HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH-D₃) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1 ml of human plasma was treated with 2 ml of a mixture of ethanol–acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250 μL of a solution of methanol 5 mmol l⁻¹ phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200 μl aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol l⁻¹ phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile–phosphate buffer 5 mmol l⁻¹, pH 6.5; 20:80 (v/v); solvent B: methanol–acetonitrile–tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265 nm. The method was linear in the range 3.0–32.0 ng ml⁻¹ with a limit of quantification of 3.0 ng ml⁻¹. Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was ≤2.80%. The proposed method permitted the simultaneous determination of Vitamin D₃ and 25-OH-D₃ in 16 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samples h⁻¹. The method was successfully applied for the determination of Vitamin D₃ and 25-OH-D₃ in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D₃ and 25-OH-D₃ concentrations in plasma were found from 4.30–40.70 ng ml⁻¹ (19.74 ± 9.48 ng ml⁻¹) and 3.1–36.52 ng ml⁻¹ (7.13 ± 7.80 ng ml⁻¹), respectively. These results were in good agreement with data published by other authors.     

A rapid and sensitive immunonanogold resonance scattering spectral probe for complement 3

Gold nanoparticles in size of about 10 nm was used to label goat anti-human complement 3 (anti-C3) to obtain a sensitive and selective immunoresonance scattering spectral probe for C3. It was based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na₂HPO₄–citric acid buffer solutions and in presence of polyethylene glycol (PEG). The resonance scattering (RS) intensity at 560 nm enhanced greatly with C3. Well linear relationships between the enhanced RS intensity (I_RS) and the C3 concentration in the range of 8.33–200 ng ml⁻¹ were obtained, with a detection limit of 2.8 ng ml⁻¹ and the limit of quantification 8.51 ng ml⁻¹ for C3. The convenient and selective and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.     

Efficiency and mechanism of new poly(acryl-phenylamidrazone phenylhydrazide) chelating fiber for adsorbing trace Ga, In, Bi, V and Ti from solution

A new po1y(acrylphenylamidrazone phenylhydrazide) chelating fiber is synthesized from polyacrylonitrile fiber and used for preconcentration and separation of trace Ga(III), In(III), Bi(III), V(V) and Ti(IV) from solution (5–50 ng ml⁻¹ Ti(IV) or V(V) and 50–500 ng ml⁻¹ Ga(III), In (III) or Bi(III) in 1000–100 ml of solution can be enriched quantitatively by 0.15 g of fiber at a 4 ml min⁻¹ flow rate in the pH range 5–7 with recoveries >95%). These ions can be desorbed quantitatively with 20 ml of 4 M hydrochloric acid at 2 ml min⁻¹ from the fiber column. When the fiber which had been treated with concentrated hydrochloric acid and washed with distilled water until neutral was reused eight times, the recoveries of the above ions by enrichment were still >95%. Two-hundred-fold to 10 000-fold excesses of Cu(II), Zn(II), Ca(II), Mn(II), Cr(III), Fe(III), Ba(II) and Al(III) caused little interference in the determination of these ions by inductively coupled plasma-atomic emission spectrometers (ICP-AES). The relative standard deviations for enrichment and determination of 50 ng ml⁻¹ Ga, In or Bi and 10 ng ml⁻¹ V or Ti are in the range 1.2–2.7%. The contents of these ions in real solution samples determined by this method were in agreement with the certified values of the samples with average errors <3.7%.     

A simple and sensitive assay of nucleic acids based on the enhanced resonance light scattering of zwitterionics

A new assay of nucleic acids at nanogram level was established based on the enhanced resonance light scattering (RLS) signals of two zwitterionics cocamidopropyl hydroxysultaine (HSB) and lauryl betaine (BS-12). Under optimum conditions, the weak RLS signal of HSB is enhanced by nucleic acids, and the enhanced RLS intensity is proportional to the concentration of nucleic acids in the range of 0.02–7.3 mg l⁻¹ for calf thymus DNA and 0.01–8.6 mg l⁻¹ for fish sperm DNA. The detection limits were 1.5 ng ml⁻¹ for calf thymus DNA and 1.9 ng ml⁻¹ for fish sperm DNA. Plasmid DNA extracted from K-12-HB101 colt was determined with satisfactory results.     

