Biomedical subjects

M D Luque de Castro

Publications and source records attributed to M D Luque de Castro.

At least 19 recordsLinked to original sources

PubMed1999

Comparison of continuous subcritical water extraction and hydrodistillation of marjoram essential oil.

Continuous subcritical water extraction (CSWE) and hydrodistillation were compared for the extraction of essential oil from marjoram leaves. Ground marjoram leaves (0.4 g) were subjected to dynamic extraction with water at 50 bar, 150 degrees C and 2 ml/min for 15 min. Hydrodistillation was performed treating 140 g of marjoram leaves with 1000 ml of water for 3 h. When CSWE was used the compounds were removed from the aqueous extract by a single extraction with 5 ml of hexane, detected by gas chromatography-flame ionization detection (GC-FID) and identified by mass spectrometry, electronic impact. The CSWE method is quicker (15 min versus 3 h), provides a more valuable essential oil (with higher amounts of oxygenated compounds and no significant presence of terpenes) and allows substantial savings of costs, in terms both of energy and plant material. The efficiency (in terms of volume of essential oil/1 g of plant) of CSWE is 5.1 times higher than that provided by hydrodistillation. The precision of the overall method (CSWE combined with GC-FID) is good (RSD less than 7.5%) for n = 5.

Chromatography, Gas↗

PubMed1999

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.

24,25-Dihydroxyvitamin D 3↗

PubMed1999

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%.

Chromatography, High Pressure Liquid↗

PubMed1999

Fully automated determination of vitamins A and E in milk-based products using a robotic station-HPLC arrangement.

An automated method, which includes an extraction procedure based on the AOAC official method of analysis for vitamins A and E in milk-based infant formula, was developed. The whole procedure is carried out automatically in a robotic station coupled to a liquid chromatograph with a UV detector for individual separation and quantification of the vitamins. Different commercial products (milk, infant formula and nutritional supplements) were analysed, and the data were compared with those obtained using the manual AOAC extraction method. Relative standard deviations using the automated method were 5.92 and 2.57% for vitamin A and E, respectively.

Animals↗

PubMed1999

Determination of formaldehyde in liquid, solid and semisolid pharmaceuticals and cosmetics by flow injection-pervaporation.

A spectrophotometric method is proposed for the determination of formaldehyde in liquid, solid and semisolid cosmetic and pharmaceutical samples, employing, for the first time in this field, the coupling of a continuous flow configuration to a pervaporation unit. The method is based on the reaction of the analyte with pararosaniline in acidic medium and subsequent formation of a coloured product (alkylsulfonic acid chromophore) with sodium sulfite, which was monitored spectrophotometrically. The method was applied to samples in which the formaldehyde content is regulated by law.

Cosmetics↗

PubMed1998

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.

Atrazine↗

PubMed1998

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.

Cholecalciferol↗

PubMed1998

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).

Chromatography, High Pressure Liquid↗

PubMed1998

Reverse micelle formation for acceleration of the supercritical fluid extraction of cholesterol from food samples.

Reverse micelle formation is presented as a new strategy for improving the extraction of polar species with supercritical (SC) CO2. The addition of a reverse-micelleforming reagent prior to SFE accelerates the quantitative extraction of the analyte. The effect was used to develop a method for the determination of cholesterol in solid foods. The proposed method involves the addition of a microemulsion of a nonionic surfactant (Triton X-100) to the sample and dynamic extraction with SC-CO2 at 383 bar and 40 degrees C for 20-40 min. The method was validated using a certified reference material (NIST-SRM 1845) and was subsequently used to analyze low-cholesterol (whole bread, oat biscuits, orange biscuits) and high-cholesterol foods (semiskimmed and whole milk) with excellent results (RSD < 11% in all instances).

Animals↗

PubMed1997

Sub- and supercritical fluid extraction of trichloropyridinol from soil prior to immunoassay.

A comparative study on the extraction of TCP (3,5,6-trichloro-2-pyridinol, a metabolite of chlorpyrifos) from soil with CO2 and H2O is reported. The polarity of the analyte requires the presence of both a cosolvent (methanol) and an ion-pair reagent [(1R)-(-)-10-camphorsulfonic acid ammonium salt] for 95% extraction in 30 min when supercritical CO2 at 40 degrees C and 383 bar is used as extractant. Subcritical water (250 degrees C and 200 bar) enables complete extraction within 15 min without additives. Quantitation of the target analyte is performed by specific immunoassay using a non-commercial monoclonal antibody which provides a linear determination range between 0.005 and 5 micrograms/g, with coefficients of variation of 5.3 and 4.9% for the SC-CO2 and sub-H2O extractions, respectively.

Chromatography, Gas↗

PubMed1997

Continuous cleanup/preconcentration procedure of hydroxyvitamin D3 metabolites in plasma as an alternative to batch solid-phase extraction.

