Rapid chromatographic determination of caseins in milk with photometric and fluorimetric detection using a hydrophobic monolithic column.

Reverse-phase liquid chromatographic methods using a hydrophobic C18 monolithic column and on-line photometric and fluorimetric detection for the determination of the major casein (CN) proteins in milk are presented. The separation of αs1-CN, αs2-CN, ß-CN and κ-CN was achieved in only five minutes. Fluorimetric detection enabled better analytical results than photometric detection. Thus, the dynamic ranges of the calibration graphs and detection limits obtained using fluorimetric detection were (mgmL(-)(1)): αs1-CN (0.74-10.0, 0.22), αs2-CN (0.15-10.0, 0.045), ß-CN (0.68-10.0, 0.20) and κ-CN (0.21-10.0, 0.06). The analytical features of the photometric method, which does not allow the quantification of ß-casein, were (mgmL(-)(1)): αs1-CN (1.5-9.0, 0.45), αs2-CN (1.4-10.0, 0.43) and κ-CN (0.4-9.0, 0.12). Precision data, expressed as relative standard deviation, ranged between 0.6% and 5.3% for the fluorimetric method and between 2.4% and 6.2% for the photometric method. Both methods were applied to the analysis of three different milk samples, obtaining recoveries in the ranges of 86.6-103.2% and 92.0-106.5% using fluorimetric and photometric detection, respectively.

Determination of polyphenolic content in beverages using laccase, gold nanoparticles and long wavelength fluorimetry.

An enzymatic fluorimetric method for the determination of polyphenol compounds in beverages is described, which is based on the temporal inhibition caused by these compounds on the oxidation of the long wavelength fluorophor indocyanine green (λ(ex) 764 nm, λ(em) 806 nm), in the presence of the enzyme laccase and positively charged gold nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its fluorescence, but it is delayed by the polyphenol, obtaining a time period directly proportional to its concentration, which has been used as the analytical parameter. The behaviour of several benzenediols and benzenetriols in the system and the modification of the activity of the enzyme by its interaction with AuNPs have been studied. The system has been optimized using gallic acid as a polyphenol model, but the dynamic ranges of the calibration graphs and the detection limits for several of the polyphenols assayed were obtained (µmol L(-1)): gallic acid (0.13-5, 0.04), catechol (0.08-5, 0.01), hydroquinone (0.05-2, 0.01), hydroxyhydroquinone (0.09-5, 0.03), pyrogallol (0.17-5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of the method, expressed as RSD% and checked at two concentration levels of each analyte, ranged between 1.8 and 5.6%. The method has been applied to the determination of polyphenol content in several foodstuff samples and the results compared with those obtained with the standard Folin-Ciocalteu method.

Determination of antioxidant additives in foodstuffs by direct measurement of gold nanoparticle formation using resonance light scattering detection.

The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidants used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5s, using an integration time of 0.1s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were (µmolL⁻¹): gallic acid (0.04-0.59, 0.01), propyl gallate (0.04-1.41, 0.01), octyl gallate (0.03-0.35, 0.08), dodecyl gallate (0.02-0.30, 0.007), butylated hydroxyanisol (0.07-0.39, 0.009), butylated hydroxytoluene (0.04-0.32, 0.01), ascorbic acid (0.11-1.72, 0.03) and sodium citrate (0.07-1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%. The results obtained were validated using two reference methods.

Control of tumor markers using nanotechnology.

The relevance of tumor markers in clinical diagnosis of cancer has given rise to the development of new approaches based on the use of nanoparticles to improve the features of the immunoassays developed for their control. This article reviews the usefulness of different nanoparticles to develop direct, sandwich and competitive assays for the individual and multiplexed determination of these compounds.

Evaluation of liposome populations using a sucrose density gradient centrifugation approach coupled to a continuous flow system.

