic and chlorogenic) and flavonoid quercitrin in a methanolic extract of Melissae herba. The proposed method has been validated with correlation coefficients from 0.9842 to 0.9988, RSD values between 0.39% and 0.83% for migration times and between 0.40% and 2.05% for peak areas.

Determination of mannitol and sorbitol in infusion solutions by capillary zone electrophoresis using on-column complexation with borate and indirect spectrophotometric detection.

Capillary zone electrophoresis with indirect UV detection at 215 nm was applied for the separation and determination of mannitol (MA), sorbitol (SO) and xylitol in the form of anionic borate-polyol complexes. The separation was carried out in a fused silica capillary (total length 60 cm, effective length 50 cm, I.D. 50 microm) at 25 kV. The optimized background electrolyte was 200 mM borate buffer (pH 9.3, adjusted with triethylamine) containing 10 mM 3-nitrobenzoate as the chromogenic co-ion. The separation took approximately 13 min. The rectilinear calibration range was 0.2-2 mg mL(-1) for MA and SO when using xylitol (1 mg mL(-1)) as the internal standard. The limit of detection at a S/N of 3 was approximately 30 microg mL(-1) for either analyte. The method was used for the assay of MA or SO in pharmaceutical infusion solutions. The RSD values were 0.15% or 1.07% (n=6) when determining 100 mg mL(-1) of MA or 50 mg mL(-1) of SO in commercial infusion solutions. The results were in good agreement with those of pharmacopoeial iodimetric titration.

Analysis of selected constituents in methanolic extracts of Hypericum perforatum collected in different localities by capillary ITP-CZE.

The on-line combination of CZE with capillary ITP (ITP-CZE) was used for the separation and quantification of selected flavonoids and phenolic acids in Hypericum perforatum leaves and flowers collected in six different localities in Slovakia. The leading electrolyte in the ITP preseparation step was 10 mM HCl with Tris as counterion (pH* 7.2). The terminating electrolyte was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The BGE in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 65 mM boric acid, pH* 8.3. The content of methanol in all electrolytes was 20% v/v. The total time of the analysis (including the preseparation step) was approximately 35 min. The rectilinear calibration ranges were between 0.125 and 5.0 microg/mL with kaempferol as internal standard. The correlation coefficients ranged between 0.9912 (for quercitrin and chlorogenic acid) and 0.9988 (for isoquercitrin). The RSD values are between 0.86 and 7.78% (n = 6) when determining rutin and quercetin (4 microg/mL). The optimized method was employed for the assay of flavonoids in medicinal plant extract of different collections of Hypericum perforatum haulm. The variability of the content of the active components depending on the place of collection was confirmed.

Determination of ibuprofen and flurbiprofen in pharmaceuticals by capillary zone electrophoresis.

Capillary zone electrophoresis with spectrophotometric detection was used for the determination of ibuprofen (IB) and flurbiprofen (FL) in pharmaceuticals. The separation was carried out in a fused silica capillary (60 cm x 100 microm i.d. effective length 45 cm) at 30 kV with UV detection at 232 nm. The optimized background electrolyte was 20mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) with 20mM imidazole and 10mM alpha-cyclodextrin of pH 7.3. 2-Naphthoxyacetic acid was used as internal standard. A single analysis took less than 5 min. Rectilinear calibration ranges were 2-500 mg l(-1) for IB and 1-60 mg l(-1) for FL. The relative standard deviations (R.S.D.) values (n=6) were 1.53% for IB and 1.29% for FL (for 200 mg l(-1) IB and 10 mg l(-1) FL). This validated method has been successfully applied for the routine analysis of 10 commercially available pharmaceutical preparations (syrup, tablets, cream and gel).

Development of a stacking-CZE method for the analysis of phenolic acids.

Eight phenolic acids were analyzed by capillary zone electrophoresis. On-line analyte preconcentration was carried out by hydrodynamic injection of large volume of sample followed by removal of the bulk of the low conductivity sample matrix by polarity switching. The optimal electrolyte system consisted of 50mM sodium tetraborate of pH 9.0 (adjusted with 0.1 M phosphoric acid) containing 2% of alpha-cyclodextrin. The separations were carried out with a fused silica capillary (effective length 50 cm, i.d. 50 microm) and monitored at 200 nm. Under optimized preconcentration conditions (sample injection 99 s at 100 mbar and the polarity switching time 1.0 min) linear calibration ranges (0.1-2.0 microg/ml, R=0.9979-0.9995), favourable limits of detection (0.01-0.025 microg/ml) and good repeatability of the peak areas (R.S.D.: 2.76-5.69%, n=6) were achieved.