Application of a new resin functionalised with 6-mercaptopurine for mercury and silver determination in environmental samples by atomic absorption spectrometry

Polystyrene–divinylbenzene (8%) has been functionalised by coupling it through an ---N=N--- group with 6-mercaptopurine. The resulting chelating resin has been characterised by using elemental analysis, thermogravimetric analysis and infrared spectra. The resin is highly selective for Hg(II) and Ag(I) and has been used for preconcentrating Hg(II) and Ag(I) prior to their determination by atomic absorption spectrometry. The maximum sorption capacity for Hg(II) and Ag(I) was found to be 1.74 and 0.52 mmol g⁻¹, respectively, over the pH range 5.5–6.0. The calibration range for Hg(II) was linear up to 10 ng ml⁻¹ with a 3σ detection limit of 0.02 ng ml⁻¹; the calibration range for Ag(I) was linear up to 5 μg ml⁻¹ with a detection limit of 29 ng ml⁻¹. The recoveries of the metals were found to be 99.7±3.8 and 101.3±4.1% at the 95% confidence level for both Hg(II) and Ag(I). In column operation, it has been observed that Hg(II) and Ag(I) in trace quantities can be selectively separated from geological, medicinal and environmental samples.     

Transients and cooperative action of β-carotene, vitamin E and C in biological systems in vitro under irradiation

Using N^•₃ species as specific electron acceptor a defined ascorbate radical: AH^•↔A^•−+H⁺ (λ_max=360 nm, =3400 dm³ mol⁻¹ cm⁻¹) is observed. The attack of DMSO^•+ on vit.E results in a vit.E^• radical (k=1×10⁹ dm³ mol⁻¹ s⁻¹; λ_max=425 nm, =2400 dm³ mol⁻¹ cm⁻¹; 2k=4.7×10⁸ dm³ mol⁻¹ s⁻¹). Vit.E-acetate leads to the formation of a radical cation (vit.E-ac^•+). β-carotene reacts also with DMSO^•+ forming a radical cation, β-car^•+ (k=1.75×10⁸ dm³ mol⁻¹ s⁻¹; λ_max=942 nm, =14 600 dm³ mol⁻¹ cm⁻¹), which probably leads to the formation of a dimer radical cation, (β-car)^•+₂ (k=2.5×10⁷ dm³ mol⁻¹ s⁻¹).

Using E.coli bacteria (AB1157) as a model system in vitro it was found that all three vitamins are rather efficient radiation protecting agents. They can also increase the activity of cytostatica, e.g., mitomycin C (MMC), by electron transfer process. The mixture of vit.E-ac and β-car acts contradictory, but adding vit.C to it a strong cooperative enhancement of the MMC activity is observed once again. A relationship between the pulse radiolysis and the radiation biological data is found and discussed. A possible explanation of the previously reported trials concerning the role of vit.E and β-car on the increased occurence of lung and other types of cancer in smokers and drinkers is presented.     

Resonance Rayleigh scattering, second-order scattering and frequency doubling scattering methods for the indirect determination of penicillin antibiotics based on the formation of Fe3[Fe(CN)6]2 nanoparticles

