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F Tagliaro

Publications and source records attributed to F Tagliaro.

At least 55 records · Page 3Linked to original sources
PubMed1994

Reversed-phase high-performance liquid chromatographic determination of cocaine in plasma and human hair with direct fluorimetric detection.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine with direct fluorimetric detection, particularly intended for the routine analysis of hair and blood samples, is described. Benzoylecgonine, eluting before cocaine in a completely resolved peak, is also detectable. Detection is based on the weak native fluorescence of cocaine and benzoylecgonine, depending on the benzene ring present in both molecules. Hair samples (20-200 mg) were incubated overnight in 2 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method; the dried residue reconstituted with 500 microliters of 0.05 M NaH2 PO4 (PH 5.2) was injected. Blood plasma samples (200 microliters) were mixed with 150 microliters of 0.1 M Na2 HPO4 (pH 8.9) and extracted with 5 ml of chloroform-2-propanol (9:1); the organic phase was evaporated and the residue dissolved and injected as above. Isocratic reversed-phase liquid chromatography was carried out on a column (150 x 4.6 mm I.D.) packed with spherical 5-microns poly(styrene-divinylbenzene) particles; the mobile phase was 0.1 M potassium phosphate (pH 3)-methanol-tetrahydrofuran (70:25:5). The excitation and emission wavelengths were set at 230 and 315 nm, respectively. Under the described conditions, cocaine eluted in a symmetrical peak with a capacity factor of about 5. The limit of detection was about 1 ng/ml (0.2 ng injected), with a signal-to-noise ratio of 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1994

Biomedical applications of capillary electrophoresis.

After having grown through the stages of technique development and instrumentation availability, capillary electrophoresis has reached the stage of applications. This review attempts to show the potential of this technique for biomedical analysis. Rather than going into a detailed description of the technical details of the separation conditions suitable for the separation of a particular category of compounds, the focus is on the general principles and areas in which this technique can be applied and the prospects for the future. Particular emphasis is placed on the separation of complex matrices and their simplification, a daily task in biomedical laboratories. In addition, methods for the optimization of separation conditions are considered. Considerable prospects for capillary electrophoresis lie in profiling. The applicability of the technique in peptide and protein mapping is discussed in some detail. Finally, three other topics are dealt with, namely enzymic activity microassays, drug-protein binding assays and monitoring of drugs in body fluids.

Animals↗
PubMed1994

Free solution capillary electrophoresis of calcitonins and calcitonin tryptic digests.

The aim of the present work was the development of a simple capillary electrophoretic strategy, complementary to high-performance liquid chromatography, for the separation of different calcitonins (CTs) and calcitonin tryptic digests. Capillary electrophoresis was carried out with a manual capillary electropherograph with "on column" UV absorbance detection at 200 nm. The separation was accomplished in a 70 cm x 50 microns I.D. bare silica capillary. About 6 nl was loaded into the capillary by means of a split-flow system. Except in particular cases, electric fields of 300 V/cm were used at constant voltage. Separations were carried out in 0.05 M citrate buffer pH 2.5 or, alternatively, in 0.05 M borate buffer pH 9.5. A complete resolution of salmon, ASU1,7-eel, and human calcitonins was obtained in citrate and borate buffers. Other CT analogues could be separated only in one of the two buffers. Capillary electrophoresis in citrate buffer was also successful in the separation of the four final trypsin cleavage fragments of salmon calcitonin and, at least tentatively, of the nine intermediate cleavage products.

Analgesics↗
PubMed1994

Capillary electrophoretic profiling of rat hair: a tool for alopecia areata diagnosis.

Capillary electrophoretic profiling of hair fractions obtained by 0.25 M HCl treatment of the tissue with subsequent extraction of the solubilized fraction with chloroform-isopropanol (9:1, v/v) revealed clear differences between hair obtained from alopecia areata affected laboratory rats (both in hair obtained from non-affected areas and hair growing on once hairless patches) and controls. Differences were observed both in the organic-phase extractable material as well as in the aqueous phase after extraction. The separations were carried out in 25 mM borate buffer pH 9.2 for the chloroform-isopropanol extractable fraction while profiling of the aqueous phase was done in the same buffer at pH 10. Untreated fused-silica capillaries, 40-45 cm to the detector of I.D. 50 and 75 microns were used at a running voltage of 20 kV. Detection was done either by UV absorbance at a fixed wavelength of 200 nm or by using a diode array detector.

Alopecia Areata↗
PubMed1994

Capillary zone electrophoresis determination of phenylalanine in serum: a rapid, inexpensive and simple method for the diagnosis of phenylketonuria.

