Non-denaturating isoelectric focusing gel electrophoresis for uranium–protein complexes quantitative analysis with LA-ICP MS

A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)–protein complexes with laser ablation–inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U–protein complex standards (i.e., U–bovine serum albumin and U–transferrin) allowing the assessment of U recovery to 64.4?±?0.4 %. This methodology enabled the quantification of U–protein complexes at 9.03?±?0.23, 15.27?±?0.36, and 177.31?±?25.51 nmol U L^?1 in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L^?1 during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L^?1. Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U–protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation.

The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin,Dopamine and Melatonin

Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L⁻¹; R ² = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L⁻¹ and the LOQs of 15.4–70.5 nmol L⁻¹ for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.

     

Electrochemically controlled solid-phase micro-extraction of proline using a nanostructured film of polypyrrole, and its determination by ion mobility spectrometry

We report on a fast, simple and accurate method for the determination of proline in urine samples by employing a nanostructured film of conducting polypyrrole for electrochemically controlled solid-phase microextraction, and ion mobility spectrometry (IMS) for detection. This method has the advantages of simple sample preparation and a sensitivity of IMS to proline that is higher than that for other amino acids. The calibration curve is linear in the range of 0.5–60 μg L^?1 (4–521 nmol L^?1), and the detection limit is 0.2 μg L^?1. The electrochemical potentials for uptake and release were optimized. The method was successfully applied to the clean-up and quantitation of trace amounts of proline in urine samples.

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.     

Electrochemical Characterization of and Stripping Voltammetry at Screen Printed Electrodes Modified with Different Brands of Multiwall Carbon Nanotubes and Bismuth Films

A screen-printed electrode sensor has been fabricated by modifying the carbon ink surface with different brands of multiwall carbon nanotubes (MWCNTs) and bismuth film (BiF) for the determination of traces of lead, cadmium and zinc ions by square wave anodic stripping voltammetry. The MWCNTs, from three different sources, were functionalized and dispersed in Nafion (MWCNT-Nafion) solution and placed on screen printed electrodes (MWCNT-Nafion/SPE); bismuth films were then prepared by ex-situ plating of bismuth onto the MWCNT-Nafion/SPE electrodes. The electrochemical characteristics of BiF/MWCNT-Nafion/SPE/ were examined by electrochemical impedance spectroscopy and showed differences; the charge transfer resistance tends to decrease with negative applied potentials. After optimizing the experimental conditions, the square-wave peak current signal is linear in the nmol L^?1 range. The lowest limit of detection found for the separate determination of lead, cadmium and zinc were 0.7 nmol L^?1, 1.5 nmol L^?1, and 11.1 nmol L^?1, respectively, with a 120 s deposition time.     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

LC Analysis of Aliphatic Primary Amines and Diamines After Derivatization with 2,6-Dimethyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester

A method for sensitive simultaneous analysis of aliphatic primary amines and diamines has been developed and validated. The compounds were analyzed by reversed-phase high-performance liquid chromatography after pre-column derivatization with 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester as fluorescent probe. The derivatization reaction was performed at 50 °C for 40 min in 0.1 mol L^?1 borax buffer solution (pH 7.5). The resulting fluorophores were separated to baseline on a C₁₈ column and fluorimetrically detected at λ _ex/λ _em = 326/409 nm. Detection limits were in the range 0.50–0.02 nmol L^?1. The method was successfully used for analysis of aliphatic amines in water, human urine, and serum.     

Determination of Paraquat by Cucurbit[7]uril Sensitized Fluorescence Quenching Method

A method for the determination of paraquat by cucurbit[7]uril (CB[7]) fluorescence quenching was developed. The assay was based on the reaction of the CB[7] with acridine orange. The fluorescence intensity of acridine orange regularly increased with the addition of CB[7]. However, while an appropriate amount of paraquat was added to the CB[7]- acridine orange system, the fluorescence intensity of the system was quenched which was employed to determine paraquat. Under the optimum conditions, a linear range of 3.0–800 nmol L^?1 and a detection limit of 1.61 nmol L^?1 for paraquat were obtained. The simple strategy reported here offers great practical potential for the determination of pesticide residues in agricultural products.     

