Search

The data collected in this study describe an initial attempt to systematically introduce the qualitative and quantitative analysis of adulterants present in seized street drugs in Italy with the aim of improving surveillance and data sharing and for this purpose, the implementation of validated and standardized procedures are essential., (Copyright © 2016 John Wiley & Sons, Ltd.)

In Italy, driving under the influence of drugs determines the suspension of the offender's driver's license. To regain the license the person must be drug free during an observation period. People whose license has been revoked or suspended can obtain, or re-obtain their driver's license subject to the judgment of a medical commission. The exclusion of illicit drug use is determined by means of toxicological analysis, mainly on urine or hair matrices. We reported the results of several years of experience of the forensic toxicology laboratory of the University of Macerata in the use of hair analysis for the assessment of past exposure to drugs in people suspected of driving under the influence of drugs. From 2004 to 2013, 8612 hair samples, were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ(9)-THC) using gas chromatography/mass spectrometry (GC/MS) method. We used a cutoff (SoHT or national guidelines) to determine the positive data, regardless of the hair sample concentrations. 1213 samples resulted positive, 71.7% were positive for cocaine and metabolites, 19.8% for morphine and metabolites, 8.5% for Δ(9)-THC. We also studied the timeframe of the abuse, as well as gender and age distribution of positive subjects. Moreover, we analyzed the possible deterrent effect of the hair analysis on driving under the influence of psychoactive substances., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The diffusion and trends in use of each substance is a basic information in policy planning of strategies aiming at deterrence of drug abuse or in the organization of the fight against drug trafficking. The actual diffusion of illicit drugs in a population is hardly measurable, but, among the various measures available, the analysis of waste water plants represents one of the most reliable source of data. We analyzed waste water in order to monitor illicit drug use by local population. We investigated the use of cocaine and heroin in the city of Florence, Italy, over a 1-year (July 2006-June 2007) period using state-of-the-art measuring techniques from waste water samples. Cocaine, benzoylecgonine, and morphine were determined in water samples by gas chromatography-mass spectrometer, and the amount of illicit substance was estimated. Data indicate for cocaine a bimodal distribution (December and March), while heroin showed a main peak in April. The heroin-to-cocaine use ratio in terms of estimated doses per month ranged from 0.11 to 0.76, representing new evidence of wider distribution of cocaine than heroin in Florence. Waste water analysis can become a valuable tool in monitoring use of illicit drugs over time. In particular, it can highlight changes in the magnitude and relative use of illicit drug at a population level thereby becoming useful to develop strategies against drug trafficking and abuse. If routinely performed, it can be part of Epidemiologic Surveillance Programmes on drug abuse.

HAIRVEQ is a proficiency testing program for hair analysis of illicit drugs organized by the Istituto Superiore di Sanità (Rome, Italy) and the Institut Municipal d'Investigació Mèdica (Barcelona, Spain). The aim of the three exercises performed in 2006 was the evaluation of 32 laboratories' performance when analyzing the same hair sample containing opiates, cocaine and methadone, after carrying out some specific educational interventions. In the first round, the sample was sent to be analyzed following laboratory routine methodology. In the second round, standard operating procedures (SOP) for hair testing including sample preparation, method validation and qualitative and quantitative data evaluation, and an open hair sample for SOP training were also sent together with other hair samples including the one used for performance evaluation. After the second round, a workshop was held with participant laboratories to discuss methodological issues and interpretation of obtained results. An additional amount of open samples was distributed to the laboratories for implementing the SOPs. In the third round, the same unknown sample containing opiates, cocaine and methadone was resent for the final evaluation of laboratory performance. In the first round, 11 incorrect qualitative results (10 false negative and 1 false positive) were reported by seven laboratories (22%), in the second round, a reduction in the number of incorrect results was observed (4 false negatives and 1 false positive were reported by four laboratories, 13%) and in the third round, 5 false positives and 5 false negatives were reported by seven laboratories (22%). Concerning quantitative results, the scatter was similar between the three rounds and similar to the ones reported by other proficiency tests in hair analysis. More educational actions should be addressed to a group of laboratories, which did not yet show satisfying qualitative and quantitative results.

