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M Marigo

Publications and source records attributed to M Marigo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Complementary use of capillary zone electrophoresis and micellar electrokinetic capillary chromatography for mutual confirmation of results in forensic drug analysis.

The purpose of this work was to compare different CE separation modes namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) for the analysis of drugs of forensic interest in order to assess the mutual degree of independence and consequently the possibility of complementary use for mutual confirmation of results. A panel of drugs including caffeine, morphine, barbital, pentobarbital, codeine, nalorphine, lidocaine, procaine, heroin, flunitrazepam, acetylcodeine, papaverine, amphetamine, narcotine, cocaine, diazepam, tetracaine, narceine, 6-monoacetylmorphine acetylcodeine and thebaine, were separated according to a MECC and two CZE methods. The MECC separation was carried out in a bare silica capillary (50 micron I.D.) with a buffer composed of 25 mM borate (pH 9.24)--20% methanol--100 mM sodium dodecyl sulphate; the applied voltage was 20 kV. The first CZE method (CZE1) was carried out in 50 mM phosphate buffer (pH 2.35) at 20 kV with a bare silica capillary (50 micron I.D.), and the second (CZE2) with 50 mM borate (pH 9.24) at 12 kV with the same capillary. The three methods were effective in the separation of the test drug mixture, but MECC was the only able to resolve all the components. Relative (to flunitrazepam), migration time RSDs ranged from 0.3 to 2.8% for the three methods were compared with Spearman's test and with principal component analysis, CZE1 and CZE2 were significantly and directly correlated (r = 0.749, p < 0.002), whereas MECC and CZE2 were also significantly, but inversely correlated (r = -0.865, p < 0.001). MECC and CZE1 (limitedly to the basic drugs) appeared non-correlated (r= -0.131, p = 0.630) and therefore the two techniques are suitable for combined use to increase the discriminatory power.

Chromatography↗
PubMed1995

Capillary electrophoresis: a new tool in forensic toxicology. Applications and prospects in hair analysis for illicit drugs.

Capillary electrophoresis, the modern approach to instrumental electrophoresis, is probably the most rapidly expanding analytical technique that has appeared in recent years. In the hands of forensic toxicologists, capillary electrophoresis (CE) represents a powerful new analytical tool, which has proved suitable for the investigation of illicit drugs in seized preparations and also in complex biological matrices, among which is hair. CE can be applied according to different separation mechanisms, and among those that are toxicologically relevant are capillary zone electrophoresis and micellar electrokinetic capillary chromatography, which display different selectivities. For the investigation of hair for drugs of abuse, capillary electrophoresis proved effective, providing simultaneous determinations of different drugs without derivatization, with acceptable sensitivity (typically better than 1 ng of drug per mg of hair). The possibility of carrying out determinations of the same analytes, based on different separation mechanisms (capillary zone electrophoresis and micellar electrokinetic chromatography) with the same instrumentation, simply changing the buffer composition, provides an interesting possibility of 'internal' confirmation of the results.

Electrophoresis↗
PubMed1995

Suicide attempted by burning: a 10-year study of self-immolation deaths.

In a 10-year retrospective survey of 699 admissions to the Burns Centre of Verona, 31 patients (4.4 per cent) had self-inflicted burns. No sex difference was found, the mean age was 38 years, with 58 per cent of the patients in the 20-39-year age group. The mean burned surface area (BSA) was 41 per cent, and 12 patients (38.7 per cent) died: seven within the first 24 h. The other five patients died within 40 days (mean survival: 18 days). A large number of patients had a history of previous psychiatric treatment, six patients had previously attempted suicide. Four patients were drug addicts, three of whom were affected by AIDS and one had been HIV-positive for a long time. The patients had continuous psychiatric treatment during the long hospital stay. After discharge the patients were assigned to territorial staff for psychiatric treatment. Our experience shows that these patients have a higher mortality rate and they need constant psychiatric support, which can be useful in preventing further suicide attempts.

Adult↗
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

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↗
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

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↗
PubMed1991

Forensic applications of molecular genetic analysis: an Italian collaborative study on paternity testing by the determination of variable number of tandem repeat DNA polymorphisms.