A continuous automatable cleanup procedure coupled on-line with a liquid chromatograph and UV detector for hydroxyvitamin D3 metabolites [24,25-(OH)2, 1,25-(OH)2 and 25-(OH)] as an alternative to batch solid-phase extraction is reported. The method, based on continuous solid-phase cleanup/preconcentration of the analytes, requires only the passage of the sample through a single minicolumn, which also results in a preconcentration effect which increases the sensitivity. The proposed method is also compared with a conventional batch, two-step solid-phase extraction method previously improved by the authors. The method has been checked by applying it to plasma samples spiked with the target analytes (linear range between 0.05 and 100 ng/ml with coefficient of variation values lower than 6.5%) and acceptable recoveries ranging between 94.6 and 101% have been obtained. The sampling frequency was 4 h-1.

24,25-Dihydroxyvitamin D 3↗

PubMed1997

Development and validation of a flow-injection method for the determination of albumin tannate, the active component of a pharmaceutical preparation.

A flow-injection analysis method for the determination of albumin tannate in tablets is reported. After optimization of the variables involved, the method has been characterized and validated in terms of calibration using three procedures: repeatability and reproducibility; ruggedness; and selectivity. Finally, it has been applied to real samples (tablets).

Albumins↗

PubMed1997

Fully robotic method for characterization of toxic residues.

A fully automated method for the determination of toxic residues was developed using a robotic station. The robot performs the weighing of the solid sample and applies the standard leaching method based on continuous stirring with discontinuous pH monitoring and control by addition of acid solution for 24 h; it then filters the sample and prepares the different dilutions for application of the luminescence test which is monitored by aspiration of the solutions into the flow cell of a luminescence detector. The data from both the sample mass and dilution step are used together with those from the detector by the computer for calculation of the toxicity. The two manual procedures usually applied in routine laboratories were developed by the robotic station; the results obtained by both methods were compared with those provided by the manual method and showed excellent agreement. The main advantage of the proposed method is that it is fully automated as opposed to the constant human attendance (for at least 28 h) required by the manual method.

Hazardous Substances↗

PubMed1996

Precipitation flow injection immunoassay for human immunoglobulin G.

The development of a precipitation flow injection immunoassay is described. This approach is based on the immunoprecipitin reaction, whereby the precipitate formed by binding of the sample antigen (HIgG) to fluorescein-labelled antibodies is retained on a filter built in on-line in a flow-injection system. After dissolving the precipitate with sodium hydroxide solution the liberation of fluorescein-labelled antibodies results in a fluorescence signal that is directly proportional to the concentration of HIgG. The instrumental set-up is very simple and fully automated. One assay cycle takes about 10 min. The RSD ranged between 1 and 3%, depending on concentration. The detection limit was about 140 fmol. The recovery from spiked serum samples was between 96 and 102%.

Automation↗

PubMed1995

Enzymatic determination of bicarbonate in serum by flow injection analysis.

An automated method for the determination of bicarbonate in human serum based on the enzymatic reaction between the analyte and phospho(enol)pyruvate (PEP) in the presence of PEP carboxylase is proposed. The analytical reaction is coupled with a derivatization reaction in which the NADH consumed is fluorimetrically monitored (lambda ex = 340 nm, lambda em = 460 nm). A stopped-flow/flow-injection approach is used in which the enzymes (PEP carboxylase and malate dehydrogenase) are immobilized on controlled-pore glass. The linear determination range is between 25 and 300 mmol/l (r2 = 0.9973). The %C.V. for the within- and between-run studies, performed at three concentration levels, ranges between 1.0 and 3.6% and the sampling frequency is 20 per h.

Analysis of Variance↗

PubMed1995

Determination of inorganic ions of clinical interest: state-of-the-art and trends.

An overview is presented of techniques, analysers and methods currently available for the determination of inorganic ions of interest in clinical laboratories; methods include those based on activation and inhibition of enzymatic reactions by these target analytes. The foreseeable, trends in this area are also discussed.

Chemistry Techniques, Analytical↗

PubMed1995

Development and validation of chromatographic methods (HPLC and GC) for the determination of the active components (benzocaine, tyrothricin and menthol) of a pharmaceutical preparation.

Methods are reported for the determination of tyrothricin and benzocaine by HPLC and menthol by GC in the analysis of throat lozenges (tablets) containing all three compounds. After optimization of the variables involved in both HPLC and GC the methods have been characterized and validated according to the guidelines of the Spanish Pharmacopoeia, and applied to both the monitoring of the manufacturing process and the quality control of the final product.

Anesthetics, Local↗

PubMed1995

Continuous-flow assay with immobilized enzymes for determining of inorganic phosphate in serum.

An automated method for the determination of inorganic phosphate based on flow-injection analysis and on the use of immobilized enzymes is reported. The method features a linear range between 0.1 and 20 mumol/L with a CV < 2.1% and 3.4% for the within-run and between-run studies, respectively, and a sampling throughput of 40 h-1. The sensitivity of the method makes a 1:250 dilution of the serum samples feasible, thus making undetectable the interferences from analytes commonly present in serum. The method shows an excellent correlation with conventional automated analyzers based on the same enzymatic reaction (Hitachi, r = 0.988) but with the catalyst in solution, and with the Kodak Ektachem method (r = 0.974) based on the use of dry reagents and formation of the phosphomolybdo heteropolyacid.

Autoanalysis↗