Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

Size dependence of SiO2 particles enhanced glucose biosensor

A wide size range of SiO₂ particles were synthesized and were used as enzyme immobilization carriers to fabricate glucose biosensors. The size of the particles was in the range of 17-520 nm. These biosensors could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.2). Particle size could affect the performance of SiO₂ modified glucose biosensors drastically. The smaller particles had higher performance. The smallest SiO₂ modified biosensor could work well in the glucose concentration range of 0.02-10 mM with a correlation coefficient of 0.9993. Its sensitivity was 2.08 μA/mM and the detection limit was 1.5 μM glucose.     

Thermokinetic studies on the activation of bovine liver arginase by manganese ions

The activation of bovine liver arginase, which catalyzes the hydrolysis of l-arginine to l-ornithine and urea, by manganese ions was studied by thermokinetic methods at 37 °C in 40 mM sodium barbiturate-HCl buffer solution (pH 9.4). Full activation of arginase, by incubation with 0.1 mM Mn²⁺, resulted in increased of V_max, and a higher sensitivity of the enzyme to product and l-lysine inhibition, with no change in the K_m for arginine. Upon addition of 0.1 mM Mn²⁺ to the reaction, the inhibitory constants of product (K_P) and l-lysine (K_I) decreased from 1.18 to 0.70 mM and from 5.60 to 3.10 mM, respectively. We suggest that the exogenous manganese ions in reaction recovered the activity of arginase, which was lost in dissolving and dilution, without effecting on the mechanism of the reaction.     

Determination of various alcohols based on a new immobilized enzyme fluorescence capillary analysis

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) has been proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactor (IE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD⁺) as an oxidizer is mixed with alcohol sample solution, it was sucked into the IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λ_ex = 350 nm and λ_em = 459 nm. The experimental conditions are as follows: The reaction time is 20 min; temperature is 40 °C; the concentrations of phosphate buffer solution (pH 7.5) and NAD⁺ are 0.1 mol L⁻¹ and 5 mmol L⁻¹, respectively; immobilization concentration of ADH is 10 U L⁻¹. The determination range of ethanol is 2.0-15.0 g L⁻¹ (F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L⁻¹; and relative standard deviation is 1.9%. IE-EFCA method is applicable for the determination of the samples containing alcohol in medicine, industry and environment.     

Simultaneous determination of two active ingredients in Flos daturae by capillary electrophoresis with electrochemiluminescence detection

The coupling of Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the simultaneous determination of the two major active ingredients (atropine and scopolamine) in Flos daturae. Parameters related to the separation and detection were discussed and optimized. It was proved that 20 mM phosphate buffer at pH 8.48 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.2 V. Under the optimized conditions: ECL detection at 1.2 V, 20 mM phosphate buffer at pH 8.48, 5 mM Ru(bpy)₃²⁺ and 50 mM phosphate buffer at pH 7.48 in the detection reservoir, detection limits of 5 × 10⁻⁸ mol/l for atropine and 1 × 10⁻⁶ mol/l for scopolamine were obtained. Relative standard derivations of the ECL intensity and the migration time were 5.16 and 0.71% for atropine and 5.07 and 1.22% for scopolamine, respectively. Developed method was successfully applied to determine the amounts of both alkaloids in Flos daturae. A baseline separation for atropine and scopolamine was achieved within 11 min.     

Glutaraldehyde activated eggshell membrane for immobilization of tyrosinase from Amorphophallus companulatus: application in construction of electrochemical biosensor for dopamine

Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. K_m and V_max values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min⁻¹, respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 °C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 °C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at −0.19 V versus Ag/AgCl (3 M KCl). Linearity was observed within the range of 50-250 μM with a detection limit of 25 μM.     

Simultaneous determination of nine flavonoids in Anaphalis margaritacea by capillary zone electrophoresis

A capillary zone electrophoresis method was developed for simultaneous determination of nine flavonoids, including two rare flavonols, in Tibetan medicine Anaphalis margaritacea. Baseline separation was performed at pH 9.6 with 25 mM Na₂B₄O₇ and 10 mM NaH₂PO₄ buffer solution, 20 kV as driving voltage and 275 nm as detection wavelength. Repeatability tests showed that the R.S.D. of both intra- and inter-day migration times and peak areas were less than 5%. Recovery results ranged from 87.9% to 106.1%. Samples of A. margaritacea extracts were analyzed using the validated method, which is useful for its quality control.     

