Claudia Mortali

A survey of Italian and Spanish neonatologists and paediatricians regarding awareness of the diagnosis of FAS and FASD and maternal ethanol use during pregnancy

BackgroundEthanol is the most widely used drug in the world and a human teratogen whose consumption among women of childbearing age has been steadily increasing. There are no Italian or Spanish statistics on ethanol consumption during pregnancy nor any information regarding prevalence of fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). There is also a reasonable suspicion that these two diseases are underdiagnosed by professionals from the above-reported countries. The objectives of this study were: 1) to evaluate the experience, knowledge and confidence of Italian and Spanish neonatologists and paediatricians with respect to the diagnosis of FAS and FASD, and 2) to evaluate professionals awareness of maternal drinking patterns during pregnancy.MethodsA multiple-choice anonymous questionnaire was e-mailed to Italian neonatologists registered in the mailing list of the corresponding Society and administered to Italian and Spanish paediatricians during their National Congress.ResultsThe response rate was 16% (63/400) for the Italian neonatologists of the National Society while a total of 152 Spanish and 41 Italian paediatricians agreed to complete the questionnaire during National Congress. Over 90% of the surveyed physicians declared that FAS is an identifiable syndrome and over 60% of them identified at least one of the most important features of FAS. Although over 60% Italian responders and around 80% Spanish responders were aware that ethanol use in pregnancy is dangerous, approximately 50% Italian responders and 40% Spanish ones allowed women to drink sometimes a glass of wine or beer during pregnancy.Neonatologists and paediatricians rated confidence in the ability to diagnosis FAS and FASD as low, with over 50% responders feeling they needed more information regarding FAS and FASD identification in newborn and child.ConclusionsItalian and Spanish neonatologists and paediatricians do not feel confident about diagnosing FAS and FASD. More training is needed in order to accurately diagnose ethanol use during pregnancy and correctly inform pregnant women on the consequences on the newborn.

Development and validation of a liquid chromatography–tandem mass spectrometry assay for hair analysis of atomoxetine and its metabolites: Application in clinical practice ☆

Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate. A procedure based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine - 4 hydroxyATX - and N-desmethylatomoxetine - des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2ml 1M NaOH at 45°C. Then, analytes were extracted from alkaline solution with two different 2ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water-60% 5mM ammonium acetate, 50mM formic acid, 4mM trifluoroacetic acid in acetonitrile-water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring. The method was linear over the concentration range 0.2-50ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0ng/mg hair and from 0.3 to 1.0ng/mg, respectively. Notwithstanding the absence of any dose-hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.

Pediatric exposure to drugs of abuse by hair testing: monitoring 15 years of evolution in Spain

Hair testing is a useful tool to investigate the prevalence of unsuspected chronic exposure to drugs of abuse in pediatric populations and it has been applied to three different cohorts of children from Barcelona, Spain along fifteen years to evaluate eventual changes in this exposure. Children were recruited from three independent studies performed at Hospital del Mar (Barcelona, Spain) and approved by the local Ethics Committee. Hair samples were collected from the first 187 children cohort (around 4 years of age) in 1998, from the second 90 children cohort (1.5–5 years of age) in 2008 and from the third 114 children cohort (5–14 years of age) in 2013. Hair samples were analysed for the presence of opiates, cocaine, amphetamines, and cannabis by validated methodologies using gas or liquid chromatography-mass spectrometry. Familiar sociodemographics and eventual consumption of drugs of abuse by parents, and caregivers were recorded. Hair samples from 24.6% children in 1998 were positive for any drug of abuse (23.0% cocaine), 25.5% in 2008 (23.3% cocaine), and 28.1% in 2013 (20.1% cocaine and 11.4% cannabis). In none of the cohorts, parental sociodemographics were associated with children exposure to drugs of abuse. The results of the three study cohorts demonstrated a significant prevalence of unsuspected pediatric exposure to drugs of abuse which mainly involved cocaine maintained along fifteen years in Barcelona, Spain. We recommend to be aware about unsuspected passive exposure to drugs of abuse in general population and to use general or selected hair screening to disclose exposure to drugs of abuse in children from risky environments to provide the basis for specific social and health interventions.

Determination of different recreational drugs in sweat by headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME GC/MS): Application to drugged drivers.

A procedure based on headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography/mass spectrometry (GC/MS) has been developed for the determination of most commonly used drugs of abuse in sweat of drivers stopped during roadside controls. DrugWipe 5A sweat screening device was used to collect sweat by a specific pad rubbed gently over forehead skin surface. The procedure involved an acid hydrolysis, a HS-SPME extraction for drugs of abuse but Δ(9)-tetrahydrocannabinol, which was directly extracted in alkaline medium HS-SPME conditions, a GC separation of analytes by a capillary column and MS detection by electron impact ionisation. The method was linear from the limit of quantification (LOQ) to 50ng drug per pad (r(2)≥0.99), with an intra- and inter-assay precision and accuracy always less than 15% and an analytical recovery between 95.1% and 102.8%, depending on the considered analyte. Using the validated method, sweat from 60 apparently intoxicated drivers were found positive to one or more drugs of abuse, showing sweat patches testing as a viable economic and simple alternative to conventional (blood and/or urine) and non conventional (oral fluid) testing of drugs of abuse in drugged drivers.

