Determination of Picoxystrobin and Pyraclostrobin by MEKC with On-Line Analyte Concentration

Micellar electrokinetic capillary chromatography was used for the determination of picoxystrobin and pyraclostrobin. The background electrolyte consisted of borate buffer (40 mmol L⁻¹ pH 8.5), SDS (30 mmol L⁻¹) and acetonitrile (15% in volume). Runs were made at 25 °C with 25 kV applied potential. The developed method was applied to analyte fortified urine samples. On-line analyte concentration, combined with a capillary of a longer optical path length, allowed limits of quantification of 8.6 × 10⁻⁸ mol L⁻¹ for picoxystrobin and 1.8 × 10⁻⁷ mol L⁻¹ for pyraclostrobin.

     

GC–MS Method Development for the Analyses of Thiophenes from Solvent Extracts of Tagetes patula L

A method for isotachophoretic determination of potassium and ammonium cations in fertilizers and silage was developed. A capillary of 0.4 mm i.d. and 100 mm effective length made of fluorinated ethylene–propylene copolymer was filled with an electrolyte system consisting of 10 mmol L⁻¹ RbOH + 0.1% (w/v) hydroxyethylcellulose, adjusted to pH 9.0 with l-histidine (leading electrolyte) and 10 mmol L⁻¹ lithium citrate (terminating electrolyte). Using contactless conductivity detection, the calibration curves in the tested concentration range up to 0.5 mmol L⁻¹ were linear for both cations. The concentration detection limits for potassium and ammonium were 2.9 and 2.7 μmol L⁻¹, respectively. RSD values of step lengths (n = 6) were 1.3% for potassium and 1.5% for ammonium. The separation time was about 20 min. Similar results were obtained with cesium cation used as the leading ion, however, in the system with rubidium better resolution of other cations present in tested matrices was reached. The elaborated method is simple to perform, sufficiently sensitive and accurate and can be recommended as an alternative procedure to the methods used so far for the determination of potassium and ammonium.

     

Simultaneous Analysis of Isoniazid and Its Impurities by CZE

An alternative methodology for simultaneous determination of isoniazid, isonicotinic acid, 4-cyanopyridine, ethyl isonicotinate and isonicotinamide by capillary zone electrophoresis, within an analysis time of 11 min, is proposed. An electrolyte composed by 50 mmol L⁻¹ of acetic acid/sodium acetate buffer and 12.5 mmol L⁻¹ of Cu²⁺, an analyte dilution in 1 mmol L⁻¹ of Brij 35 and 12.5 mmol L⁻¹ of Cu²⁺ and +20 kV voltage were optimized. After evaluating some figures of merit, such as linearity, precision, recovery and quantification limit, the method was successfully applied to the isoniazid analysis in tablets. The contents found were 99.3 mg of isoniazid and 0.3 mg of isonicotinic acid, corresponding to 0.3 % of impurity regarding the content of the active ingredient in the pharmaceutical formulation.

     

Determination of Fatty Acids (C1 – C10) from Bryophytes and Pteridophytes

A simple and mild method for the determination of fatty acids (C₁ – C₁₀) based on a condensation reaction using 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) as labeling reagent with capillary zone electrophoresis has been developed. The detection was performed with a diode array detector at 254 nm. A 58.5 cm × 50 μm i.d. (50 cm effective length) untreated fused-silica capillary was used. To optimize the separation conditions, the background electrolyte concentration, column temperature, voltage and other factors were evaluated. The optimal separation conditions were as follows: 30 mmol L⁻¹ borate buffer (pH 9.5), 15 mmol L⁻¹ β-CD, temperature at 20 °C, pressure 50 mbar and injection time 8 s. Under the established conditions, 10 fatty acid derivatives could be well-separated within 17 min. The linearity was in the range of 0.07–5.0 μmol L⁻¹. Detection limits (at a signal-to-noise ratio of 3) were in the range of 0.027–0.042 μmol L⁻¹. The fatty acids from the extracted Funaria Hedw. and Selaginella samples were determined with satisfactory results.

