Mario Marigo

Death from heroin overdose: findings from hair analysis

BACKGROUND Morphine analysis of hair is used in forensic toxicology to study the addiction history of heroin addicts. To clarify the features underlying fatal heroin intake, we measured hair morphine content in a group of deceased heroin addicts, to verify a possible correlation between fatal heroin overdoses and the addiction behaviour of these individuals before death. METHODS 91 deaths were attributed to heroin overdose in Verona, Italy, in 1993-96. We analysed the hair of 37 of these individuals, and of 37 active heroin addicts, 37 former heroin users abstinent from the drug for several months, and 20 individuals with no evidence of exposure to opioids. From each individual, a hair sample of about 150 mg was analysed by RIA and high-performance liquid chromatography, to measure the morphine content. FINDINGS The mean morphine content in the hair of the addicts who had died was 1.15 ng/mg (SD 2.35 ng/mg; range 0-12.25 ng/mg) compared with 6.07 ng/mg (4.29; 1.15-17.0) in the active heroin addicts, 0.74 ng/mg (0.93; 0.10-3.32) in the abstinent former addicts, and values below the detection limit in the non-exposed group. Hair morphine content among those who had died was significantly lower than that in active heroin consumers (p<.00001), but not significantly different from that in the former addicts (p=0.978). INTERPRETATION Although our findings may be subject to selection bias, since suitable hair samples were available for only 37 of the 91 addicts who had died, these findings support the theory of high susceptibility to opioid overdose after periods of intentional or unintentional abstinence, due to loss of tolerance. Medical staff running detoxification programmes should be aware of the risk inherent in relapse to heroin after a period of abstinence. Moreover, occasional heroin use without a build-up of tolerance could also give a high risk of overdose.

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.

Optimization of a simple method for the chiral separation of phenethylamines of forensic interest based on cyclodextrin complexation capillary electrophoresis and its preliminary application to the analysis of human urine and hair

Because of the forensic importance of the chiral analysis of amphetamine and other phenethylamines for investigating their synthetic pathways and the metabolic patterns of these compounds, a capillary electrophoresis method has been developed based on the chiral selectivity of beta-cyclodextrin. The influence of different experimental conditions, such as cyclodextrin nature and concentration, voltage, temperature and buffer concentration and pH, on analytical performance has been studied. The optimized analytical conditions are: capillary: bare fused silica, 50 microns I.D., 40 cm effective length; buffer: 150 mM phosphate pH = 2.5, 15 mM beta-cyclodextrin; voltage: 10 kV; temperature: 17.5 degrees C; detection: UV absorption at 200 nm wavelength. Under these conditions, amphetamine, methamphetamine and ephedrine have been easily separated, with baseline resolution of the respective enantiomers. Sensitivity was better than 300 ng per ml. The average precision of migration times of the three analytes was good with RSD = 0.45% and 0.58% in intra-day and day-to-day tests, respectively. Reproducibility of peak heights was also good, with RSD = 2.51% and 3.14% in intra-day and day-to-day tests, respectively. The preliminary analysis of amphetamine in human urine and hair samples, subjected to a simple work-up procedure based on liquid-liquid extraction, showed clean blank electropherograms, excellent chiral resolution and sensitivity, suitable for the analysis of real samples from amphetamine users.

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.

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE-mass spectrometry in forensic toxicology is finally given.

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 Spearmans 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.

Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).

Field-amplified sample stacking capillary zone electrophoresis applied to the analysis of opiate drugs in hair

The present paper describes the methodological optimization and validation of capillary zone electrophoresis (CZE) for the determination of major opiates (morphine, codeine, 6‐monoacetylmorphine, acetylcodeine, heroin) in hair samples by using a field‐amplified sample stacking injection before the separation in a binary running buffer (0.1 M sodium phosphate, pH 2.5, with 40% ethylene glycol). The applied potential was 20 kV, at 25°C. Detection was by UV absorption at the fixed wavelength of 214 nm or by recording the full spectrum between 190—400 nm, thus improving the analytical selectivity and identification power of CZE. Hair samples were liquid/liquid extracted; dried extracts, reconstituted with a low‐conductivity solvent (0.1 mM phosphoric acid, with 80% 1‐propanol), were injected by electromigration at 10 kV for 99 s, after a 0.5 mm plug of water. Under the described conditions, the limit of detection (with a signal‐to‐noise ratio of 3) in hair extracts was 100 pg/mL for codeine, 75 pg/mL for morphine and 6‐monoacetylmorphine (6‐MAM), 150 pg/mL for ethylmorphine, and 0.75 ng/mL for acetylcodeine and heroin. The precision of the method was validated for standards in pure solution by using internal standardization, providing for intraday and day‐to‐day assays, in terms of migration times, relative standard deviation (RSD) values ≤0.2%, and in terms of peak areas, RSD values <5.71%.

Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking

The present paper describes the methodological optimisation and validation of a capillary zone electrophoresis method for the determination of morphine, cocaine and 3,4-methylenedioxymethamphetamine (MDMA) in hair, with injection based on field-amplified sample stacking. Diode array UV absorption detection was used to improve analytical selectivity and identification power. Analytical conditions: running buffer 100 mM potassium phosphate adjusted to pH 2.5 with phosphoric acid, applied potential 10 kV, temperature 20 degrees C, injection by electromigration at 10 kV for 10 s, detection by UV absorption at the fixed wavelength of 200 nm or by recording the full spectrum between 190 and 400 nm. Injection conditions: the dried hair extracts were reconstituted with a low-conductivity solvent (0.1 mM formic acid), the injection end of the capillary was dipped in water for 5 s without applying pressure (external rinse step), then a plug of 0.1 mM phosphoric acid was loaded by applying 0.5 psi for 10 s and, finally, the sample was injected electrokinetically at 10 kV for 10 s. Under the described conditions, the limit of detection was 2 ng/ml for MDMA, 8 ng/ml for cocaine and 6 ng/ml for morphine (with a signal-to-noise ratio of 5). The lowest concentration suitable for recording interpretable spectra was about 10-20-times the limit of detection of each analyte. The intraday and day-to-day reproducibility of migration times (n = 6), with internal standardisation, was characterised by R.S.D. values < or = 0.6%; peak area R.S.D.s were better than 10% in intraday and than 15% in day-to-day experiments. Analytical linearity was good with R2 better than 0.9990 for all the analytes.

High-performance liquid chromatographic determination of morphine in biological samples: An overview of separation methods and detection techniques

High-performance liquid chromatogrpahy with electrochemical detection at present suits most of the needs of toxicologists for the determination of morphine and some related compounds in biological samples, although fluorescence detection is still a useful alternative. Chemiluminescence detection may be promising, but needs further optimization of its coupling with HPLC to give the best performances. Morphine detection by absorbance spectrophotometry does not seem to allow the degree of sensitivity and selectivity from matrix interferences that is required in most instances. However, this approach is useful when morphine congeners undetectable by alternative means (i.e., heroin and morphine-3-glucuronide) are to be determined or when a general toxicological screening is required.