Drugs of abuse analysis in urine and hair for the evaluation of the driving fitness. An epidemiological study

La guida sotto l\u2019effetto di sostanze stupefacenti rappresenta un grave problema per la sicurezza stradale. La legge Italiana vieta il rilascio o la conferma della patente di guida a chiunque si trovi in stato di dipendenza o faccia un consumo abituale di sostanze stupefacenti o psicotrope. Ci\uf2 ha rilevanti ripercussioni sul piano sociale e professionale, dal momento che oggi la patente di guida \ue8 diventato uno strumento essenziale, oltre che per la mobilit\ue0, anche per lo svolgimento di numerose attivit\ue0 professionali. La normativa prevede che la valutazione di queste condizioni di consumo abituale e/o di dipendenza da sostanze d'abuso sia affidata alle commissioni mediche provinciali per le patenti, che si avvalgono di accertamenti chimico-tossicologici eseguiti sulle urine e/o sui capelli. Questo studio si \ue8 proposto di analizzare i risultati degli accertamenti chimico-tossicologici effettuati, su richiesta della Commissione Medica Provinciale per le patenti di Verona, dal laboratorio di Tossicologia Forense del Dipartimento di Sanit\ue0 Pubblica e Medicina di Comunit\ue0 dell'Universit\ue0 di Verona con l\u2019obiettivo di: (i) valutare frequenze relative di positivit\ue0 ed eventuali trend per le diverse classi di sostanze d\u2019abuso nel periodo 2003-2008; (ii) definire, nell\u2019ambito della popolazione esaminata, i principali fattori di rischio per la positivit\ue0 all'accertamento; (iii) identificare l\u2019approccio diagnostico pi\uf9 efficace per la valutazione di idoneit\ue0 alla guida in rapporto all\u2019uso di droga. Durante il periodo esaminato la cocaina \ue8 stata in assoluto la sostanza stupefacente riscontrata pi\uf9 frequentemente. Il confronto tra l'analisi delle urine e dei capelli ha confermato la complementariet\ue0 delle due matrici biologiche e, dunque, la necessit\ue0 di effettuare entrambe le analisi al fine di massimizzare la sensibilit\ue0 epidemiologica dell'accertamento. Questo studio ha inoltre dimostrato come l'accertamento di idoneit\ue0 alla guida costituisca un efficace deterrente al consumo di droghe dal momento che soltanto circa un quarto dei soggetti positivi al primo controllo risulta nuovamente positivo al secondo controllo.Driving under the influence of drugs is a serious problem for road traffic safety. According to the Italian Road Traffic Code, the driving licence must not be issued to anyone who abuses, is addicted to, or suffers for dependence to illicit or psychotropic drugs. The diagnosis of such clinical conditions is performed by Provincial Medical Commissions of the Public Health Service also on the basis of drugs of abuse testing results on urine and/or hair samples. This study aimed at examining test results obtained by the Forensic Toxicology laboratory of the Department of Public Health & Community Medicine, University of Verona, upon request of the local Medical Commission, over the period 2003-2008 with the purposes of (i) defining trends in drug abuse in the examined population (ii) identifying specific risk factors for testing positive and for relapse, (iii) selecting the most effective and efficient analytical strategy to detect illicit drugs use. During the study period, cocaine was the most frequently detected illicit drug. The comparison of results from urine and hair testing confirmed the complementary features of these two biological substrates and the importance to have both data in order to increase the sensitivity in detecting illicit drug use. Moreover, this study showed that testing for driving fitness is an effective deterrent to illicit drug use, as only about one quarter of subjects testing positive at the first testing are still positive at the second testing

Use of synthetic cannabinoids to cheat the toxicological screenings

Use of synthetic cannabinoids to cheat the toxicological screening

Assessment of fitness to drive in correlation with narcotic and psychotropic drug use. Epidemiologic study in Verona

Driving under the influence of drugs is a serious problem for road traffic safety. According to the Italian Road Traffic Code, the driving licence must not be issued to anyone who abuses, is addicted to, or suffers for dependence to illicit or psychotropic drugs. The diagnosis of such clinical conditions is performed by Provincial Medical Commissions of the Public Health Service also on the basis of drugs of abuse testing results on urine and/or hair samples. This study aimed at examining test results obtained by the Forensic Toxicology laboratory of the Department of Public Health & Community Medicine, University of Verona, upon request of the local Medical Commission, over the period 2003-2008 with the purposes of (i) defining trends in drug abuse in the examined population (ii) identifying specific risk factors for testing positive and for relapse, (iii) selecting the most effective and efficient analytical strategy to detect illicit drugs use. During the study period, cocaine was the most frequently detected illicit drug. The comparison of results from urine and hair testing confirmed the complementary features of these two biological substrates and the importance to have both data in order to increase the sensitivity in detecting illicit drug use. Moreover, this study showed that testing for driving fitness is an effective deterrent to illicit drug use, as only about one quarter of subjects testing positive at the first testing are still positive at the second testing