     

Optimization and Validation of a Capillary MEKC Method for Determination of Proteins in Urine

Proteinuria, i.e. increased excretion of proteins in urine, is a common sign indicating renal or urinary tract diseases. In this study, a fast and simple procedure for urine sample preparation and capillary micellar electrokinetic chromatographic analysis is presented, without any sample pretreatment prior to the analysis. The developed MEKC method was employed for simultaneous determination of albumin (ALB), haemoglobin (HGB), and myoglobin (MYO) in human urine samples obtained from patients with diagnosed proteinuria. Optimum conditions for detection and separation of ALB, HGB, and MYO are 50 mmol L⁻¹ borate buffer containing 20 mmol L⁻¹ SDS (pH 9.3), injection 40 mbar × 20 s, voltage 25 kV, temperature 30 °C, and detection wavelength 200 nm. The method was shown to be specific, accurate, linear (correlation coefficients r ² > 0.99), and precise (RSD below 3.75 and 7.23% for migration time and peak area, respectively). Multi-variable-at-a-time (MVAT) approach for robustness testing shows no significant variations in accuracy, specificity, and precision as RSD values were lower than 5 and 10% for migration time and peak area, respectively. The presented method is applicable for routine analyses of urine samples as a screening method for patients with excess ALB, HGB, and MYO.

     

Simultaneous Determination of Toxic Alkaloids in Blood and Urine by HPLC–ESI–MS/MS

A sensitive and selective method for simultaneous determination of 29 toxic alkaloids in human blood and 31 in urine using high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry was developed and validated. The samples were diluted with 0.1 mol L⁻¹ HCl, and the target alkaloids were purified by solid phase extraction. The separation of 31 alkaloids was carried out on a C18 column using a gradient mobile phase with 10 mmol L⁻¹ ammonium formate in water with 0.1% formic acid and methanol at the rate of 0.25 mL min⁻¹. The triple-quadrupole mass spectrometer equipped with an electrospray source in the positive mode was set up in the dynamic multiple reactions monitoring mode (dynamic MRM) to detect the ion transitions of 31 alkaloids. The calibration curves were linear over a range of 0.5–400, 1–400, or 4–400 μg L⁻¹ for target alkaloids in human blood and urine, and the correlation coefficients (r ²) was higher than 0.9943. The limit of determination and limit of quantification were 0.2–1 and 0.5–4 μg L⁻¹ for blood and urine, respectively. The only exceptions were sanguinarine and chelerythrine in human blood. All the target alkaloids were stable under the test condition. In addition, the solvent effect and reconstituted solution were investigated. The method was validated and proved to be accurate and precise over the studied concentrations and suitable for poisoning diagnosis and forensic toxicology.

     

Electroanalytical Characteristics of Lercanidipine and its Voltammetric Determination in Pharmaceuticals and Human Serum on Boron-Doped Diamond Electrode

The electrooxidative behavior and determination of lercanidipine (LRC) were investigated in aqueous acetonitrile medium at a boron-doped diamond electrode using voltammetric techniques. The LRC in selected supporting electrolyte presents a well-defined anodic response at 0.944 V, studied by the proposed method. The linear response was obtained in the ranges of 4 × 10^?6 to 2 × 10^?4 mmol L^?1 range in 0.5 mmol L^?1 sulfuric acid supporting electrolyte and 1 × 10^?5 to 8 × 10^?5 mmol L^?1 range in spiked serum sample for square wave voltammetric technique. No electroactive interferences from the excipients and endogenous substances were found in the pharmaceutical dosage form and in the biological sample, respectively.     

Sensitive Analysis of Wilforidine in Human Plasma by LC-APCI-MS/MS

Wilforidine is a potentially efficient medicine to cure autoimmune diseases. In this paper, a sensitive and selective liquid chromatographic method coupled with atmospheric -pressure chemical ionization mass spectrometry (LC–APCI–MS/MS) has been developed for quantification of wilforidine in human plasma. Samples were deproteinized with acetonitrile and cleaned by solid-phase extraction. The chromatographic separation was performed on an analytical RRHD C₁₈ column (50 × 2.1 mm) using ammonium acetate solution (10.0 mmol L⁻¹)/acetonitrile (30/70, v/v) as the mobile phase at a flow rate of 0.7 mL min⁻¹. Detection was carried out by the positive multiple reaction monitoring mode with transitions of m/z 780 → 684 for wilforidine, and 646 → 586 for aconitine (internal standard), respectively. The calibration curve was linear (r = 0.9991) in the concentration range of 0.5–100.0 μg L⁻¹ with a lower limit of quantification of 0.5 μg L⁻¹ in plasma. Intra- and inter-day relative standard deviations were less than 6.8 and 13.1 %, respectively, and the recoveries were between 88.0 and 96.0 %. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of wilforidine in human plasma.