Recent trends in the determination of polyphenols by electromigration methods.

An overview mapping recent trends in the determination of polyphenols of natural origin (mostly flavonoids) and their synthetic derivatives by electromigration methods is presented. The overview (covering the period of the recent 5 years and comprising 61 references) is focused on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with various detection methods. Techniques comprising on-line pre-separation such as isotachophoresis (ITP)-CZE and flow-injection-CZE, chiral separations and CZE evaluation of antioxidation activity are also discussed.

Separation and determination of clotrimazole, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography.

In this study, micellar electrokinetic chromatography (MEKC) method was developed for the determination of clotrimazole (CLO), methylparaben (MP) and propylparaben (PP) in a pharmaceutical preparation. Separation was carried out in a fused silica capillary (60 cm x 75 microm i.d.) at 25 kV with UV detection at 212 nm. Optimized background electrolyte (BGE) was 15 mM phosphate buffer (pH 7.2) containing 30 mM sodium dodecyl sulfate (SDS) as a surfactant. Rectilinear calibration ranges were 50-500 mg l(-1) for CLO, 10-100 mg l(-1) for MP and 2.5-25 mg l(-1) for PP. The total analysis time was < 12 min.

Use of molybdate as novel complex-forming selector in the analysis of polyhydric phenols by capillary zone electrophoresis.

Molybdate was examined as a complex-forming additive to the CE background electrolytes (BGE) to affect the selectivity of separation of polyhydric phenols such as flavonoids (apigenin, hyperoside, luteolin, quercetin and rutin) and hydroxyphenylcarboxylic acids (ferulic, caffeic, p-coumaric and chlorogenic acid). Effects of the buffer concentrations and pH and the influence of molybdate concentration on the migration times of the analytes were investigated. In contrast to borate (which is a buffering and complex-forming agent generally used in CE at pH > or =9) molybdate forms more stable complexes with aromatic o-dihydroxy compounds and hence the complex-formation effect is observed at considerably lower pH. Model mixtures of cinnamic acid, ferulic acid, caffeic acid and 3-hydroxycinnamic acid were separated with 25 mM morpholinoethanesulfonic acid of pH 5.4 (adjusted with Tris) containing 0.15 mM sodium molybdate as the BGE (25 kV, silica capillary effective length 45 cm x 0.1mm I.D., UV-vis detection at 280 nm). With 25 mM 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulphonic acid/Tris of pH* 7.4 containing 2mM sodium molybdate in aqueous 25% (v/v) methanol as the BGE mixtures of all the above mentioned flavonoids, p-coumaric acid and chlorogenic acid could be separated (the same capillary as above, UV-vis detection at 263 nm). The calibration curves (analyte peak area versus concentration) were rectilinear (r>0.998) for approximately 8-35 microg/ml of an analyte (with 1-nitroso-2-naphthol as internal standard). The limit of quantification values ranged between 1.1 mg l(-1) for p-coumaric acid and 2.8 mg l(-1) for quercetin. The CE method was employed for the assay of flavonoids in medicinal plant extracts. The R.S.D. values ranged between 0.9 and 4.7% (n=3) when determining luteolin (0.08%) and apigenin (0.92%) in dry Matricaria recutita flowers and rutin (1.03%) and hyperoside (0.82%) in dry Hypericum perforatum haulm. The recoveries were >96%.

Enantiomeric purity determination of tamsulosin by capillary electrophoresis using cyclodextrins and a polyacrylamide-coated capillary.

The chiral separation of racemic tamsulosin hydrochloride (TH) was carried out using cyclodextrin (CD)-mediated capillary electrophoresis (CE) with DAD at 200 nm. The best separation of enantiomers of the studied compound was achieved at 20 kV with 30 cm x 50 microm I.D. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm) and running buffer with sulfated-beta-CD (S-beta-CD) as chiral selector. Other selected native or derivatized CDs were also tested: beta-CD (5, 15 mmol l(-1)), carboxymethyl-beta-CD (5, 30 mmol l(-1)), dimethyl-beta-CD (15 mmol l(-1)) and hydroxypropyl-beta-CD (5, 30 mmol l(-1)). Several parameters such as capillary pretreatment, buffer type and concentration, pH of background electrolyte, methanol content, separation temperature and voltage, were optimized. The excellent baseline separation of chiral TH was successfully achieved within 12 min using 100 mmol l(-1) phosphate buffer with pH 2.5 containing 1.7 mmol l(-1) S-beta-CD. Rectilinear calibration range was 50.0-500.0 mumol l(-1) of each enantiomer (r = 0.9993-0.9996). The method was applied to the assay of R-TH in Omnic, capsules (nominal content 0.4 mg per capsule) with R.S.D. 2.75% (n = 6), recovery 99.3-101.7% and it was suitable for the chiral purity control of the active enantiomer in the pharmaceutical.