Quantitative Analysis of Sertraline in Human Serum by LC with Fluorescence Detection After Pre-Column Derivatization with 4-Chloro-7-nitrobenzofurazan

An accurate and sensitive reversed-phase high-performance liquid chromatographic method for analysis of sertraline in human serum, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent, is described. The drug and an internal standard (azithromycin) were extracted from serum by use of a mixture of diethyl ether and chloroform, and subjected to pre-column derivatization with the reagent. Analysis of the resulting derivatives was performed on a 250 mm × 4.0 mm cyano column with 63:37 (v/v) methanol–sodium phosphate buffer (0.05 M, pH 3.7) containing 2 mL L^?1 triethylamine as mobile phase. Detector response was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. The calibration plot was linear over the concentration range 2–640 ng mL^?1. The lower limits of detection and quantification were 0.5 and 2 ng mL^?1, respectively. The method was validated for specificity, sensitivity, linearity, precision, accuracy, and stability and shown to be accurate (intra-day and inter-day accuracy from 0.3 to 4.2%) and precise (intra-day and inter-day precision from 2.4 to 15.5%). The drug was detected at concentrations as low as 2 ng mL^?1 in 0.5 mL serum and the method described can be easily applied to human single-dose pharmacokinetic studies of sertraline.     

A Highly Efficient ZrO2 Nanoparticle Based Electrochemical Sensor for the Detection of Organophosphorus Pesticides

A sensitive electrochemical method for square‐wave voltammetric detection of organophosphate (OP) compounds was developed based on zirconia (ZrO₂) nanoparticles modified electrode. The electrode was fabricated using electrochemical deposition and characterized by scanning electron microscopy (SEM), which confirmed the successful formation of nanoparticles. Due to the strong affinity of ZrO₂ with the phosphoric group, nitroaromatic OPs can strongly bind to the surface of ZrO₂ nanoparticles (ZrO₂NPs). Under optimized operational conditions, SWV was employed for Omethoate (a model of OP compounds) detection with 5 min absorption, which showed a wide detection range from 98.5 pmol·L^?1 to 985 nmol·L^?1, with a detection limit as low as 52.5 pmol·L^?1. This electrochemical sensor has good selectivity, stability and reproducibility, and great potential in the detection of OP compounds in agriculture area.     

LC with Fluorimetric Detection for Sensitive Analysis of Reduced Flubendazole in Biological Samples

A validated LC method is proposed for analysis of flubendazole and its metabolites in biological samples of Haemonchus contortus. Two detectors were used—photodiode-array and spectrofluorimetric. The native fluorescence of reduced flubendazole, the key substance investigated during biological experiments, was used for its fluorimetric detection with a very low limit of quantification (0.63 nmol L^?1).     

Single Laboratory Validation of a Method for the Determination of Total Inorganic Arsenic by Hydride Generation Atomic Absorption Spectrometry

The purpose of this study consists in reporting of single laboratory validation of a method for the determination of total inorganic arsenic by hydride generation atomic absorption spectrometry from natural and residual water samples. Applicability, fitness for purpose, selectivity, and sensitivity were discussed. A calibration study was realized, linear working range (0.4–4 μg·L^?1), detection (0.11 μg·L^?1), and quantification (0.38 μg·L^?1) limits being determined. It was also proven that the method is accurate and precise. Following the bottom-up approach measurement, uncertainty was estimated (method validation data were used).     

Occurrence of cytostatic compounds in hospital effluents and wastewaters,determined by liquid chromatography coupled to high-resolution mass spectrometry

The occurrence of 26 commonly used cytostatic compounds in wastewaters was evaluated using an automated solid-phase extraction (SPE) method with liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Detection was optimized using Oasis HLB SPE cartridges at pH 2. Two hospital effluents and their two receiving wastewater treatment plants were sampled over five days. In hospital effluents, eight cytostatics were detected at levels up to 86.2 μg L^?1 for ifosfamide, 4.72 μg L^?1 for cyclophosphamide, and 0.73 μg L^?1 for irinotecan, the three most relevant compounds identified. Cyclophosphamide and megestrol acetate were found in wastewaters at concentrations up to 0.22 μg L^?1 for the latter. The predicted environmental concentrations (PEC) in sewage effluents of ifosfamide (2.4–4.3 ng L^?1), capecitabine (11.5–14.2 ng L^?1), and irinotecan (0.4–0.6 ng L^?1), calculated from consumption data in each hospital, published excretion values for the target compounds, and wastewater elimination rates, were in agreement with experimental values.     