     

In-Capillary Derivatization and Preconcentration for CE of Metal Ions as Their Phenanthroline Complexes

A simple and automated method involving in-capillary derivatization and in-capillary preconcentration was developed for the simultaneous determination of metal ions by capillary zone electrophoresis. Fe(II), Zn(II), Cu(II) and Cd(II) were derivatized using 1,10-phenanthroline as the derivatizing agent. The in-capillary derivatization and in-capillary preconcentration via large volume injection were performed sequentially as follows: 60 mmol L^?1 1,10-phenanthroline was first hydrodynamically injected (0.2 psi) for 2 s; metal ions were introduced by hydrodynamic injection (0.5 psi) for 60 s; 0.2 mol L^?1 acetate pH 5.5 containing 20 % methanol was used as the running buffer. Four metal ions can be determined within 8 min using 16 kV. The resulting preconcentration factors were in the range 12–21. Good linearity was obtained for concentrations of 0.1–8.0 mg L^?1 (r ² > 0.990). The mean recoveries of the metal ions evaluated by fortification of wine samples were in the range 90–102 %. The limits of detection ranged from 0.05 to 0.2 mg L^?1. The proposed method can be applied for directly determining metal ions in wine samples.     

LC Method for Determination of Ginkgolic Acids in Mice Plasma and Its Application to a Pharmacokinetic Study

A sensitive and simple LC method for the quantification of ginkgolic acids in mice plasma has been developed. Following acetonitrile deproteinization, samples were separated on a SinoChrom ODS-AP C₁₈ column. The mobile phase was 3% (v/v) acetic acid water solution–methanol (8:92, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was at 310 nm. Calibration curve was linear over the range of 0.25–50 μg mL⁻¹ with intra- and inter-day precisions (RSD%) of less than 9.5%. The extraction recovery ranged from 87.0 to 90.2% (RSD 2.4–6.4%) for ginkgolic acids. The method was successfully applied to the pharmacokinetic study of ginkgolic acids in mice after oral dosing of 1.0 g kg⁻¹.

     

Simultaneous Determination of Organic Acids in Wine Samples by Capillary Electrophoresis and UV Detection: Optimization with Five Different Background Electrolytes

A simple technique is described for the routine capillary electrophoretic determination of organic acids in wine samples. Several aromatic and non‐aromatic compounds, including phthalic acid, benzoic acid, sorbic acid, boric acid, and phosphate, were evaluated as background electrolytes in order to obtain the highest resolution and detection sensivity. Factors that affect capillary electrophoretic separation such as the concentration and pH of the background electrolyte (BGE), the concentration of the electroosmotic flow modifier (EOF), and methanol addition to the electrolyte were investigated systematically. Tartaric, malic, succinic, acetic, and lactic acids were determined simultaneously in approximately six minutes using an electrolyte containing 3 mM phosphate and 0.5 mM myristyltrimethylammonium bromide (MTAB) as electroosmotic flow modifier at pH 6.5. This method is quantitative, with recoveries in the 90–102% range and linear up to 50 mg L^–1. The precision is better than 1% and the procedure shows the appropriate sensibility, with detection limits between 0.015 and 0.054 mg L^–1. The proposed method was successfully employed for the determination of organic acids in wine samples by direct sample injection after appropriate dilution and filtration.     

Determination of BTEX Compounds by Dispersive Liquid–Liquid Microextraction with GC–FID

Rapid, inexpensive, and efficient sample-preparation by dispersive liquid–liquid microextraction (DLLME) then gas chromatography with flame ionization detection (GC–FID) have been used for extraction and analysis of BTEX compounds (benzene, toluene, ethylbenzene, and xylenes) in water samples. In this extraction method, a mixture of 25.0 μL carbon disulfide (extraction solvent) and 1.00 mL acetonitrile (disperser solvent) is rapidly injected, by means of a syringe, into a 5.00-mL water sample in a conical test tube. A cloudy solution is formed by dispersion of fine droplets of carbon disulfide in the sample solution. During subsequent centrifugation (5,000 rpm for 2.0 min) the fine droplets of carbon disulfide settle at the bottom of the tube. The effect of several conditions (type and volume of disperser solvent, type of extraction solvent, extraction time, etc.) on the performance of the sample-preparation step was carefully evaluated. Under the optimum conditions the enrichment factors and extraction recoveries were high, and ranged from 122–311 to 24.5–66.7%, respectively. A good linear range (0.2–100 μg L⁻¹, i.e., three orders of magnitude; r ² = 0.9991–0.9999) and good limits of detection (0.1–0.2 μg L⁻¹) were obtained for most of the analytes. Relative standard deviations (RSD, %) for analysis of 5.0 μg L⁻¹ BTEX compounds in water were in the range 0.9–6.4% (n = 5). Relative recovery from well and wastewater at spiked levels of 5.0 μg L⁻¹ was 89–101% and 76–98%, respectively. Finally, the method was successfully used for preconcentration and analysis of BTEX compounds in different real water samples.