A simple, rapid, and quantitative capillary zone electrophoresis method for phenylalanine analysis in serum has been developed, with the aim of providing an analytical tool, as an alternative to liquid and gas chromatography, for the routine laboratory diagnosis of phenylketonuria. Electrophoresis was carried out in a 65 cm long, 50 microns wide bare silica capillary, using 0.025 M borate (adjusted to pH 10 with 1 M NaOH) at a potential of 20 kV, with in-column UV detection at 214 nm. Under these conditions, the three aromatic amino acids (tryptophan, phenylalanine and tyrosine) migrated according to the pKs of the respective amine (and hydroxyl) groups. The efficiency of separation was about 150,000 plates/column for phenylalanine. Diprophylline was adopted as internal standard. The injection of ethanol-deproteinized normal control serum gave rise to only a few major peaks not interfering with phenylalanine; phenylalanine in serum at normal concentrations appeared in a clean region of the electropherogram as a symmetrical peak with a migration time of about 11 min. The sensitivity was > or = 3 micrograms/mL, with s/n ratio = 3. The linearity, in the range of 5-175 micrograms/mL, was described by the equation y = 1.407-0.583 x, r2 = 0.9998. Accuracy and precision were satisfactory, with intra-day and inter-day coefficients of variation lower than 4% and 7%, respectively. The injection of sera from five phenylketonuria patients gave electropherograms clearly showing huge peaks of phenylalanine, thus allowing an easy laboratory diagnosis of phenylketonuria.

Capillary Action↗
PubMed1993

Capillary electrophoresis for the investigation of illicit drugs in hair: determination of cocaine and morphine.

Toxicological analysis of hair is becoming a popular method for investigating past, chronic use of illicit drugs. Several analytical methods using immunometry, chromatography and mass spectrometry have been reported. In this work, capillary electrophoresis was first used for the determination of illicit drugs, such as cocaine and morphine, in the hair of heroin and cocaine users. After rapid washing, hair samples were incubated overnight in 0.25 M HCl at 45 degrees C and the mixtures were extracted with ready-to-use Toxi-tubes A. The organic phase was evaporated and the residue dissolved in a suitable amount of electrophoresis buffer. Free zone capillary electrophoretic determinations of morphine, the main heroin metabolite, and cocaine were accomplished in 0.05 M borate buffer (pH 9.2) at a potential of 15,000 V, with UV detection at 214 and 238 nm, respectively. The use of the less selective wavelength of 200 nm allowed the simultaneous detection of both compounds. Efficient separations (up to 350,000 theoretical plates) and accurate and precise determinations (intra-day R.S.D.s in the range 3-5%) of cocaine and morphine in hair extracts were easily achieved. The analytical sensitivity was sufficient to determinate as little as 0.15 ng/mg of cocaine and morphine in hair using 100-mg samples. Interferences from more than 90 therapeutic drugs and drugs of abuse were excluded.

Chromatography, High Pressure Liquid↗
PubMed1993

Immune response to opiates: new findings in heroin addicts investigated by means of an original enzyme immunoassay and morphine determination in hair.

In the early 1970's, some papers appeared reporting an immune response to opiates in animals treated with morphine and in heroin addicts, but further researches failed to confirm these results in humans. The aim of the present work is investigating with a newly developed enzyme immunoassay the existence of specific antibodies to morphine in a group of opiate chronic users, controlled by means of the toxicological analysis of hair. Twenty five opiate addicts inpatients for detoxication treatments were investigated for the presence of morphine specific antibodies and for the morphine content in hair, as a marker of addiction to opiates. Antibodies to morphine were investigated using an original ELISA method using a morphine-human serum albumin conjugate immobilized into the wells of polystyrene microtiter plates. Morphine determinations in hair were accomplished by a radioimmunologic screening followed by HPLC confirmation of positive results. The group of opiate users, in which all the subjects resulted positive for morphine content in hair, showed in the ELISA test an average D OD% value significantly higher than the control group (p < 0.001); in particular, 16 out of 25 addicts could be classified positive for anti-morphine antibodies, which were identified as IgM. Inhibition studies demonstrated Ka's for morphine ranging from 10(4) to 10(10) M-1 and a high cross reactivity for codeine. The presence of circulating antibodies specific to morphine in chronic users of opiates is strongly supported by the present findings.

Adult↗
PubMed1993

High-sensitivity low-cost methods for determination of cocaine in hair: high-performance liquid chromatography and capillary electrophoresis.