In the last years the interest in monitoring drug exposure with human sweat as alternative biological fluid, is increasing. Sweat collection is convenient, less invasive and difficult to adulterate compared to traditional specimens. The objective of this study was to determine the excretion profile of methadone and other drugs into human sweat. Pharmscope sweat patches (Medical Europe Diagnostic, Madrid, Spain) were used on heroin abusers under methadone treatment. Sweat patches were applied to 10 heroin addicts and 3 drug free volunteers admitted into the study. Sweat patches were worn for about 1 week; urine, saliva and hair samples were collected at the time of the removal of patches. After the extraction, sweat eluates were directly analyzed by GC/MS for the presence of nicotine, cotinine, caffeine, methadone, EDDP and cocaine. The extracts were subsequently derivatized to detect benzoylecgonine, ecgonine methyl ester, morphine, codeine and 6-acetylmorphine. No false positive results were obtained on the drug free samples. All the patches showed positive results for methadone. Cocaine was detected in two cases. Mainly the parent drug was identified rather than the metabolites. The results obtained show the usefulness of sweat as complementary specimen to saliva and urine providing a longer detection window. Moreover, sweat testing offers the advantage of being a non-invasive means of obtaining information about drug exposure.

Since 2002, the Istituto Superiore di Sanità, Rome, Italy, in cooperation with Institut Municipal d'Investigaciò Mèdica, Barcelona, Spain, has set up an external proficiency testing program (HAIRVEQ) to evaluate reliability in hair testing for drug abuse by laboratories from the Italian National Health Service. The results obtained in the last 2 rounds (2004-2005) by 26 laboratories and the evolution of the performance in hair testing for drugs of abuse by laboratories that have participated during the whole external proficiency testing program are presented. The 3 hair samples from the last exercise (2005) were also included in the proficiency test organized by the Society of Hair Testing (SoHT) and 17 international laboratories reported results. Samples analyzed in both exercises were real hair samples from drug consumers. In 2004, 2 identical samples were sent containing cocaine and opiates. One sample was a pulverized specimen and the second one was cut in short segments. In 2005, 2 samples, one containing MDMA and another containing cocaine, were included together with one blank sample. In 2004, approximately 42% of HAIRVEQ laboratories reported an erroneous qualitative result. The scatter of quantitative results was high, although no statistical differences, except for codeine, were found between results reported for the hair specimen if pulverized or reduced in short cuts. In 2005, 47 incorrect qualitative results were reported by HAIRVEQ laboratories, whereas only 5 were informed by SoHT laboratories. Concerning quantitative results, the ones from HAIRVEQ laboratories were comparable, although more dispersed, than those reported by SoHT laboratories. The scatter in quantitative results remained quite high and similar to those of the previous years; nonetheless, an improvement in the qualitative performance was observed. Considering the few number of laboratories showing a satisfying performance, guidelines have to be provided focused on method validation and qualitative and quantitative data evaluation.

For the first time in Europe, the "Meconium Project" aimed to estimate the prevalence of drug use by pregnant women and the effects of exposure to illicit drugs during pregnancy on the fetus and infant. Between October 2002 and February 2004, 1151 (79%) dyads among the 1439 mother-infant dyads from the Hospital del Mar, Barcelona, Spain, met eligibility criteria and agreed to participate in the study. We present preliminary results on the first 830 meconium samples and 549 mother-infant dyads, for which statistical analysis of socio-economic and demographic characteristics and newborn somatometry was completed. The meconium analysis showed an overall 7.9% positivity for drugs of abuse, with 6-monoacetylmorphine and cocaine being the analytes, most frequently found in samples positive for opiates and cocaine. Structured interview disclosed 1.3, 1.8 and 1.3% of mothers exposed to opiates, cocaine and both drugs, while only one mother declared ecstasy consumption. Meconium analysis showed that prevalence of opiates, cocaine and combined drugs exposure was 8.7, 4.4 and 2.2%, respectively, and confirmed the case of ecstasy use. Arecoline, the main areca nut alkaloid, was found in meconium specimens from four Asiatic newborns, whose mothers declared beetle nut consumption during pregnancy. Parental ethnicity was not associated with drug use, nor was the social class, although a higher tendency toward drug consumption was observed in professional and partly skilled mothers. Drug consuming mothers showed a higher number of previous pregnancies and abortions (p<0.05) when compared to non-consumer mothers (meconium negative test), probably due to a lack of family planning. Consumption of opiates and cocaine during pregnancy was associated with active tobacco smoking, a higher number of smoked cigarettes and cannabis use. Exposure status and smoking behavior correlated with significantly lower birth weight in newborns from mothers exposed only to cocaine and to opiates and cocaine simultaneously. Of the four newborns exposed to arecoline, one showed a low birth weight, low intrauterine growth, hyporeflexia and hypotonia.