Simple Determination of 4-Methylimidazole in Soft Drinks by Headspace SPME GC–MS

A simple method to detect 4-methylimidazole in soft drinks is described. This method is based on headspace solid-phase micro-extraction and gas chromatography–mass spectrometry (HS-SPME GC–MS). The HS-SPME parameters (selection of fiber, extraction temperature, heating time, and pH) were optimized and selected. Under the established condition, the detection and the quantification limit were 1.9 and 6.0 μg L^?1 using 4 mL of the liquid sample, respectively. The relative standard deviation for five independent determinations at 100.0 and 500.0 μg L^?1 was less than 8 %. The calibration curve was y = 0.6027x–0.0033 with a linearity of r ² = 0.997. Using the proposed method, the levels of 4-MEI were detected in a range from 94.0 to 324.8 μg L^?1. The comparison of liquid chromatography tandem mass spectrometry (LC–MS/MS) with the proposed method was performed and the agreement with LC–MS/MS for all samples was acceptable.     

Simultaneous determination of eight antibiotics from distinct classes in surface and wastewater samples by solid-phase extraction and high-performance liquid chromatography–electrospray ionisation mass spectrometry

A reliable multiclass method has been developed and validated for the determination of eight antibiotics from distinct classes (sulfonamides, macrolides, fluoroquinolones, tetracyclines, cephalosporins and dihydrofolate reductase inhibitors) in wastewater – influent and effluent – and surface water from Porto Alegre, Brazil. The pre-concentration and clean-up was conducted with a simple and fast protocol using solid-phase extraction allowing a 100-fold concentration factor. The proposed method was validated by using spiked blank wastewater samples in terms of linearity, repeatability, reproducibility, recovery, matrix effects and limits of detection and quantification. Recovery was obtained in the range of 66–149%. Method limit of quantification ranged between 1.6 and 61.7 ng L^?1. Samples (n = 16) were taken from January to August 2011 in one wastewater treatment plant, which uses conventional biological treatment. Sulfamethoxazole and trimethoprim show higher concentration, ranging from >10 to <6500 ng L^?1, whereas erythromycin presented the lower amount. Differences between influent and effluent profiles were discussed. Surface water samples (n = 8) were collected in Arroio Diluvio, in four sampling points, in February 2012. From the eight antibiotics analysed, five were detected: sulfamethoxazole, trimethoprim, azythromicyn, ciprofloxacin and norfloxacin, in a concentration range of 376–572 ng L^?1, 27–94 ng L^?1, 24–40 ng L^?1, 16–66ng L^?1 and 30–54 ng L^?1, respectively.     

A novel,simple and robust method for the analysis of sulfonamides in fish farming water samples using column switching liquid chromatography

A novel method for the online extraction and preconcentration of four sulfonamides was developed using column switching liquid chromatography. Sulfadiazine, sulfathiazole, sulfamethoxypyridazine and sulfamethoxazole were analysed in water samples and preconcentrated in a C18 guard column. Suitable validation parameters were obtained, such as precision, accuracy and relative recovery, in accordance with the validation guidelines of the Food and Drug Administration. Low limits of detection (0.05–0.09 µg L^?1) and quantification (0.30 µg L^?1, for all of them) were obtained. The quadratic polynomial model was used to adjust the calibration data, and the coefficients of determination were higher than 0.999 for all the analytes. The method was shown to be robust to the assayed parameters according to Youden’s test. The proposed method was successfully used to determine sulfonamides in 11 different fish farming water samples, in which sulfadiazine (0.732 µg L^?1), sulfamethoxazole (0.531 µg L^?1), sulfathiazole (0.546–1.856 µg L^?1) and sulfamethoxypyridazine (0.369–1.509 µg L^?1) were found.     

Analysis of Hexanal and Heptanal in Human Blood by Simultaneous Derivatization and Dispersive Liquid–Liquid Microextraction then LC–APCI–MS–MS