A method for the evaluation of liposome size populations using sucrose density gradient centrifugation coupled with a continuous flow system is presented. Liposomes, prepared using different methods (rapid solvent evaporation, rehydration, and detergent removal) and modified by assaying several procedures (shaking, sonication and extrusion) were evaluated according to the type of liposome, size and polydispersity. The preparation of liposomes was carried out in the presence of the fluorophor cresyl violet. Extracts of the liposomes were homogenised and centrifuged at 20,073 x g at 4 degrees C for 30 min using sucrose density gradient centrifugation programmes, which provide efficient liposome separation in different sizes. The results of the separation procedure were tested by aspiration of the extracts into a continuous flow system in which the liposomes were disrupted by the continuous mixing with a Triton X-100 solution, prior to their translation to the detector. The luminescence provided by the liberation of the encapsulated fluorophor indicates the distribution of liposomes in each density gradient stage. Three zones were obtained: zone alpha, containing giant unilamellar and multivesicular vesicles, zone beta, with large and medium size liposomes, and zone gamma, which contained small size liposomes. The precision of the separation zones obtained, expressed as RSD%, was lower than 5.6% in all instances. The method provides a relative rapid way to evaluate the liposome polydispersity and size after using conventional methods of synthesis and mechanical modifications.

Stopped-flow kinetic method for the fluorimetric determination of DNA traces in biological samples based on the interaction long-wavelength fluorophor, surfactant and nucleic acid.

A simple and rapid method for the determination of DNA, involving the interaction between a surfactant, a long-wavelength fluorophor (LWF) and the nucleic acid, is presented. Different chemical systems based on the local effective charge of the surfactant/LWF system with DNA were tested, choosing cetyltrimethyl ammonium bromide (CTAB) and indocyanine green (ICG) for the development of the method. The fluorescence of ICG increases in the presence of CTAB, but it rapidly decreases in the presence of deoxyribonucleic acid. The initial reaction-rate (v(0)) and signal at a prefixed-time (DeltaIF(20)) are monitored at 780 and 802 nm as excitation and emission wavelengths, respectively, using stopped-flow mixing technique, which makes the method applicable to automate routine analysis. Each measurement was obtained in about 30 s, being the integration time 0.1 s. The dynamic range of the calibration graph was 10-1500 ng mL(-1), with a detection limit of 5 ng mL(-1). The precision of the method, expressed as relative standard deviation, ranged between 2.1% and 4.5%. After a sample treatment consisting on a conventional extraction, the method was applied to the determination of DNA in several samples from different biological materials.

Nanostructures as analytical tools in bioassays.

We critically evaluate the usefulness of different nanostructures described as labels, nanoscaffolds or separation media in immunoassays and nucleic-acid hybridization assays. Many of the great number of publications describe only theoretical aspects of using these nanostructures or nanoparticles, but do not verify their applicability in the presence of potential interferents that can be present in the sample matrix. We attempt a systematic study of the advantages and the limitations of using these new reagents in bioassays, the different assay formats for individual and multiplexed detection, and the capability of these assays in analyzing real samples.

Chromatographic determination of flumequine in food samples by post-column derivatisation with terbium(III).

The potential usefulness of terbium(III) as reagent for the luminescent determination of flumequine residues in food samples has been studied using both fluorescence (FL) and time-resolved (TR) modes and both batch (B) and integrated liquid chromatography (LC)/derivatisation approaches. The system was optimised in each instance to establish the analytical features of the four methods. The dynamic ranges of the calibration graphs, obtained with standard solutions of flumequine, were (ng mL(-1)): B-FL 0.18-600; B-TR 2.4-150; LC-FL 3.7-1000 and LC-TR 52-3000. The detection limits were also obtained giving the following values (ng mL(-1)): B-FL 0.055; B-TR 0.7; LC-FL 1.1 and LC-TR 15. The precision, expressed as the percentage of relative standard deviation, was equal or lower than 5.1% in all instances. The LC methods, which avoid the interference of other quinolone antibiotics, were applied to the analysis of chicken muscle and liver, and whole milk samples. The sample pre-treatment only consisted of a deproteinisation step. The validation procedure for the analysis of samples was carried out using EC recommendations, and the decision limit and detection capability were calculated. The recoveries obtained ranged from 95.0% to 103.8%.