Chiral analysis of methorphan in opiate-overdose related deaths by using capillary electrophoresis

An enantioselective CE-based determination of methorphan and its main metabolites in blood is described. Enantiomeric separations were carried out in 50cm 750\u3bcm (ID) uncoated fused silica capillaries, using a background electrolyte composed of 150mM sodium phosphate pH 4.4 added with 5mM 2-(hydroxypropyl)-\u3b2-cyclodextrin and methanol 20% (v/v), at a constant voltage of 25kV. Sample injections were performed under field amplified sample stacking conditions. Detection was by recording UV absorbance at the wavelength of 200nm. Linearity of response was assessed within a concentration range from 25 to 500ng/mL for dextrometorhan, levomethorphan and their main metabolites (namely dextrorphan and levorphanol, respectively). Folcodine was used as internal standard. Under these conditions, the limit of quantification resulted 25ng/mL for each one of the analytes. The intra-day and inter-day precision, in terms of coefficient of variation (CV) were below 3.7% and 14.9 % for migration times and peak areas, respectively. The present method was successfully applied to the analysis of post-mortem blood samples from ten subjects died for heroin overdoses. Among the samples "positive" for methorphan (n=4), the d-enantiomer was found in concentrations ranging from 214 to 1282ng/mL. The concentration of its main metabolite dextrorphan in the same samples ranged from 49 to 389ng/mL

Calculation of LogP based on migration data in MEKC: application to the new synthetic cannabinoids.

Since 2004, a number of \u201cherbal blends\u201d containing synthetic cannabinoid analogues have appeared in the market, as a form of \u201clegal\u201d alternatives to Cannabis. These products are available online through the \u201ce-commerce\u201d and in \u201csmart shops\u201d under several forms and various brand names. A particular warning related to the diffusion of synthetic cannabinoids is based on one hand on their high toxicological potential, and on the other hand on the insensitivity of the current screening tests for cannabis towards these molecules. Moreover, because of their recent introduction, the literature regarding these compounds is still limited. In particular any experimental information on their octanol/water partition coefficient (logP) values is still lacking. Indeed, hydrophobicity value is an important parameter to investigate drug absorption, bioavailability and metabolism of molecules, as well as their toxicity. As a measure of molecular hydrophobicity, the logarithm of the partition coefficient between 1-octanol and water (logP) is widely used. The aim of the present study was to calculate the LogP of synthetic cannabinoids by using micellar electrokinetic chromatography (MEKC) with UV detection. Samples were analyzed using a fused silica capillary (30 mm x 40 cm) and a 25 mM sodium borate buffer pH 8, containing30 mMSDS and n-propanol 20%. After having constructed a calibration line using 10 appropriate standards with known LogP, an EOF marker and a micelle marker, a good correlation was found between the MEKC retention data of further 5 compounds with known LogP. The same calibration line was used to calculate the unknown LogP of the new synthetic cannabinoids, namely JWH-200, AM-694, JWH-250, JWH-073, JWH-015, JWH-018, JWH-081, JWH-122, JWH-019, JWH-210. The resulting Log P were in the range from 2.9 to 5.15. An acceptable agreement was found between the experimental data and a few LogP values calculated with XLogP3-AA reported in the literature

Finger-prick dried blood spot and capillary electrophoresis: a challenge for alcohol abusers screening analysis

Introduction and aim Finger-prick related DBS (fpDBS) is a new and innovative specimen collection in clinical practice to develop a screening method for forensic toxicology study [1].Despite this, still very limitedly employed is the fpDBS specimens for capillary electrophoresis (CE) analyses. Transferrin glycoforms are a suitable parameter in alcohol abuse investigation and in particular the carbohydrate deficient transferrin (CDT), defined as the percentage distribution of less glycosilated transferrin, is increased after chronic sustained alcohol intake [2]. The aim of this study was to verify if the use of fpDBS could be suitable for CDT screening CE analysis. Method Human serum specimens were collected from volunteer subjects. fpDBS sample collection was performed by pricking the little finger, absorbing the capillary blood drops on DBS cards and letting it to air dry. Once dried, samples required a treatment, which included three easy steps: A) sample resuspension from fpDBS with an acid solution (to remove interfering circulating proteins, mainly haemoglobin); B) removing the paper from the sample solution and pH check (3<pH<4); C) sample neutralization (pH 7-8). The final sample was centrifuged and the supernatant was diluted with an iron rich solution before CE analysis. The CE running buffer was borate (pH 7.9) with diaminobutane (7.5 mmol/L), and the separations were performed within a 30 \u3bcm i.d. uncoated fused-silica capillary (length 60cm) using a constant voltage (30kV) and detection at 200 nm. The study was executed analysing fpDBS and parallel serum samples collected from each investigated subject and %CDT levels were measured using both high performance liquid chromatography (HPLC) and CE techniques. Results and conclusion The comparison between fpDBS and parallel serum CDT level, analysed by the traditional CE and HPLC methods, demonstrated significant correlation r2>0.85. In addition, statistical analysis confirmed that the concentration differences measured in DBS specimens were not relevant. Our results demonstrate how the acid treatment allows analysis in CE despite the small volumes and the large amount of various interfering compounds of whole blood. Therefore, the CE technique use coupled to the fpDBS procedure for CDT analysis expresses a simplified and inexpensive tool designed for use in population screening. In conclusion, the fpDBS CE procedure appear suitable in the forensic alcohol abuse investigations. References [1] CP Stove, AS Ingels, PM De Kesel, WE Lambert. Crit Rev Toxicol. 42 (2012) 230-43. [2] JR Delanghe, ML De Buyzere. Clin Chim Acta. 406 (2009) 1-7