Free solution capillary electrophoresis and high performance liquid chromatography were applied to the analysis of cocaine in hair. Capillary electrophoresis conditions were as follows. Background electrolyte: 0.050 M borate, pH 9.2; capillary: bare silica, 40 cm long, 50 micrograms i.d.; potential: 15 kV; detection: UV at 238 nm wavelength. In addition, the separation was accomplished in a 50 cm x 75 microns capillary with an instrument with a photodiode array detector, recording on-line UV spectra of peaks from 200 to 400 nm. The isocratic high performance liquid chromatographic method with fluorimetric detection used 230 nm (ex.) and 315 nm (em.) wavelengths. Cocaine separation was carried out under conditions summarized as follows: column packed with spherical polystyrene-divinylbenzene 5 microns particles, mobile phase 0.1 M potassium phosphate (pH 3.0)/methanol/THF (70:25:5) at a flow rate of 0.5 ml/min and at room temperature. Liquid-liquid and solid-liquid sample preparation methods were used. Typically, levels of cocaine in hair as low as 0.15-0.30 ng/mg were detected by capillary electrophoresis, while HPLC allowed the determination of concentrations lower by one order of magnitude (0.015 ng/mg). Intra- and inter-assay precision data of the two techniques were comparable and characterized by relative standard deviations in the range from 3 to 7%. On the other hand, the sample throughput of capillary electrophoresis (7-10 injections/h) was higher than high performance liquid chromatography (2 injections/h). A good correlation of the results (r2 = 0.92) between the two techniques was observed in the assay of real cases.

Chromatography, High Pressure Liquid↗
PubMed1992

Chromatographic methods for blood alcohol determination.

This review is focused on the different chromatographic strategies for blood alcohol determination which can be adopted for clinical and/or forensic purposes. Particular attention is paid to gas chromatography and to high-performance liquid chromatography. However, other analytical techniques in common use, such as chemical and enzymic methods, are also briefly presented, together with some, at present unusual or experimental, approaches, such as enzymic reactors and catalytic electrodes, which are suitable for application in column liquid chromatography. Finally, mention is made of the methods for the determination of acetaldehyde, the major ethanol metabolite, and of some proposed markers of chronic alcohol abuse, such as acetaldehyde-protein adducts and carbohydrate-deficient transferrin. In order to give the background of knowledge for the rational choice of an analytical strategy, an updated outline of ethanol metabolism and toxicology is presented, together with basic information for the interpretation of the results. Problems concerning blood sampling and storage are also discussed.

Chromatography↗
PubMed1992

Methods for chromatographic determination of amanitins and related toxins in biological samples.

Methods for the chromatographic determination of amanitins, toxins of Amanita phalloides (Fr.), Link mushrooms and related toxins are reviewed; particular emphasis is given to high-performance liquid chromatographic methods. The main chemical and toxicological aspects are discussed, but the focus of the present review is on the analytical problems arising in a laboratory charged with the setting up of a procedure which can direct the appropriate clinical management of an intoxicated patient or solve a forensic case.

Amanita↗
PubMed1992

Immunoreactive "calcitonin-like" material in heroin addicts: varying reactivity with different antibodies.

High levels of immunoreactive calcitonin (iCT) in the blood of heroin addicts were previously reported. As it is well known that multiple forms of calcitonin exist in the blood and in tissues, the purpose of the present study was to investigate the immunological nature of the CT-like immunoreactive material found in the blood of these subjects. We investigated 25 addicts, who had been using heroin for more than one year and were hospitalized for a 2 week detoxication program. Blood samples were drawn at the start of the program (when the subjects were still on heroin) and after 5 and 12 days of abstinence from heroin. Twenty-five healthy subjects served as controls. We used 2 commercial RIA kits, calibrated against the same reference material (WHO 70-234), but employing different antisera. One antiserum substantially confirmed the previous findings of increased levels of calcitonin during heroin use, but the other seemed to exclude any change in the hormone concentrations. This suggests that the "calcitonin like" material found in heroin addicts contains some epitopes similar to those found in the calcitonin standard which are detected by the first antiserum. However it lacks other epitopes which are also present in calcitonin standard and which are recognized by the second antiserum. Therefore, this substance seems to be different from the standard human calcitonin 1-32. A possible involvement of a calcitonin analogue (precursor or metabolite) in the biochemical changes occurring during chronic opiate use is suggested.

Adolescent↗
PubMed1991

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor.

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm x 3 mm I.D.) in series, packed with 10 microns HEMA-S 1000 packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 microns), packed in a 30 mm x 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-microliters volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the "front" of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125-4 micrograms injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 micrograms injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).

Alcohol Oxidoreductases↗
PubMed1991

Improved high-performance liquid chromatographic determination with amperometric detection of alpha-amanitin in human plasma based on its voltammetric study.

A sensitive and simple method is described for the selective determination in human plasma of alpha-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C18 silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene-divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer-acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for alpha-amanitin was 3-200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n = 5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n = 5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of alpha-, beta- and gamma-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for alpha- and gamma-amanitins at the amperometric detector, and for beta-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.

Amanitins↗
PubMed1991

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection.

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.

Alcohol Oxidoreductases↗