Flow-injection spectrophotometric determination of cyanate in bioremediation processes by use of immobilised inducible cyanase.

A new flow injection (FI) method for photometric monitoring of cyanate in bioremediation processes using immobilised native cyanase is described. The method is based on the catalytic reaction between cyanate and bicarbonate to produce ammonia and carbon dioxide in the presence of an inducible native cyanase, immobilised in a reactor packed with glass beads. Two degrees of purification of the biocatalyst were used-heated cell-free extract and purified extract of cyanase from Pseudomonas pseudoalcaligenes CECT 5344. The ammonia produced by the enzymatic reaction is finally monitored photometrically at 700 nm using a modification of the conventional Berthelot method. The method furnishes different calibration curves depending on the degree of purification of the cyanase, with linear ranges between 1.23 and 616.50 micromol L(-1) ( r(2)=0.9979, n=7) and between 1.07 and 308.25 micro mol L(-1) ( r(2)= 0.9992, n=7) for the heated cell-free extract and the purified cyanase extract, respectively. No statistically significant differences between the samples were found in the precision study evaluated at two cyanate concentration levels using one-way analysis of variance. A sampling frequency of 15 h(-1) was achieved. The method was used to monitor cyanate consumption in a cyanate bioremediation tank inoculated with Pseudomonas pseudoalcaligenes CECT 5344 strain. The correlation between cyanate degradation and ammonia production was tested using a conventional method. Finally, the method was applied to different samples collected from the bioremediation tank using the standard addition method; recoveries between 85.9 and 97.4% were obtained.

Determination of 3,5,6-trichloro-2-pyridinol (TCP) in water by a continuous competitive immunoassay system based on the streptavidin-biotin interaction.

A competitive continuous immunoassay system for the determination of 3,5,6-trichloro-2-pyridinol (TCP), the major degradation product of the insecticide chlorpyrifos, in water is described. The immunoassay system is based on the transient retention of the specific LIB-MC2 monoclonal antibody anti-TCP as a biotinylated derivative using the streptavidin-biotin interaction. The permanent immobilization of streptavidin on controlled-pore-glass provides an adequate active support for the transient retention of the biotinylated monoclonal antibody anti-TCP. In a subsequent step, the immuno-competitive reaction between the biotinylated LIB-MC2 and the TCP/hapten-POD mixture takes place. This competitive assay relies on the determination of the biocatalytic action of peroxidase, retained in the active support, on a derivatization reaction which yields a fluorescent product. The method exhibits a determination range of 0.01-200 microg L(-1) of TCP (r2=0.9919, n=9) with a precision, expressed as RSD, lower than 4.2% and a sampling frequency of 3 h(-1). The approach has been applied to the determination of TCP in water with recoveries of 89.7-105.6%.

Continuous determination of chloroquine in plasma by laser-induced photochemical reaction and fluorescence.

A flow injection fluorimetric method is proposed for the determination of chloroquine based on the photochemical derivatisation in an alkaline medium of the analyte and fluorescence generation after irradiation with a pulsed Nd:YAG laser operated at 355 nm. Chemical, hydrodynamic and laser variables were studied in order to obtain the best conditions for quantification. A linear range from 25 to 600 micrograms/L was achieved, with a correlation coefficient of 0.997 (n = 8), an RSD of 4.3% (n = 11) and a detection limit of 8 micrograms/L (3 sigma). The sample throughput was 10 h-1. The method was successfully applied to the determination of chloroquine in human plasma. The increase of sensitivity with respect to the method based on monitoring the intrinsic fluorescence of chloroquine itself was 1.7 times.

Inhibition-based determination of metrifonate in liquid and solid samples using the triple integration chemical hydrolysis-pervaporation-enzymic derivatisation.