Under the HCl solution and heating condition, penicillin antibiotics such as amoxicillin (AMO), ampicillin (AMP), sodium cloxacillin (CLO), sodium carbenicillin (CAR) and sodium benzylpenicillin (BEN) could react with Fe(III) to produce Fe(II) which further reacted with Fe(CN)₆³⁻ to form a Fe₃[Fe(CN)₆]₂ complex. By virtue of hydrophobic force and Van der Waals force, the complex aggregated to form Fe₃[Fe(CN)₆]₂ nanoparticles with an average diameter of 45 nm. This resulted in a significant enhancement of resonance Rayleigh scattering (RRS) and non-linear scattering such as second-order scattering (SOS) and frequency doubling scattering (FDS). The increments of scattering intensity (ΔI) were directly proportional to the concentrations of the antibiotics in a certain range. The detection limits for the five penicillin antibiotics were 2.9–6.1 ng ml⁻¹ for RRS method, 4.0–6.8 ng ml⁻¹ for SOS method and 7.4–16.2 ng ml⁻¹ for FDS method, respectively. Among them, the RRS method exhibited the highest sensitivity and the AMO system was more sensitive than other antibiotics systems. Based on the above researches, a new highly sensitive and simple method for the indirect determination of penicillin antibiotics has been developed. It can be applied to the determination of penicillin antibiotics in capsule, tablet, human serum and urine samples. In this work, the spectral characteristics of absorption, RRS, SOS and FDS spectra, the optimum conditions of the reaction and the influencing factors were investigated. In addition, the reaction mechanism was discussed.     

Spectral studies on the interaction of Amido black 10B with DNA in the presence of cetyltrimethylammonium bromide

The interaction of Amido black 10B (AB) with DNA in basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV–vis, CD spectra, and RLS imaging. The interaction has been proved to give a ternary complex of CTMAB–DNA–AB in Britton–Robinson buffer of pH 11.55, which exhibits strong negative Cotton effect at 233.3 nm and 642.8 nm, and strong RLS signals characterized at 469 nm. Experiments showed that the enhanced RLS intensities (ΔI_RLS) against the mixture of AB and CTMAB are proportional to the concentration of fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA), respectively over the range of 0.03–1.0 and 0.05–1.5 μg ml⁻¹, with the limits of determination (3σ) of 7.3 ng ml⁻¹ for fsDNA and 7.0 ng ml⁻¹ for ctDNA.     

Development of an enzyme-linked immunosorbent assay for pentachlorophenol

Pentachlorophenol (PCP) is a hazardous pollutant with toxicity and potential carcinogenic properties being a serious threat to the environment. In this work, the development of an immunoassay for PCP is presented. A hapten was synthesised and conjugated to protein for rabbit immunisation. Three polyclonal antibodies were obtained and the best results were achieved in the antibody-coated format using antiserum R3. Calibration range was 0.3–30.5 ng ml⁻¹, with an average I₅₀ value of 2.9 ng ml⁻¹ and a detection limit of 0.1 ng ml⁻¹. The specificity of the assay was tested against PCP structurally related compounds. The method is highly specific for PCP and shows low cross-reactivity (CR) for chlorine-containing phenols, nitrophenols, benzenic and piridinic compounds. The good recoveries achieved with different water samples indicate that this assay can be a good alternative method for the determination of PCP in this kind of samples.     

Indirect determination of ascorbic acid by solid-phase spectrophotometry

Solid-phase spectrophotometry (SPS) technique, in the visible region, was used for the spectrophotometric determination of ascorbic acid based on the reducing effect on iron(III) ion, followed by formation of the iron(II)-ferrozine chelate. The chelate is easily sorbed on a dextran-type anion-exchange gel and the absorbance of the resin at 567 and 800 nm, packed in a 1 mm cell, is measured directly. The apparent molar absorptivity using 100 ml of sample was 2.1×10⁷ l mol⁻¹ cm⁻¹ and it allowed the determination of ascorbic acid in the range 5–90 ng ml⁻¹; the detection limit was 0.91 ng ml⁻¹ and the RSD 0.91% for a concentration of 50 ng ml⁻¹ of ascorbic acid (n=10). The proposed method permits a highly sensitive and selective determination of ascorbic acid without any preconcentration and it has been satisfactorily applied for its determination in fruit juices, pharmaceuticals, urine and conservative liquids.     