Drug screening by using the Toxtyper\u2122 LC-ion trap MS: Optimization of its application on serum samples in a DUID context

The toxicological approach for monitoring Driving Under Influence of Drugs (DUID) requires analytical techniques with a broad spectrum of identification coupled to a high analytical sensitivity. In this context immunological methods are generally used, while GC or LC-MS are applied for the confirmation step. A different approach for drug screening is represented by the Toxtyper\u2122 instrumentation, an LC-MS platform equipped with a high-speed ion trap mass analyzer, provided with ready-to-use protocols and a database of as many as 4500 therapeutic, toxic/illicit drugs and metabolites. The aim of the present work was to verify its performances in real conditions of drug screening of human serum in the context of DUID. To test and compare its analytical performances, four pooled serum samples were fortified with a selected panel of 47 drugs and metabolites. The agreement between the results from the ToxtyperTM and from the confirmatory techniques currently in use at the University of Verona (GC and LC-MS) was investigated by analyzing 90 real samples chosen from those routinely analyzed. The present study highlights the suitability of the ToxtyperTM for drug screening in serum with a sensitivity compatible with the needs of the DUID for all the tested compounds, with the only exception of cannabinoids

Dextromethorphan/levomethorphan issues in a case of opiate overdose.

The paper reports the case of a heroin addict who was found dead at his home with a fresh puncture mark on his leftarm. The GC-MS analysis of post-mortem blood and of the content of the syringe found next the body showed the presence of heroin and methorphan, probably used as adulterant of heroin. However GC-MS method was not able to distinguish between the two methorphan enantiomers, which show different pharmacological activity. To solve this problem, a chiral method based on the use of capillary electrophoresis with cyclodextrins was developed. The method was successfully applied to the analysis of the postmortem blood identifying the presence of dextromethorphan at a concentration of 842 ng/mL

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids

Development and preliminary use of CDT analysis on dried blood spot (DBS) in forensic and administrative contexts

BACKGROUND-AIM Carbohydrate Deficient Transferrin (CDT) defines two minor glycoforms of transferrin (asialo and disialo-tansferrin), characterized by a reduced glycosylation degree, whose serum concentration increases after chronic sustained alcohol intake (60-80 g per day for at least 10 days). The use of finger-prick and related dried blood spots (fpDBS) is an innovative tool for blood sample collection in clinical and forensic toxicology. The aim of this work was to develop a screening method for CDT analysis based on the use of fpDBS coupled with capillary electrophoresis. METHODS Capillary blood drops collected by finger-prick were placed on DBS cards and left to air dry. Each dried fpDBS disc was sliced and suspended in acid solution. After centrifugation the sample pH was adjusted by 120 mmol HCl to pH 3-4. After overnight incubation the sample pH was neutralized and an iron rich solution was added. The resulting sample was analyzed by a validated CE method. The CDT level was expressed as %CDT (%ratio of disialo-Tf on total transferrin). The blood samples were obtained from volunteers of the forensic toxicology laboratory and from subjects submitted to blood testing for mandatory toxicological investigations. The DBS were analyzed in parallel with the sera of each investigated subject, using HPLC and CE techniques. The %CDT cut-offs used for the study were 1.80% and 1.90% for CE and HPLC, respectively. RESULTS The observed fpDBS transferrin glycoform CE patterns were comparable with serum CE CDT patterns. Moreover, a statistical correlation was demonstrated of fpDBS CDT percentage levels with both HPLC and CE % CDT (p< 0.01). This correlation was confirmed also by Passing-Bablok tests and Bland Altman test. The cut off proposed for this %CDT screening method was 1.6% demonstrating a sensitivity and specificity of about 75% and 90%, respectively. These data were calculated comparing %CDT by fpDBS CE vs serum HPLC, the latter considered the reference method. CONCLUSIONS The results of the study, even if preliminary, showed that fpDBS procedure coupled with CE for CDT analysis could express a simplified and inexpensive tool designed for use in population screening