Several variable number of tandem repeat (VNTR) DNA polymorphisms detecting different loci (YNH24/MspI or TaqI, CMM101/MspI or MLJ14/MspI, EFD64.2/RsaI or HinfI, YNZ22/TaqI, AW101/EcoRI, EKMDA2.1/PvuII and 3'-HVR/PvuII) were used in the analysis of 27 cases of disputed paternity in the Italian population. Fourteen exclusions and 17 attributions were performed. The results were compared with those obtained with immunohematologic analyses. Four exclusions and 2 attributions were made possible only by the combined use of several DNA polymorphisms, as the analyses of red-blood-cell antigens and isoenzymes, serum proteins and HLA group determinants were inconclusive. With the DNA test, 10 exclusions and 15 attributions were confirmed, with increased overall probability. In conclusion, VNTR polymorphisms were more informative, accurate and sensitive than the immunohematologic tests. Therefore, DNA analysis is the method of choice for testing genetic relationships.

Alleles↗
PubMed1990

Direct injection high-performance liquid chromatographic assay of morphine with electrochemical detection, a polymeric column and an alkaline eluent.

A simple method is described for the direct determination of morphine in untreated plasma or serum, using reversed-phase high-performance liquid chromatography with amperometric electrochemical detection. A basic eluent [0.05 mol/l phosphate buffer-isopropanol-tetrahydrofuran (88:10:2) (pH 9.5)] allows both reversed-phase chromatography of morphine under ionization control conditions and its detectability at an unprotected thin-layer glassy carbon electrode at a potential of 350 mV (vs. an Ag/AgCl reference electrode). In addition, the alkaline mobile phase promotes the ionization of serum proteins, which, being poorly retained by the hydrophobic column packing [poly(styrene-divinylbenzene) copolymer], elute early in the chromatogram, leaving a clean baseline. Up to 50 microliters of simply filtered plasma can be injected. The absolute limit of detection is 0.75 ng on-column. No interferences were observed from more than 80 opiate and non-opiate drugs. The intra- and inter-assay relative standard deviations (n = 5) were 3.2 and 6.6%, respectively, at a morphine concentration of 100 ng/ml in plasma and 0.09 and 4.2%, respectively, at the level of 500 ng/ml.

Chromatography, High Pressure Liquid↗
PubMed1990

Use of enzymatic reactors in the high performance liquid chromatographic determination of ethanol and methanol with electrochemical detection.

Despite numerous applications both in industry and in analytical chemistry, immobilized enzyme technology has encountered only limited success in modern liquid chromatography. The strict requirements of enzymatic reactors for high performance liquid chromatography (HPLC) (i.e., mechanical resistance, limited void volumes, high contact surface, high binding capacity, stable enzyme-to-matrix bond without loss of enzyme activity) are not yet fully met by commercially available products. In our laboratory, the development of post-column reactors with immobilized alcohol oxidase on-line with an HPLC/EC system has been undertaken. Three home-made reactors were produced: in the first alcohol oxidase from Candida boidinii was quasi-immobilized onto an HPLC-sized anion exchange support, in the second the enzyme was bound to a silica-based support via Schiff's base formation, and in the third a covalent linkage to an epoxy-activated hydroxyalkyl methacrylate material was chosen. Chemical immobilization proved superior to the ionic binding. The importance of limiting the post-column band spreading due to the reactor void volume, by using HPLC-size supports, was confirmed.

Chromatography, High Pressure Liquid↗
PubMed1990

Frequency distribution of the alleles of several variable number of tandem repeat DNA polymorphisms in the Italian population.

Variable number of tandem repeat (VNTR) DNA polymorphisms were studied in a random sample of 100 Italians. The following systems were used: YNH24/MspI, CMM101/MspI, MLJ14/RsaI, EFD64.2/RsaI, EFD64.2/HinfI, JCZ3.1/HinfI and AW101/EcoRI. For each system, the allele size, number and frequency were determined. The number of alleles, for the different VNTRs, varied from 21 to 44, and the heterozygosity from 70 to 90%. All the VNTRs showed a modal distribution of fragment size and overrepresentation of the lower-molecular-weight DNA fragments. All of them exhibited greater variability than that reported for the North American population. These data will be useful for individual identification purposes and for calculating the probability of random inheritance of a particular allele in the Italian population. The results are discussed in relation with the possible mechanism of generation of new-length alleles.

Alleles↗