Monitoring supercritical fluid extraction by thermal lens spectrometry with pulsed laser excitation

A dual-beam thermal lens spectrometer (TLS) with a pulse pump–probe coaxial configuration was coupled to commercially available supercritical fluid extraction equipment using a high-pressure flow cell interface. Because of its feasible critical parameters, moderate cost and good thermooptical properties, carbon dioxide was used as supercritical solvent. Using trans-β-carotene as analyte, several factors related with the extraction process under continuos flow conditions were studied in order to establish their influence in the thermal lens signal magnitude. Under the optimum experimental conditions, the relative TLS signal area showed a linear relationship with the concentration of trans-β-carotene from 1.5×10⁻⁶ to 8×10⁻⁸ M (n=5, r=0.998) in the supercritical phase. Thus, the viability of on-line detection for supercritical fluid extraction with a pulse thermal lens spectrometer was demonstrated.     

High-performance liquid chromatographic assay of phosphate and organophosphorus pesticides using a post-column photochemical reaction and fluorimetric detection

A sensitive method for the post-column reaction detection of organophosphorus pesticides is described. The method relies on photolysis of the organocompounds by irradiation with a low-pressure mercury lamp (main spectral line, 254 nm) in the presence of peroxydisulfate. The resultant orthophosphate was reacted with molybdate to form molybdophosphoric acid, which subsequently reacted with thiamine to generate thiochrome. Finally, the fluorescence intensity of thiochrome was measured at 440 nm with excitation at 375 nm. Factors affecting the rate of these reactions were optimized so that its contribution to the total band-broadening was negligible.

This detection system was used for the determination of phosphate, acephate and methamidophos, which were separated on an ODS column by isocratic reversed phase chromatography with acetonitrile–water as the mobile phase. A linear relationship between analyte concentration and peak area was obtained within the range 0.016–7.0 μg ml⁻¹ with correlation coefficients greater than 0.9995 and detection limits between 4 and 12 ng ml⁻¹. Intra- and inter-day precision values of about 1.2% R.S.D. (n = 10) and 2.1% R.S.D. (n = 30), respectively, were obtained.

Pesticide residues below ng ml⁻¹ levels could be determined in environmental waters when a preconcentration device was coupled on-line with the HPLC system. Detection limits as low as 0.01 ng ml⁻¹ were achieved for only 250 ml of sample. In the analyses of vegetables and grains, the detection limit was about 1 μg kg⁻¹.     

Development and application of a capillary electrophoresis-electrochemiluminescent method for the analysis of enrofloxacin and its metabolite ciprofloxacin in milk

Enrofloxacin (ENR) is a fluoroquinolone developed exclusively for the use in veterinary practice for the treatment of respiratory and gastrointestinal infections, and ciprofloxacin (CIP) is its main active metabolite. Their contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. We developed a sensitive and rapid method for the determination of ENR and CIP by capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The method is based on the detection of aliphatic tertiary or secondary amino moieties in ENR and CIP with end-column tris(2,2-bipyridyl)ruthenium(II) electrochemiluminescence. Parameters that affect separation and detection were optimized. Under the optimized conditions, the calibration functions were linear in the range of 0.03–1 μg ml⁻¹ for ENR and 0.05–1.2 μg ml⁻¹ for CIP. The detection limits of ENR and CIR were 10 ng ml⁻¹ and 15 ng ml⁻¹, respectively, based on the signal-to-noise ratio of 3. The relative standard derivations of the peak height and the migration time for ENR and CIP were less than 4.13%. The developed method was successfully applied to determine ENR and CIP in milk with a solid-phase extraction procedure.     