     

Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L⁻¹ tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL⁻¹ for cytosine (C), 0.01 ng mL⁻¹ for 5-methylcytosine (5-mC), and 0.02 ng mL⁻¹ for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

     

Carbon‐nanotube Modified Screen‐printed Electrode for the Simultaneous Determination of Nitrite and Uric Acid in Biological Fluids Using Batch‐injection Amperometric Detection

A portable electroanalytical system applied for rapid and simultaneous determination of uric acid (UA) and nitrite (NIT) in human biological fluids (urine, saliva and blood) is reported. The system is based on batch‐injection analysis with multiple‐pulse amperometric (BIA‐MPA) detection using screen‐printed electrodes (SPEs) modified with multi‐walled carbon nanotubes. Sample dilution in optimized electrolyte (0.1 mol L⁻¹ Britton‐Robinson buffer pH 2) followed by injection of 100 μL on the electrode surface using an electronic micropipette is performed. UA is detected at +0.45 V and both UA+NIT at +0.70 V. Linear calibration plots for UA and NIT were obtained over the range of 1–500 μmol L⁻¹ with detection limits of 0.05 and 0.06 μmol L⁻¹, respectively. For comparison, a differential‐pulse voltammetric (DPV) method was optimized, and linear calibration plots for UA and NIT were obtained over range of 1–30 μmol L⁻¹ and 1–40 μmol L⁻¹ with detection limits of 0.1 and 0.3 μmol L⁻¹, respectively. BIA‐MPA is highly precise (RSD<1.3 %), fast (160 h⁻¹) and free from sample‐matrix interferences as recovery values ranged from 77 to 121 % for spiked samples (short contact time of sample aliquot with SPE). Contrarily, recovery tests conducted using DPV did not provide adequate recovery values (>150 %), probably due to the longer contact time of the SPE with the biological samples during analysis leading to a severe interference of sample matrices.     

Rapid Determination of DDT and Its Metabolites in Environmental Water Samples with Mixed Ionic Liquids Dispersive Liquid–Liquid Microextraction Prior to HPLC-UV

In this paper, a novel mixed ionic liquids-dispersive liquid–liquid microextraction method was developed for rapid enrichment and determination of environmental pollutants in water samples. In this method, two kinds of ionic liquids, hydrophobic ionic liquid and hydrophilic ionic liquid, were used as extraction solvent and disperser solvent, respectively. DDT and its metabolites were used as model analytes and high-performance liquid chromatography with ultraviolet detector for the analysis. Factors that may affect the extraction recoveries, such as type and volume of extraction solvent (hydrophobic ionic liquid) and disperser solvent (hydrophilic ionic liquid), extraction time, sample pH and ionic strength, were investigated and optimized. Under the optimum conditions, the linear range was 1–100 μg L⁻¹, limits of detection could reach 0.21–0.49 μg L⁻¹, and relative standard deviation was 6.01–8.48 % (n = 7) for the analytes. Satisfactory results were achieved when the method was applied to analyze the target pollutants in environmental water samples with spiked recoveries over the range of 85.7–106.8 %.

     

Rapid Determination of Trifluoromethanesulfonate and p-Toluenesulfonate by Ion-Pair Chromatography Using a Reversed-Phase Silica-Based Monolithic Column: Application to the Analysis of Ionic Liquids

A method of ion-pair chromatography was developed on a reversed-phase silica-based monolithic column for the fast and simultaneous determination of trifluoromethanesulfonate (CF₃SO₃ ⁻) and p-toluenesulfonate (C₇H₇SO₃ ⁻). The analysis was performed using a mobile phase of tetrabutylammonium hydroxide + citric acid + acetonitrile on the Chromolith Speed ROD RP-18e column with direct conductivity detection. The effects of the eluent, column temperature and flow rate on the retention of the anions were investigated. The experimental phenomenon was discussed according to hydrophobic interaction and ion-exchange mechanism in the separation. The optimized chromatographic conditions were selected. The optimized eluent for the separation consisted of 0.2 mmol L⁻¹ tetrabutylammonium hydroxide + 0.10 mmol L⁻¹ citric acid + 9% acetonitrile (pH 5.5). The flow rate was set at 6.0 mL min⁻¹. The column temperature was 25 °C. Under the optimal conditions, the better separation of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ was achieved without any interference by other anions (Cl⁻, Br⁻, I⁻, NO₃ ⁻, SO₄ ²⁻, ClO₃ ⁻, BF₄ ⁻ and PF₆ ⁻). The detection limit (S/N = 3) was 0.28 and 0.71 mg L⁻¹ for CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻, respectively. The method has been applied to the determination of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ in ionic liquids. The spiked recoveries of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ were 101.1 and 100.2%, respectively.