An innovative continuous flow approach consisting of the triple integration of chemical hydrolysis, analytical pervaporation and enzyme inhibition-based reaction for the determination of metrifonate is presented. The method is based on chemical degradation of the pesticide, a separation step consisting of analytical pervaporation of its volatile metabolite and inhibition action of this on the acetylcholinesterase catalysis. The subsequent derivatisation reaction is a two-step reaction involving choline oxidase (ChOD) and horseradish peroxidase (POD) with fluorimetric detection (lambda (ex )=310 nm and lambda (em )=415 nm ) of the dimer formed by the action of hydrogen peroxide. The efficiency of the inhibitory effect was increased using an open-closed flow system. Applied to liquid samples, the method has a linear determination range of 0.0025-0.15 g l(-1)(n=8, r=0.9993) with a precision, expressed as RSD, of 3.2-6.7% and a sampling frequency of 3 h(-1). When applied to solid samples the method shows a linear determination range of 0.0026-0.13 g kg(-1) (n=5, r(2)=0.9981, RSD 2.7-7.7%) and a sampling frequency of 2h(-1). The approach has been applied to the determination of metrifonate in natural water and spiked soil samples with recoveries ranging between 94.3 and 107.8% for liquid samples and between 86.5 and 99.6% for solid samples.

Determination of anti-canine IgG using a continuous filtration/dissolution system based on the formation of a high-molecular size immunocomplex.

A method for the determination of monoclonal antibody anti-canine-IgG based on a continuous filtration/dissolution system is presented as prototype for further developments. The basis of the system is the continuous formation of a high-molecular immunocomplex, which is temporally retained on a microfilter located prior to the detector. The immunochemical method consists of the development of a sandwich type heterogeneous non-competitive reaction to yield a high molecular immunocomplex, as a result of the affinity interaction between streptavidin and biotincanine IgG and the immunoreaction between canine IgG and mAb anti-canine IgG, which occurs in solution. Goat anti-mouse IgG labelled with peroxidase is used as tracer. The extension of the immunoreaction is monitored fluorimetrically via the condensation product between 4-hydroxyphenylacetic acid and hydrogen peroxide in the presence of the peroxidase retained on the filter. The method provides a dynamic range from 10(-4) to 500 mug l(-1) with an IC(50) of 0.554 mug 1(-1) (for a biotin-IgG dilution of 1:250, chi(2)=0.6085, r(2)=0.9991, n=14) and a precision, expressed as R.S.D.%, lower than 4.7%. After modifications, the method here proposed can be extended for monitoring analytes of interest in the agrochemical, food and environmental areas, as far as permitted by the availability to produce the corresponding monoclonal antibody.

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection.

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.

Determination of vitamin D3 metabolites: state-of-the-art and trends.

The steps involved in the methods for the determination of vitamin D3 metabolites (namely, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3) mainly in clinical samples are critically reviewed. Sample pretreatment (e.g. deproteinization, saponification, liquid liquid and liquid solid extraction, etc.) as a function of both type of sample and detection system, quantitation based on protein saturation and liquid as well as gas chromatography are discussed. The chemical principles on which the methods are based and the derivatization procedures, which facilitate separation and/or detection, are also commented upon. Finally, the future prospects of the research on methods for the determination of these metabolites are outlined.

Near infrared thermal lens spectrometry for the real-time monitoring of supercritical fluid extraction.

A commercially available supercritical fluid extractor provided with carbon dioxide was coupled to a dual-beam thermal lens spectrometer with a pumpprobe coaxial configuration, pumped by a pulsed Nd-YAG laser operating at the fundamental wavelength of 1064 nm. As a preliminary step, several compounds were studied in batch regime using carbon tetrachloride as solvent, in order to observe the influence of overtones and combinations involving distinct chemical bonds on thermal lens spectrometry (TLS). Several factors related with supercritical fluid extraction (SFE) under hydrodynamic conditions were studied in order to establish their influence both in the extraction yield and thermal lens signal magnitude obtained. The advantages and limitations of the hyphenated SFE-TLS technique proposed are discussed, and the possibility of on-line detection in SFE with a pulse thermal lens spectrometer was demonstrated.