A new analytical approach, simultaneous derivatization and dispersive liquid–liquid microextraction followed by liquid chromatography–atmospheric-pressure chemical ionization tandem mass spectrometry, has been developed for analysis of hexanal and heptanal in human blood. In the derivatization and extraction procedure a solution of 2,4-dinitrophenylhydrazine (derivatization reagent) in 85 μL acetonitrile (dispersive solvent) and 50 μL tetrachloromethane (extraction solvent) was rapidly injected into the aqueous sample containing hexanal and heptanal. Within a few seconds the aldehydes were derivatized and simultaneously extracted. After centrifugation, the hydrazones in the sediment phase were analyzed by LC–APCI–MS–MS. Derivatization and extraction conditions were investigated systematically. Under the optimum conditions enrichment factors for hexanal and heptanal in a 1-mL sample were 63 and 73, respectively. The calibration plots were linear in the ranges 0.5–100 and 100–1,000 nmol L^?1, respectively, and the respective limits of detection (LOD) were 0.17 and 0.076 nmol L^?1. Reproducibility and recovery were good. The experimental results were compared with those obtained by use of solid-phase extraction and polymer monolithic microextraction. Because sample derivatization, extraction, and concentration were combined in a single step, the proposed method enabled simple, rapid, inexpensive, and efficient analysis of aldehydes in blood. The method has great potential for clinical analysis of biologically relevant aldehydes.     

Optimization of the Extraction Conditions and Simultaneous Quantification of Six Flavonoid Glycosides in Flos Chrysanthemi by RP-LC

A sensitive, accurate and reliable reversed-phase liquid chromatographic method coupled with DAD (278 nm) was established for simultaneous quantification of six compounds in 20 cultivars of Flos Chrysanthemi. The method was carried out by using a Kromasil 100-5 C₁₈ column with methanol–acetonitrile—1.414 × 10^?2 mol L^?1 aqueous phosphoric acid as a gradient mobile phase. The contents of the six flavonoid glycosides in Flos Chrysanthemi could be determined within 120 min. The linear calibration ranges for these were 0.42–126.00, 11.44–220.00, 0.53–530.00, 4.80–195.00, 11.00–220.00, and 0.12–200.00 μg mL^?1. Their recoveries were 95.33–105.33% with RSDs from 0.10 to 2.00%. Their lower limits of quantification were 0.420, 1.144, 0.250, 0.480, 0.242, and 0.120 μg mL^–1. The method can be used for analysis of the six flavonoid glycosides in Flos Chrysanthemi.     

Determination of organophosphate pesticides in sea and surface water with ultrasound-assisted dispersive liquid–liquid micro-extraction coupled to GC-MS/MS analysis

A sensitive enrichment method for the detection and quantification of 20 organophosphate pesticides in sea water and surface water was improved and validated. The compounds were extracted using ultrasound-assisted dispersive liquid–liquid microextraction and were analysed with an optimised Gas Chromatography - Tandem Quadrupole Mass Spectrometry (GC-MS/MS) method. Calibrations and validations were conducted for spiked local pond and Baltic Sea water. Recoveries of the organophosphate compounds from the different matrices ranged from about 25% to 70% and from 30% to 130% for pond and sea water, respectively. Obtained limit of detection (LoD) for both matrices was about 3 ng L^?1 and the limit of quantification (k = 3) was 12 and 7 ng L^?1 for pond and sea water, respectively. Valid extractability at the LoD level could be proven.     

Determination of Veterinary Pharmaceuticals in Production Wastewater by HPTLC-Videodensitometry

An SPE-HPTLC method for simultaneous identification and quantification of seven pharmaceuticals in production wastewater was optimized and validated. The studied compounds were enrofloxacine, oxytetracycline, trimethoprim, sulfamethazine, sulfadiazine, sulfaguanidine and penicillin G/procaine. The method involves solid-phase extraction on hydrophilic-lipophilic balance cartridges with methanol and HPTLC analysis of extracts on CN modified chromatographic plates followed by videodensitometry at 254 and 366 nm. Optimization of chromatographic separation was performed by systematic variation of the mobile phase composition using genetic algorithm approach and the optimum mobile phase composition for TLC separation was 0.05 M H₂C₂O₄:methanol = 0.81:0.19 (v/v). Linearity of the method was demonstrated in the ranges from 1.5 to 15.0 μg L⁻¹ for enrofloxacine, 100–500 μg L⁻¹ for oxytetracycline, 150–600 μg L⁻¹ for trimethoprim, 300–1100 μg L⁻¹ for sulfaguanidine and 100–400 μg L⁻¹ for sulfamethazine, sulfadiazine and penicillin G/procaine with coefficients of determination higher than 0.991. Mean recoveries ranged from 74.6 to 117.1% and 55.1 to 108.0% for wellspring water and production wastewater, respectively. Only sulfaguanidine showed lower results. The described method has been applied to the determination of pharmaceuticals in wastewater samples from pharmaceutical industry.