Assay of acebutolol in pharmaceuticals by analytical capillary isotachophoresis.

Acebutolol [N-{3-acetyl-4-[(2-hydroxy-3-(isopropylamino)propoxy]phenyl} butanamide] is a cardioselective beta-blocker with a potent anti-hypertensive and antiarrhythmic effect. The optimised operational system of electrolytes for the newly developed ITP separation of acebutolol consisted of 10mM potassium acetate +10mM acetic acid (pH 4.65) as the leading electrolyte and 10mM beta-alanine with pH approximately 4 (adjusted with acetic acid) as the terminating electrolyte. The driving and detection currents were 75 and 20 microA, respectively and the analysis took approximately 13 min. Under these conditions the effective mobility of acebutolol was determined as 20.7 x 10(-9) m2 V(-1) s(-1). The calibration dependence was rectilinear in the range 0.14-1.4 mg ml(-1) of acebutolol base (r = 0.9995); relative standard deviation (RSD) values were 1.1% and 1.2% (n = 6) when determining 0.42 and 0.98 mg ml(-1) of acebutolol in a pure standard solution. The method, with the limit of detection (LOD) of 0.04 mg ml(-1) and limit of quantification (LOQ) of 0.12 mg ml(-1), was applied to the assay of acebutolol in Sectral tablets, Acecor tablets, Apo-acebutol tablets (nominal content 400 mg of acebutolol per tablet) and Acebirex tablets (nominal content 200 mg of acebutolol per tablet) with RSD = 0.7-1.7% (n = 6). No interference from any excipients present in the tablets was observed. The recoveries ranged from 98.8% to 102.4% as found by the standard addition technique.

Assay of phenolic compounds in red wine by on-line combination of capillary isotachophoresis with capillary zone electrophoresis.

The on-line combination of capillary zone electrophoresis (CZE) with capillary isotachophoresis (ITP) increases significantly the separation capability and sensitivity of capillary electrophoresis. This technique was used for separation and quantification of fourteen selected natural constituents in red wine belonging to flavonoids and phenolic acids. The leading electrolyte (LE) in the ITP pre-separation step was 10 mM HCl of pH* 7.2 with Tris as counterion, the terminating electrolyte (TE) was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The background electrolyte in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 15 mM boric acid and 5 mM beta-cyclodextrin of pH* 8.5. The content of methanol in all electrolytes was 20% (v/v). For exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of leading and terminating ITP electrolytes picric acid was used as coloured marker. The R.S.D. values (n = 6) ranged between approximately 0.1% (for 0.25 microg ml(-1) rutin) and approximately 11% (for 0.25 microg ml(-1) of quercitrin). Detection limits were 30 ng mi(-1) for phenolic acids, quercitrin and rutin, 100 ng ml(-1) for quercetin, kaempferol and epicatechin and 250 ng ml(-1) for catechin. A single analysis took 45 min.

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers.

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.

On-line coupling of capillary isotachophoresis and zone electrophoresis for the assay of phenolic compounds in plant extracts.

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column coupling configuration was optimized in a mode where the electrolyte for the CZE step is different from the leading and terminating ITP electrolytes. Two colored markers, picric acid and 1-nitroso-2-naphthol, were used for exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of the leading and terminating ITP electrolytes entering the CZE capillary together with the analytes, thus controlling the duration of transient ITP migration in the CZE capillary and ensuring good separation of the analytes and reproducibility of the migration times (relative standard deviations 1%). ITP-CZE was applied to the simultaneous assay of several cinnamic acid derivatives and flavonoids in methanolic extracts of Sambucus flowers and Crataegus leaves and flowers. The preconcentrating and cleansing effect of the ITP step allowed injection of relatively large sample volumes (30 microL). The limits of detection were approximately 20-50 ng x mL(-1) and 100 ng x mL(-1) for the acids and flavonoids, respectively ( thick similar 200-times lower compared to conventional CE) with spectrophotometric detection at 254 nm. The ITP-CZE exhibited satisfactory linearity and precision when using CZE buffer of pseudo "pH" 9.0; 1-nitroso-2-naphthol was employed as the internal standard. The separation took approximately 35 min. The ITP-CZE results for rutin, hyperoside, and vitexin-2-O"-rhamnoside were in good accordance with those obtained previously by high-performance liquid chromatography.