Marzia Bernini

Hair analysis for driving licence in cocaine and heroin users.: An epidemiological study

Diagnosis of drug exposure is strongly supported by analysis of hair samples. In the province of Brescia, Italy, for regranting driving license to drug addicts or occasional abusers, a control programme was adopted including analysis of illicit drugs in two hair segments (0-3 and 3-6 cm) and in urine. From January 1998 to April 1999, upon request of the Local Medical Commission, 697 hair samples were tested in our laboratory. One hundred and eighty subjects resulted positive in hair for one or two of the controlled drug classes (73.3% for cocaine, 10% for opiates, 16.7% for both). Positive subjects were classified by residence, age, sex and license category. Seventy-two subjects were called back after 6-12 months and submitted to a second hair and urine analysis: in 34 cases the result of the first analysis was confirmed (19 negatives, 15 positives for one or both drug classes). Another 37 cases tested positive at the first control and negative at the second, suggesting the hypothesis that a strict control may have a significant deterrent function. The high percentage of negative results at the second control may be explained by the prevalence of cocaine users in the examined population. Our results allow us to conclude that the strict application of control rules lead to a decrease of social risk behaviours.

Ethyl glucuronide in vitreous humor and blood postmortem specimens: analysis by liquid chromatography-electrospray tandem mass spectrometry and interpreting results of neo-formation of ethanol

INTRODUCTION The determination of ethyl glucuronide (EtG), a stable and sensitive marker that is specific to alcohol intake, finds many applications both in the forensic toxicology and clinical fields. AIM The aim of the study is to examine the possibility of using a cadaveric biological matrix, vitreous humor (VH), to determine EtG as a marker of recent ethanol use. METHODS The blood, taken from the femoral vein, and the VH were obtained from 63 autopsy cases. Analysis of the EtG was performed using an LC/MS/MS system. Analyses of the ethanol and putrefaction biomarkers, such as acetaldehyde and n-propanol, were performed using the HS-GC/FID technique in both the matrices. RESULTS In 17 cases, both ethanol and EtG were absent in both matrices.Nineteen cases presented ethanol in blood from 0.05 to 0.30 g/L, EtG-Blood concentration from 0.02 to 3.27 mg/L, and EtG-VH concentration from 0.01 mg/L to 2.88 mg/L. Thirteen cases presented ethanol in blood > 0.05 g/L but EtG concentration in blood and VH lower than 0.01 mg/L, are part of these 8 samples presented acetic aldehyde and n- propanol in blood or VH, means identification of putrefaction indicators. Fourteen cases presented ethanol in blood > 0.46 and EtG concentration in blood and VH higher than 0.01 mg/L. CONCLUSIONS The determination of EtG in biological material is important in those cases where the intake of ethanol appears doubtful, as it allows us to exclude the possibility of any post-mortem formation of ethanol.

Death after 25C-NBOMe and 25H-NBOMe consumption

A teenager male was found dead in a waterway after he was spotted jumping off into the water stream. The boy looked agitated and confused after a party with friends. At the gathering place, investigators seized packages of blotter papers. A complete autopsy and a histological evaluation of the main tissues were performed; although the death occurred by drowning, the prosecutor requested toxicological exams, in order to evaluate the potential role of drugs of abuse in the episode. Blood (both peripheral and central) and urine samples as well as seized blotter papers were collected and analyzed as follows. The blotter paper, analyzed through a GC-MS method, revealed the presence of 25-NBOMes. A liquid chromatography tandem mass spectrometric (LC-MS/MS) system was used to identify and quantify 5 different 25-NBOMes (namely 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe) in blood and urine. 25E-NBOMe was used as internal standard (IS). 1mL of urine and 1mL of blood (both peripheral and cardiac) were diluted in 2mL phosphate buffer at pH 6.0, containing IS and purified on a solid phase extraction (SPE) cartridge. LOD and LOQ for the five 25-NBOMes were calculated at 0.05 and 0.1ng/mL respectively. Linearity, accuracy, precision, ion suppression, carry over and recovery were tested and all parameters fulfilled the acceptance criteria. Blood and urine provided positive results for 25C-NBOMe and 25H-NBOMe. Eventually, the seized blotter papers were analyzed by means of LC-MS/MS and the presence of the two NBOMes was confirmed: 25C-NBOMe and 25H-NBOMe were measured at the concentration of 2.80 and 0.29ng/mL in peripheral blood, of 1.43 and 0.13ng/mL in central blood and of 0.94 and 0.14ng/mL in urine, respectively. THC and THCCOOH were also detected in biological fluids, at the concentration of 15.5 and 56.0ng/mL in peripheral blood, 9.9 and 8.5ng/mL in central blood, respectively. NBOMes can produce severe hallucination even at very low doses, and the 25C-NBOMe levels measured in the subjects blood are considered potentially toxic.