Highly selective and sensitive copper(II) membrane coated graphite electrode based on a recently synthesized Schiff’s base

A PVC membrane electrode based on bis-2-thiophenal propanediamine (TPDA) coated directly on graphite is described. The electrode exhibits a Nernstian response for Cu²⁺ over a very wide concentration range (1.0×10⁻¹ to 6.0×10⁻⁸ M) with a detection limit of 3.0×10⁻⁸ M (2.56 ng ml⁻¹). It has a fast response time and can be used for at least 2 months without any major deviation. The proposed sensor revealed very good selectivities for Cu²⁺ over a wide variety of other metal ions and could be used in the pH range of 3.0–7.0. It was successfully used for direct determination of copper in black tea and as an indicator electrode in potentiometric titration of copper ion.     

Flow injection determination of nitrite by fluorescence quenching

A simple, sensitive and selective fluorimetric method for the determination of nitrite ion in waters using a merging zones flow injection system is described. The fluorimetric determination is based on the measurement of the quenching effect produced by nitrite on proflavine (3,6-diaminoacridine) fluorescence (λ_ex/λ_em=290/519 nm).

The optimum experimental conditions were investigated by merging 0.5 ml of the sample and 0.5 ml of a solution of 5 mg l⁻¹ of proflavine (in 0.1 M HCl) in a flow injection system, on-line connected to a flow-cell placed in the conventional sample compartment of a spectrofluorimeter. The selected carrier solution and final flow rate were 0.1 M HCl and 0.5 ml min⁻¹, respectively. A reaction coil of 2 ml was used. As a result of the simplicity of this system, a sample throughput of about 50 samples h⁻¹ can be achieved with the proposed methodology.

The detection limit was 1.1 ng ml⁻¹ (3σ criterion) of nitrite. The repeatability for five sample injections containing 100 ng ml⁻¹ of nitrite was ±0.3% and the observed linear range extended up to 400 ng ml⁻¹. Also, the effect of interferences from various metals and anions commonly present in waters was also studied.

The method was successfully applied to the determination of low levels of nitrite in different water samples (river, fountain, tap and commercial drinking waters).     

A flow-injection renewable surface sensor for the fluorimetric determination of vanadium(V) with Alizarin Red S

Vanadium(V) is determined by a simple bead injection spectroscopy–flow-injection analysis (BIS–FIA) system with spectrofluorimetric detection using a commercially available flow cell (Hellma 176-QS). The 500 μl of a homogeneous bead suspension of an anionic resin (Sephadex QAE A-25) previously loaded with the fluorogenic reagent 1,2-dihydroxyanthraquinone-3-sulfonic acid (Alizarin Red S) was injected to fill the flow cell. Next, V(V) is injected into the carrier and reacts with the immobilized reagent on the active solid support placed in the flow cell to form a fluorescent chelate in the absence of surfactant agents. The complex is so strongly retained on the beads that the regeneration of the solid support becomes extraordinarily difficult, so needing the renovation of the sensing surface which is achieved by means of bead injection. At the end of the analysis, beads are automatically discarded from the flow cell and transported out of the system by reversing the flow.

The measurement of fluorescence is continuously performed at an excitation wavelength of 521 nm and an emission wavelength of 617 nm. Using a low sample volume of 800 μl, the analytical signal showed a very good linearity in the range 2–60 ng ml⁻¹, with a detection limit of 0.45 ng ml⁻¹ and a R.S.D. (%) of 4.22 for 50 ng ml⁻¹ of V(V) concentration (n = 10). The sensor showed both a good selectivity, which could also be increased by the addition of EDTA and F⁻ as masking agents, and applicability to the determination of V(V) in waters, physiological samples (serum and urine) and mussel tissues.     

Preparation of dendrimer-like polyamidoamine immobilized silica gel and its application to online preconcentration and separation palladium prior to FAAS determination

A amino-terminated G 4.0 dendrimer-like polyamidoamine (PAMAM) immobilized silica gel (PAMAMSG) was prepared with a divergent method by repeating two processes: (1) Michael addition of methyl acrylate (MA) to surface amino groups; and (2) amidation of the resulting esters with ethylenediamine (EDA) from γ-aiminopropyl silica gel (APSG) core. It was then used for the first time as microcolumn packing for the flame atomic absorption spectrometry (FAAS) determination of trace or ultra trace Pd(II), after flow injection (FI) online preconcentration and separation process. A limit of detection (LOD) of 3.9 ng ml⁻¹ was achieved when 0.200 μg ml⁻¹ Pd(II) was preconcentrated in 0.2 mol l⁻¹ HCl medium with a sampling flow rate of 6.0 ml min⁻¹ for 60 s and the relative standard deviation (R.S.D.) was 1.7%. The proposed method was successfully used for the determination of Pd in two metallurgical samples.     