Segmental hair analysis to assess effectiveness of single-session motivational intervention to stop ethanol use during pregnancy

BACKGROUND This study aimed to test the effectiveness of single-session motivational intervention to stop ethanol use during pregnancy using segmental hair analysis of ethyl glucuronide to objectively verify drinking behavior before and after intervention. METHODS 168 pregnant women attending Hospital del Mar (Barcelona, Spain) for antenatal visit were included in the study and randomly assigned to one of two conditions: single-session motivational intervention (MI; N=83) or single-session educational control condition (ECC; N=85). Ethyl glucuronide was measured in maternal hair divided into three segments of 3 cm each corresponding to the three different gestation trimesters by a validated liquid chromatography tandem mass spectrometry method. Concentrations of EtG<7 pg/mg, between 7 and 30 pg/mg and ≥30 pg/mg in each segment were used to assess total abstinence, repetitive moderate drinking and chronic excessive consumption in the previous three months. RESULTS About a third of pregnant women self-reporting no ethanol consumption during gestation showed hair EtG values corresponding to ethanol drinking. Single-session MI helped in decreasing alcohol consumption during pregnancy as assessed by lower hair EtG concentrations in 2nd and 3rd trimesters. However, it did not significantly increase complete abstinence in pregnant women who previously showed hair EtG compatible with ethanol consumption. CONCLUSIONS Pregnant women did not correctly self reported ethanol consumption during gestation, while hair EtG was essential to correctly identify drinking patterns. Single-session MI was not enough to stop ethanol use during pregnancy. Interventions at any visit during pregnancy are strongly recommended.

Hair testing of propofol by liquid chromatography tandem mass spectrometry and azo-coupling derivatization

Propofol is a short-acting intravenous hypnotic-amnesiac agent used in the general anesthesia. In recent years propofol has gained importance for recreational use (being able to give euphoria, relaxation, sexual hallucinations and disinhibition) and for suicidal purposes. For these reasons, forensic toxicology interest in this substance has increased as well, taking into account the chance of its detection in hair. In this study, a new method in liquid chromatography-tandem mass spectrometry for hair detection of propofol was applied in two real cases for clinical and forensic purposes. This method consists in propofol derivatization with a diazonium salt from aniline and a liquid-liquid extraction with dichloromethane and ethyl acetate. Lower limit of quantification in negative ionization mode is 0.1 pg/mg. In Case 1, a segmental analysis was carried out on a sample collected from a female declaring self-administration. Propofol was found in the second and third segment at concentration of 0.21 and 0.11 ng/mg. In case 2, propofol was detected at 0.50 ng/mg in pubic hair from a male underwent a gastroscopy. In this study we validated and successfully applied a new versatile, sensitive and efficient method for hair detection of propofol even after a single administration.

Hair and urine testing to assess drugs of abuse consumption in couples undergoing assisted reproductive technology (ART).

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.

The NASOROSSO (Rednose) Project: An Italian Study on Alcohol Consumption in Recreational Places

The Nasorosso project of the Italian Youth Department and the National Institute of Health, aimed to raise awareness about drinking and driving under the influence of alcohol among club goers with a series of initiatives. Within the framework of the project, blood alcohol concentration (BAC) was measured on 106,406 individuals before and after clubbing in 66 different recreational sites from 11 Italian provinces, over 16 months. Participating individuals were interviewed regarding sociodemographic and environmental characteristics and alcohol intoxicated people were offered to be taken home. The BAC median at the club entry was 0.26 g/L with 65.3% subjects showing a BAC value under the driving legal limit of 0.5g/L. At the exit from clubs, BAC median value rose to 0.44 g/L and subjects with BAC value under the legal limit decreased to 54.9%. Being male, aged between 18 and 34 years with a diploma, being a drinker and entering the disco with a BAC already beyond the legal limit predicted a BAC value beyond 0.5 g/L at exit from the recreational place. Conversely, being a driver, being a student and exiting from the disco before 4 a.m. reduced the probability of having a BAC higher than 0.5 g/L at the end of the night. Health policies to prevent harmful use of alcohol in young people should continue to offer targeted information/ prevention; in order to steadily increase the awareness of the dangers and the damages of excessive use of alcohol.

Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry Assay for Determination of Endogenous GHB and GHB-Glucuronide in Nails

Background: The short chain fatty acid gamma-hydroxybutyric acid (GHB) is a precursor, and the metabolite of gamma-aminobutyric acid is commonly used as an illegal recreational drug of abuse. Methods: An ultra-high-performance liquid chromatography tandem mass spectrometry was developed and validated for endogenous GHB and its glucuronide in nails, to complement hair in forensic contexts for a retrospective detection of psychotropic drugs consumption. Results: GHB endogenous values for children and adolescents, adult females, and adult males in fingernails ranged from 0.3 to 3.0, 3.2, and 3.8 ng/mg, respectively, and toenails values ranged from 0.3 to 1.8, 2.0, and 2.4 ng/mg, respectively. In the three different groups, values of GHB in fingernails were statistically higher than those in toenails. GHB glucuronide could only be detected in finger nails with values ranging from 0.08 to 0.233, 0.252 and 0.243 in children and adolescents, adult females and adult males, respectively. Conclusions: The validated method was efficaciously applied to real finger and toe nails specimens from a population of males and females non GHB consumers. A preliminary cut-off of 5.0 ng/mg nail for endogenous GHB and 0.5 ng/mg for endogenous GHB-Gluc in the general population was proposed.