The Istituto Superiore di Sanità of Rome, Italy, in cooperation with Institut Municipal d'Investigaciò Mèdica of Barcelona, Spain, set up an external quality control program (HAIRVEQ) to evaluate reliability in hair testing for drug abuse by laboratories from the Italian National Health Service. Samples included in the program were real hair samples from drugs consumers. Prior to sending, hair samples were reduced to powdered form, mixed to ensure homogeneity and tested with GC/MS by four Reference Laboratories. Up to now, four different exercises have been concluded and 23 laboratories participated. Samples containing high and low concentrations of opiates, cocaine and metabolites, low concentrations of MDMA and two blank samples, were included in the intercomparison exercises performed in the first year of HAIRVEQ activities. Results show an insufficient performance of participating laboratories. About 82% of laboratories reported incorrect results on a qualitative basis (false positive and false negative results) for some of the submitted samples. More than one-half of laboratories reported quantitative results (60%). On the basis of the calculated z scores, only between 35 and 55% of results reported should be considered as satisfying. Guidelines have to be provided by Italian authorities for method validation as well as set of recommended cut-off concentrations to orientate laboratories in their quality objectives when developing analytical methodologies as tools to improve reliability and consequently performance of hair analysis.

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).

According to the laws presently in force in Italy and the guidelines of the Driving Licence Enforcement Commission of Verona, applicants for the driving licence with a history of drug abuse undergo a medical examination, during which complete anamnestic and clinical data are recorded. On this occasion, a hair sample (50-200 mg) is collected and a urinalysis program is started consisting of EMIT controls for opiates, methadone, cocaine, barbiturates, amphetamines, cannabinoids, benzodiazepines and alcohol carried out on eight seriate samples, collected at random over about 40 days under direct supervision. The positive results from urine immunoassays are confirmed by standardized GC/MS methods. The hair samples are screened for morphine and cocaine, the most abused illicit substances in our region, using commercial RIAs adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negative are confirmed by HPLC. In case of confirmed positive results, the applicant is informed: if the subject denies use of opiates or cocaine in the recent months, he or she has the chance of submitting for analysis a new hair sample, which is analyzed in parallel with the hair remaining from the previous assay. In case of persisting denial, claiming analytical interferences by other drugs or endogenous substances, further confirmation of results can be carried out by CE and/or by qualitative MS/MS. In addition, hair sampling from multiple sites (scalp, axillary, pubic hair) with different susceptibility to contamination from the external sources can be carried out to rule out the possibility of passive contamination. At present, we investigate more than 700 subjects per year. The results of this integrated diagnostic strategy are presented and discussed.

In our laboratory, analysis of human hair for drugs of abuse detection was first performed in 1980. In the last 10 years we have processed about 2000 subjects/year ('living subjects' only). In the last 3 years we have also introduced hair analysis of cocaine: at first only in clinical applications, but for the last 2 years this analysis is now routine. Our application of hair analysis includes: clinical toxicology, medico-legal and administrative agencies. Requests come for example from several Committees for Driving Licenses, Addiction Treatment Centers and Legal Authorities. Hair samples are currently collected from the occipital area at the back of the head, which appears to show less variability in hair growth rate. At present we perform hair analysis using highly sensitive radioimmunoassay screening methods for the detection of parent drug and/or metabolites. All positive cases of cocaine and opiates abuse are confirmed by gas chromatography-mass spectrometry in electron impact or chemical ionization mode. Positive cases for opiates are also analysed using a specific morphine radioimmunoassay kit. Data show that, when the opiates/morphine ratio is higher than 6, we are dealing with consumption of codeine and/or dihydrocodeine. In our routine work last year there were 177 (263 samples) positive opiates subjects out of 2244 patients; positive cocaine subjects were 290 (362 samples) out of 2001 patients. Guidelines for hair analysis in Lombardia have been established based on the experience of our laboratory. Furthermore it will be possible to apply a unique protocol for all Committees for Driving Licenses, involving hair testing in addition to urine assay.

Toxicological analysis of hair was used to detect cocaine use in a group of 615 pregnant women. Hair samples were washed, enzymatically digested, and analyzed by radioimmunoassay for cocaine. Positive results were confirmed by gas chromatography-mass spectrometry after a solid-phase extraction. Benzoylecgonine (BZE) and cocaine were detected after derivatization with pentafluoropropionic-anhydride-pentafluoropropanol. Deuterated cocaine and BZE were used as internal standards. This study demonstrated a mean frequency of 1.9% cases positive for cocaine in all the hair samples examined. The positive rate was 6% among women admitted for spontaneous abortion (66 cases), and the positive rate was 1.4% for those who carried to full term (549 cases). These data underline the usefulness of hair analysis for the diagnosis of drug abuse and demonstrate that there is significant use of cocaine in the Italian population.

The chemical-toxicological diagnosis aspects involved in 16 cases of "intravenous narcotism" deaths are discussed. Liquids and biological materials were directly tested by RIA, GLC and TLC. According to the Authors, RIA is assumed to be more speedy and more sensitive than traditional chemical technics. Therefore RIA is proposed for quantitative determination of morfina and its derivates in biological liquids and viscera, previous the qualitative confirmation by the chemical tests.