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets with high oxidase yield for analytical glucose and choline detections

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets (PBBIns-Gs) was used to modify a gold electrode to form a three-dimensional PBBIns-Gs/Au electrode that was sensitive to hydrogen peroxide (H₂O₂) in the presence of acetic acid (AcOH). The positively charged nanostructured poly(N-butyl benzimidazole) (PBBIns) separated the graphene sheets (Gs) and kept them suspended in an aqueous solution. Additionally, graphene sheets (Gs) formed “diaphragms” that intercalated Gs, which separated PBBIns to prevent tight packing and enhanced the surface area. The PBBIns-Gs/Au electrode exhibited superior sensitivity toward H₂O₂ relative to the PBBIns-modified Au (PBBIns/Au) electrode. Furthermore, a high yield of glucose oxidase (GOD) on the PBBIns-Gs of 52.3 mg GOD per 1 mg PBBIns-Gs was obtained from the electrostatic attraction between the positively charged PBBIns-Gs and negatively charged GOD. The non-destructive immobilization of GOD on the surface of the PBBIns-Gs (GOD-PBBIns-Gs) retained 91.5% and 39.2% of bioactivity, respectively, relative to free GOD for the colloidal suspension of the GOD-PBBIns-Gs and its modified Au (GOD-PBBIns-Gs/Au) electrode. Based on advantages including a negative working potential, high sensitivity toward H₂O₂, and non-destructive immobilization, the proposed glucose biosensor based on an GOD-PBBIns-Gs/Au electrode exhibited a fast response time (5.6 s), broad detection range (10 μM to 10 mM), high sensitivity (143.5 μA mM⁻¹ cm⁻²) and selectivity, and excellent stability. Finally, a choline biosensor was developed by dipping a PBBIns-Gs/Au electrode into a choline oxidase (ChOx) solution for enzyme loading. The choline biosensor had a linear range of 0.1 μM to 0.83 mM, sensitivity of 494.9 μA mM⁻¹ cm⁻², and detection limit of 0.02 μM. The results of glucose and choline measurement indicate that the PBBIns-Gs/Au electrode provides a useful platform for the development of oxidase-based biosensors.     

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N⁶-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L⁻¹). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R² > 0.999) was achieved over the concentration range 5–1000 μmol L⁻¹. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Conductometric nitrate biosensor based on methyl viologen/Nafion/nitrate reductase interdigitated electrodes

A highly sensitive, fast and stable conductometric enzyme biosensor for determination of nitrate in water is reported for the first time. The biosensor electrodes were modified by methyl viologen mediator mixed with nitrate reductase (NR) from Aspergillus niger by cross-linking with glutaraldehyde in the presence of bovine serum albumin and Nafion^® cation-exchange polymer. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, the enzyme loading and time of immobilization in glutaralaldehyde vapor were investigated with regard to their influence on sensitivity, limit of detection, dynamic range and operational and storage stability. The biosensor can reach 95% of steady-state conductance value in about 15 s. Linear calibration in the range of 0.02 and 0.25 mM with detection limits of 0.005 mM nitrate was obtained with a signal-to-noise ratio of 3. When stored in 5 mM phosphate buffer (pH 7.5) at 4 °C, the sensor showed good stability over 2 weeks.     

Ascorbic acid sensor based on ion-sensitive field-effect transistor modified with MnO2 nanoparticles

An ascorbic acid (AA) sensor based on an ion-sensitive field-effect transistor (ISFET) was prepared by modifying the sensitive area of the transducer with MnO₂ nanoparticles. An additional Nafion membrane coated on top of the sensor was used to immobilize the MnO₂ nanoparticles and restrict the amount of ascorbic acid entering the membrane. The reaction of the MnO₂ nanoparticles with ascorbic acid produced a local pH change, which was correlated with the ascorbic acid concentration and could be monitored by the ISFET. The linear range of the ascorbic acid sensor was 0.02-1.27 mM, and the detection limit was 0.01 mM. The effects of buffer concentration, pH, and ionic strength on the sensor performance were also examined. In addition, the sensor has good stability and reproducibility, and the construction and renewal of the sensor are simple and inexpensive.     

Glutathione-capped CdTe quantum dots for the sensitive detection of glucose

A simple and sensitive assay system for glucose based on the glutathione (GSH)-capped CdTe quantum dots (QDs) was developed. GSH-capped CdTe QDs exhibit higher sensitivity to H₂O₂ produced from the glucose oxidase catalyzed oxidation of glucose, and are also more biocompatible than other thiols-capped QDs. Based on the quenching of H₂O₂ on GSH-capped QDs, glucose can be detected. The detection conditions containing reaction time, the concentration of glucose oxidase and the sizes of QDs were optimized and the detection limits for glucose was determined to be 0.1 μM; two detection ranges of glucose from 1.0 μM to 0.5 mM and from 1.0 mM to 20 mM, respectively were obtained. The detection limit was almost a 1000 times lower than other QDs-based optical glucose sensing systems. The developed glucose detection system was simple and facile with no need of complicated enzyme immobilization and modification of QDs.     