Speciation of inorganic arsenic by electrochemical hydride generation atomic absorption spectrometry

A simple procedure was developed for the speciation of inorganic arsenic by electrochemical hydride generation atomic absorption spectrometry (EcHG–AAS), without pre-reduction of As(V). Glassy carbon was selected as cathode material in the flow cell. An optimum catholyte concentration for simultaneous generation of arsine from As(III) and As(V) was 0.06 mol l⁻¹ H₂SO₄. Under the optimized conditions, adequate sensitivity and difference in ratio of slopes of the calibration curves for As(III) and As(V) can be achieved at the electrolytic currents of 0.6 and 1 A. The speciation of inorganic arsenic can be performed by controlling the electrolytic currents, and the concentration of As(III) and As(V) in the sample can be calculated according to the equations of absorbance additivity obtained at two selected electrolytic currents. The calibration curves were linear up to 50 ng ml⁻¹ for both As(III) and As(V) at 0.6 and 1 A. The detection limits of the method were 0.2 and 0.5 ng ml⁻¹ for As(III) and As(V) at 0.6 A, respectively. The relative standard deviations were of 2.1% for 20 ng ml⁻¹ As(III) and 2.5% for 20 ng ml⁻¹ As(V). The method was validated by the analysis of human hair certified reference material and successfully applied to speciation of soluble inorganic arsenic in Chinese medicine.     

Homogeneous stopped-flow fluoroimmunoassay using europium as label

A competitive homogeneous fluoroimmunoassay (FIA) based on the use of europium(III) as label together with kinetic methodology, is described for the first time. This approach has been applied to the determination of skatole, a compound causing boar-taint, in pig back fat samples. The analytical signal was obtained by measuring the initial rate of the dissociation reaction of europium(III) from the tracer through the formation of an intense luminescent chelate. This initial rate is higher for the free tracer than for that corresponding to the tracer bound to the antibody, and it is directly related to the analyte concentration. Each kinetic measurement was obtained in less than 1 s by using stopped-flow (SF) mixing technique. Benzoyltrifluoroacetone, tri-n-octylphosphine and Triton X-100 were used to obtain an adequate luminescent signal. The dynamic range of the calibration graph of the method is 30–300 ng ml⁻¹ and the calculated detection limit is 11 ng ml⁻¹. The precision, obtained for two skatole concentrations, 50 and 150 ng ml⁻¹, and expressed as relative standard deviation (n=11), was lower than 6.5%. The method was applied to the analysis of pig back fat samples with recoveries ranging from 92 to 108%.     

Extending the dynamic range of copper determination in differential pulse adsorption cathodic stripping voltammetry using wavelet neural network

A wavelet neural network (WNN) model is proposed for extending the dynamic range of Cu(II) determination by differential pulse adsorption cathodic stripping voltammetry (DP-AdSV) using xylenol orange (XO) as a suitable ligand. All of voltammograms data consisting of Cu(II) and Cu(II)–XO peak currents were used in WNN model. The WNN model consisted of three layers (2-8-1) with the Morlet mother wavelet transfer function in the hidden layer. The model was able to extend the dynamic range of Cu(II) from its narrow linear range (1–50 ng ml⁻¹) to the higher dynamic range (1–1500 ng ml⁻¹). The results of the WNN model was also compared with artificial neural network (ANN) model and it was demonstrated the superiority of the WNN model relative to ANN model.