     

CZE Separation of New Drugs for Treatment of Leukemia

Imatinib, bosutinib, dasatinib, pazopanib, erlotinib, canertinib and vatalanib are new developed anticancer drugs, especially for treatment of leukemia. In this article, a fast and high throughput capillary zone electrophoresis method has been developed and validated for analysis of these new drugs in pharmaceutical formulas. The method can be easily utilized for determination of all the drugs in one run what is advantageous for the quality control in pharmaceutical industry because there is no need for changing and optimization of separation conditions when changing the analyte. The separation was performed using an uncoated fused silica capillary with 100 mmol L⁻¹ sodium phosphate buffer pH 2.75, voltage of 25 kV, hydrodynamic injection time of 5 s by 50 mbar, and detection at 214 nm. Under these conditions, the analysis took about 8 min. The validation of all the drugs resulted in recoveries in the range of 84–100 %. The method showed to be precise for all the drugs with RSDs of migration times lower than 0.9 % (interday precision). A very good linearity in the validated range (5–100 μg mL⁻¹) and the limits of detection (LODs) in the range of 0.5–2.0 (μg mL⁻¹) were achieved. Finally, we proved that the method is robust by the Youden’s test. Therefore, our method can be successfully applied for analysis of the real pharmaceutical samples.

     

An electrochemical sensor for stripping analysis of Pb(II) based on multiwalled carbon nanotube functionalized with 5-Br-PADAP

The present study serves to focus attention on the modification of multiwalled carbon nanotube with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) and its application for the development of a new, simple, and selective modified electrode in order to determine Pb(II) in standard alloys and water samples. The electrochemical method was based on open circuit accumulation of lead ions onto a 5-Br-PADAP-modified multiwalled carbon nanotube electrode and then their anodic stripping voltammetric determination. The linear range for Pb(II) ions was from 0.9 to 114.6 μg L⁻¹. The detection limit and relative standard deviation were found to be 0.1 μg L⁻¹ and 1.6%, respectively. This method demonstrates the powerful application of carbon nanotubes in the field of mercury-free electrodes in voltammetric stripping analysis.

     

Simultaneous Determination of Procaspase Activating Compound 1 and Permeability Markers in Intestinal Perfusion Samples and Application to a Rat Intestinal Absorption Study

A sensitive and simple HPLC method for simultaneous determination of PAC-1 (first procaspase-activating compound), phenol red, and permeability markers (carbamazepine and furosemide) in perfusion samples was developed and validated to assess intestinal absorption of PAC-1 using single-pass intestinal perfusion technique (SPIP) in rats. The chromatographic separation was carried out on a Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with acetonitrile–methanol–30 mmol L⁻¹ phosphate buffer (pH 3.0, 25:10:65, v/v/v) as mobile phase at a flow rate of 1.0 mL min⁻¹, and the wavelength of the UV detector was set at 281 nm. The calibration curves were linear in the ranges of 2.40–48.0 μg mL⁻¹ for PAC-1; 3.60–72.0 μg mL⁻¹ for carbamazepine; 3.20–64.0 μg mL⁻¹ for furosemide, and 4.80–96.0 μg mL⁻¹ for phenol red (r > 0.999). Both the intra- and inter-day precisions (RSD%) of all analytes were less than 6.8 % at three concentration levels, while accuracy ranged from 95.4 to 104.5 %. Data obtained in all method validation studies indicated that the method was suitable for the intended purpose. The effective permeability values (P _eff) considering water flux with the help of non-permeable marker phenol red was calculated to be 0.42 × 10⁻⁴, 0.62 × 10⁻⁴, 0.32 × 10⁻⁴ cm s⁻¹ for PAC-1; 0.72 × 10⁻⁴, 0.77 × 10⁻⁴, 0.52 × 10⁻⁴ cm s⁻¹ for carbamazepine; 0.20 × 10⁻⁴, 0.16 × 10⁻⁴, 0.12 × 10⁻⁴ cm s⁻¹ for furosemide in duodenum, jejunum and ileum, respectively. The P _eff value can be increased by co-perfusion with verapamil, indicating that absorption of PAC-1 is efficiently transported by P-glycoprotein (P-gp) in the gut wall.

     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL⁻¹ and detect 50 pg mL⁻¹ of taurine ranging normally between 65 and 179 mmol L⁻¹ (8–22 μg mL⁻¹). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.