Determination of endogenous/exogenous γ-hydroxybutyric acid (GHB) in urine

SummaryBackgroundγ-Hydroxybutyric acid (GHB) is an endogenous metabolite and a precursor of the neurotransmitter γ-aminobutyric acid. In some European countries GHB is also used as an anaesthetic agent and in the treatment of alcohol withdrawal. The purpose of the present study was to determine GHB concentrations in urine to enable discrimination between endogenous GHB and exogenous GHB derived from therapeutic administration (Alcover®, Laboratorio farmaceutico C.T.Srl, Sanremo).Methods34 urine samples from 17 alcoholics under treatment for alcohol withdrawal with GHB (group 1) and 12 urine samples from healthy volunteers without GHB intake (group 2) were collected anonymously and analysed by HPLC/MS/MS. The urine concentrations of GHB were determined from two standard curves with the following concentrations: 0.25, 0.50, 1.00, 2.50,5.00 and 10.00 μg/ml, and 5.00, 10.00, 25.00, 50.00 and 100.00 μg/ml.ResultsAcceptable linear regression was obtained for both calibration curves: R2 = 0.9950 and R2 = 0.9994, respectively. Intraday precision and accuracy were calculated as coefficient of variation (%) and bias (n = 10) on three positive control specimens at three different concentrations (0.50, 2.50 and 7.50 μg/ml). The limit of quantification was determined to be 0.50 μg/ml. The median and average concentrations of GHB in group 2 (no GHB intake) were 3.55 and 3.49 μg/ml, respectively, while 52.90% of group 1 (subjects treated with GHB) had urinary GHB concentrations less than the currently used cutoff of 10.00 μg/ml.ConclusionIn the absence of a rapid immunoassay for the detection of GHB in urine, LC/MS/MS analysis was found to be specific and reasonably fast for clinical and forensic toxicology applications. Of the subjects being treated with GHB, 52.90% were found to have a urinary GHB concentration less than 10.00 μg/ml, with GHB concentrations ranging from 2.95 to 8.50 μg/ml. Thus the widely accepted 10.00 μg/ml cut-off could be too high so that there is the risk of false-negative samples.RiassuntoPremesseL’acido γ-idrossibutirrico (GHB) é un metabolita endogeno e precursore del neurotrasmettitore γ-amminobutirrico. In Europa il GHB é anche impiegato come anestetico e come farmaco per il trattamento della dismissione dall’alcol. Lo scopo del presente studio é stato la determinazione della concentrazione urinaria di GHB per poter discriminare i livelli endogeni della molecola da quelli esogeni derivanti dalla somministrazione a scopo terapeutico di GHB (Alcover®, Laboratorio farmaceutico C.T.Srl, Sanremo).Metodi34 campioni di urina di 17 soggetti alcolisti sottoposti al trattamento con GHB per la dismissione dall’alcol (gruppo 1) e 12 campioni di urine di soggetti sani, non bevitori e non in terapia con GHB sono stati raccolti in modo anonimo e sottoposti ad analisi mediante HPLC/MS/MS. La concentrazione urinaria di GHB é stata determinata mediante l’allestimento di due curve di calibrazione: 0,25-0,5-1-2,5-5-10 μg/mL e 5-10-25-50-100 μg/mL.RisultatiEntrambe le curve di calibrazione hanno fornito coeficienti di regressione lineare accettabili: R2 = 0,9950 e R2 = 0,9994 rispettivamente. La precisione intraday e l’accuratezza sono state calcolate mediante la stima del coefficiente di variazione (%) e del bias (n=10) per tre controlli a concentrazioni diverse (0,5 μg/mL, 2,5 μg/mL, 7,5μg/mL). Il limite di quantificazione é stato determinato pari a 0,5 μg/mL. La mediana e la concentrazione media di GHB nel gruppo 2 (non assuntori di GHB esogeno) sono risultate pari a 3,55 e 3,49 μg/mL rispettivamente. Il 52,90% dei soggetti appartenenti al gruppo 1(soggetti trattati con GHB) hanno presentato una concentrazione urinaria inferiore al cut-off utilizzato pari a 10 μg/mL.ConclusioniIn assenza di un test immunochimico rapido per la determinazione di GHB urinario, l’analisi LC/MS/MS puó rappresentare una valida alternativa sia in campo clinico che tossicologico. Il 52,90% dei soggetti trattati con GHB ha presentato una concentrazione urinaria di GHB compresa tra 2,95 e 8,50 μg/mL, ossia inferiore a 10 μg/mL. Si ritiene quindi che un cut-off pari a 10 μg/mL sia troppo alto e che aumenti il rischio di ottenere campioni falsi negativi.