Micellar electrokinetic chromatography with amperometric detection and off-line solid-phase extraction for analysis of carbamate insecticides

Six selected primary carbamate insecticides, methomyl, carbaryl, carbofuran, propoxur, isoprocarb, and promecarb, were hydrolyzed in alkaline solution, resulting in electroactive derivatives detectable at a platinum (Pt) electrode poised at +0.8 V vs Ag/AgCl (3 M NaCl). The Pt electrode was inserted into a small electrochemical cell and positioned close to the capillary outlet as an end-column detector to detect the carbamate derivatives after electrophoretic separation. Based on their predicted pK_a values and aqueous solubilities, micellar electrokinetic chromatography (MEKC) was optimized for baseline separation of the derivatives using 20 mM borate, pH 10.2 containing 20 mM sodium dodecyl sulfate as a running buffer. When combined with solid-phase extraction (SPE) on octadecyl silica, a preconcentration factor of 100-fold achieved detection to 0.5 μM methomyl and to 0.01 μM for the remaining five pesticides, significantly below the level regulated by government agencies of most countries. The SPE-MEKC method when applied to the separation and analysis of spiked river water and soil samples, yielded results with excellent reproducibility, recovery and selectivity.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.     

Amperometric biosensing of carbamate and organophosphate pesticides utilizing screen-printed tyrosinase-modified electrodes

A tyrosinase (Tyr) screen-printed biosensor based on the electroreduction of enzymatically generated quinoid products was electrochemically characterized and optimized for determination of carbamates and organophosphorus pesticides. A composite electrode prepared by screen-printing a cobalt (II) phthalocyanine (CoPc) modified cellulose-graphite composite on a polycarbonate support was employed as electrochemical transducer. The Tyr biosensor was prepared by immobilization of enzyme on the composite electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. Parameters affecting the biosensor response such as response time, enzyme loading, concentration and pH of the buffer solution were optimized utilizing catechol as substrate. The maximum response for o-quinone enzymatically generated was obtained after 2 min of reaction. A good reproducibility and high operational stability were found for Tyr biosensor (60 units) at 50 mM phosphate buffer, pH 6.50. Under these conditions, the useful lifetime of biosensor was 10 days. After 15 days, the biosensor could be used with 20% of the initial value. Inhibition studies on the o-quinone steady-state current (at −0.20 V versus Ag/AgCl) were performed to investigate the inhibition kinetics of the pesticides in the enzymatic activity of mushroom tyrosinase. The results shown that the methyl parathion and carbofuran can lead to competitive inhibition process of the enzyme, while diazinon and carbaryl act as mixed inhibitors. Linear relationships were found for methyl parathion (6-100 ppb), diazinon (19-50 ppb), carbofuran (5-90 ppb) and carbaryl (10-50 ppb). Analysis of natural river water samples spiked with 30 ppb of each pesticide showed recoveries between 92.50% and 98.50% and relative standard deviations of 2%.     

Hydrogen peroxide biosensor based on hemoglobin modified zirconia nanoparticles-grafted collagen matrix

A novel method for the immobilization of hemoglobin (Hb) and preparation of reagentless biosensor was proposed using a biocompatible non-toxic zirconia enhanced grafted collagen tri-helix scaffold. The formed membrane was characterized with UV-vis and FT-IR spectroscopy, scanning electron microscope and electrochemical methods. The Hb immobilized in the matrix showed excellent direct electrochemistry with an electron transfer rate constant of 6.46 s⁻¹ and electrocatalytic activity to the reduction of hydrogen peroxide. The apparent Michaelis-Menten constant for H₂O₂ was 0.026 mM, showing good affinity. Based on the direct electrochemistry, a new biosensor for H₂O₂ ranging from 0.8 to 132 μM was constructed. Owing to the porous structure and high enzyme loading of the matrix the biosensor exhibited low limit of detection of 0.12 μM at 3σ, fast response less than 5 s and high sensitivity of 45.6 mA M⁻¹ cm⁻². The biosensor exhibited acceptable stability and reproducibility. ZrO₂-grafted collagen provided a good matrix for protein immobilization and biosensing preparation. This method was useful for monitoring H₂O₂ in practical samples with the satisfactory results.     

Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

Electrochemical behaviour of isatin at a glassy carbon electrode

A rapid, reliable and simple capillary zone electrophoresis method for the determination of organic acids in beverages was developed. The complete separation of oxalic, formic, tartaric, malic, succinic, maleic, glutaric, pyruvic, acetic, lactic, citric, butyric, benzoic, sorbic, ascorbic and gluconic acids can be achieved in less than 3.5 min with a simple electrolyte composed by phosphate as the carrier buffer (7.5 mM NaH₂PO₄ and 2.5 mM Na₂HPO₄), 2.5 mM TTAOH as electroosmotic flow modifier and 0.24 mM CaCl₂ as selectivity modifier, adjusting the pH at 6.40 constant value. Injection was performed in hydrodynamic mode (30 s) and the detection mode was UV direct at 185 nm. The running voltage was −25 kV at thermostated temperature of 25 °C. The method developed has been applied to several beverage samples with only a simple dilution and filtration treatment of the sample. The proposed method is fast because the separation time decrease two, four or, even, six times the separation times of the previous reported CZE methods. It is also simple and cheap due to a low consumption of chemicals and samples. These reasons permit it to be considered adequate for routine analysis of organic acids in beverage samples.