Determination of vitamin D(3) hydroxymetabolites in plasma at the sub-part per trillion levels using on-line cleanup/preconcentration and HPLC-fluorimetric post-column derivatisation.

A new method for the determination of the hydroxymetabolites of vitamin D(3) (24,25-(OH)(2)-D(3), 1,25-(OH)(2)-D(3) and 25-OH-D(3)) in plasma is reported. The method is based on the integration of three subsystems: continuous cleanup/preconcentration, HPL separation and post-column fluorimetric derivatisation. The derivatising subsystem is based on the dehydration reaction undergone by the secosteriod molecules in a strong-acid medium. The calibration graphs were run between 0.1 pg ml(-1) and 100 ng ml(-1) for each analyte with excellent regression coefficients (>/=0.9933) in all cases. The precision at two concentration levels was established with acceptable RSDs (%) in all instances (values between 2.1 and 5.2%). The method was also checked by applying it to human plasma samples spiked with the target analytes and the recoveries ranged between 86 and 106%.

Application of screen-printed electrodes as transducers in affinity flow-through sensor systems.

An affinity flow-through sensor system based on a heterogeneous competitive affinity assay for the determination of low molecular weight compounds is described using the examples of biotin and atrazine determination. The binding proteins, either streptavidin or a biotinylated monoclonal antibody, were immobilized on a biotinylated screen-printed electrode, where the competition between the analyte and an analyte-enzyme-conjugate took place. Determination of the bound enzyme was done through the supply of suitable enzyme substrates and electrochemical determination of an enzyme reaction product. In the assays described here, peroxidase was used as enzyme label. As hydrogen peroxide and hydroquinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrically at a reducing potential of -600 mV vs a screen-printed platinum electrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserted. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of required reagents. The system was optimized mainly through the determination of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. The method showed a linear range from 0.045 to 2 micrograms/l (r2 = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by using a biotinylated monoclonal antibody against atrazine for analyte recognition and performing a competitive assay between atrazine and a triazine-peroxidase-conjugate. The linear range was from 0.01 to 10 micrograms/l, with IC50 = 0.4 microgram/l and RSD lower than 4.6%. The method was also applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays.

Synergistic approaches based on nonchromatographic continuous separation techniques (solid-phase extraction and pervaporation) and chromatography couplings.

Approaches based on continuous separation units coupled to either liquid or gas chromatography for improving the features of analytical methods are proposed. Examples of solid-phase separation-liquid chromatography for the determination of fat-soluble vitamins and their metabolites in clinical samples, and pervaporation-gas chromatography for the determination of volatile compounds in solid environmental samples are described. The clean-up and preconcentration effect achieved by the former coupling and the easy and effective solid-sample pretreatment in the latter clearly show their utility. The use of pervaporation as an advantageous alternative to headspace is demonstrated.

Quantitation of circulating hydroxyvitamin D3 in human plasma by a continuous cleanup/concentration procedure prior to HPLC-UV detection.

A method for the determination of hydroxyvitamin D3 metabolites (25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and 1,25-didydroxyvitamin D3) based on a continuous cleanup/ preconcentration procedure coupled with HPLC and UV-detection is reported here. The method exhibits a linear range between 0.05 and 100 ng/ml (r2 = 0.9917) with CV values lower than 6.5%, and has been checked by applying it to plasma samples from a hospital with acceptable recoveries. The results compare well with those obtained by routine radioimmunoassay (y = 2.784+/-1.37 + 0.333+/-0.05 sigma(yx), r = 0.8233, n = 19 for 25-hydroxyvitamin D3). The sampling frequency was 4 h(-1); 12 analytes h(-1).