Angelo Groppi

Effect of bleaching on ethyl glucuronide in hair: An in vitro experiment

INTRODUCTION Ethyl glucuronide in hair (HEtG) has recently gained great attention, because of its high sensitivity and specificity in the diagnosis of chronic alcohol abuse. Due to its high polarity hydrophilicity, a strong hair treatment followed by a shampooing may lead to removal/degradation of this molecule from hair matrix. AIM To set up an in vitro study in order to evaluate the ability of bleaching of modifying HEtG test results. METHODS Thirty hair samples from teetotalers (n=5), social drinkers (n=4) and heavy drinkers (n=21), after an informed written consent, were collected and divided longitudinally into four aliquots. The first aliquot was kept untreated and was processed following the method routinely used in our lab for the determination of HEtG (double washing with methanol/dichloromethane, overnight incubation in water, and LC-MS/MS analysis, LLOQ: 3pg/mg). To the other three aliquots a commercially available bleaching solution was applied, according to the manufacturers instructions. One out of the three aliquots was submitted to the analysis by following the same procedure used for the untreated sample. The other two were submitted to a purification step before LC-MS/MS analysis, by using two different SPE cartridges (aminopropyl and dimethyl butylamine). RESULTS HEtG levels in the untreated samples from social drinkers and heavy drinkers ranged from 7.7 to 149.0pg/mg. All the samples from teetotalers tested negative. The treated samples processed without any SPE extraction and with aminopropyl cartridges showed a relevant ion suppression for both EtG and D(5)-EtG (IS) signals. Samples treated with the bleaching solution and extracted with dimethyl butylamine cartridge allowed to sensitively reduce ion suppression (less than 35%) and to verify that EtG, after a strong treatment like bleaching, completely disappears. CONCLUSIONS This in vitro study showed that HEtG disappears from hair matrix after a strong hair treatment. It is not clear whether the mechanism involved is chemical degradation or physical removal from the damaged keratinic matrix. However, owing to the highly hydrophilic character of the compound, the second mechanism seems more likely to occur. Finally, bleaching solutions could lead to a heavy ion suppression of this metabolite that may be avoided by using an SPE purification before instrumental analysis.

Fully-automated systematic toxicological analysis of drugs, poisons, and metabolites in whole blood, urine, and plasma by gas chromatography–full scan mass spectrometry

The availability of automated, rapid and reliable methods for the systematic toxicological analysis (STA) of drugs and poisons in biosamples is of great importance in clinical and forensic toxicology laboratories. Gas chromatography-continuous scan mass spectrometry (GC-MS) possesses a high potential in STA because of its selectivity and identification power. However, in order to develop a fully automated STA method based on GC-MS two main obstacles have to be overcome: (a) sample preparation is rather sophisticated owing to the need to isolate analytes from the aqueous matrix and to allow a correct GC repartition of polar analytes; (b) the large amount of information collected within a single analysis makes it difficult to isolate relevant analytical information (mass spectra of analytes) from the chemical noise. Using a bench-top GC-MS system equipped with a laboratory robot for sample preparation (the Hewlett-Packard 7686 PrepStation) and an original method for mass spectral purification, a fully automated STA procedure was developed involving isolation of drugs from the sample (whole blood with minimal pretreatment, plasma, urine) by means of solid-phase extraction, derivatization (trimethylsilylation) of the acidic-neutral and of the basic extracts, GC-MS analysis, processing of data, and reporting of results. Each step of the procedure, and the method for data analysis in particular, can be easily integrated with other existing STA methods based on GC-MS.

Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol

This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother-infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography-tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography-mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2 nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother-infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs.

Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption

This study was designed with the aim to compare sensitivity and specificity of ethyl glucuronide in hair (HEtG) and carbohydrate-deficient transferrin (CDT) in serum as markers of heavy drinking. Eighty-six volunteers, including teetotalers, social, and heavy drinkers, were interviewed to evaluate their ethanol daily intake (EDI) during the last 2-week and 3-month periods. HEtG determination was performed by a fully validated LC-MS-MS procedure and ranged from <LOD (2 pg/mg) to 890.5 pg/mg. CDT was measured by immunonephelometry or by HPLC. Sensitivity and specificity of the two markers as indicators of an EDI higher than 60 g/day were calculated, with cut-off at 27 pg/mg (HEtG) and 2.5% (CDT). Considering the EDI of the last 2 weeks, HEtG showed equal selectivity (0.93 for both HEtG and CDT-immunonephelometry; 0.70 for both HEtG and CDT-HPLC) and 2 times the sensitivity of either of the two CDT methods (1.00 vs. 0.44 for CDT-immunonephelometry; 0.96 vs. 0.50 for CDT-HPLC). The same difference in performances but with higher absolute sensitivity and selectivity values for HEtG were observed considering the EDI of the last 3-months (selectivity: 1.00 for both HEtG and CDT-immunonephelometry, 0.89 and 0.78 for HEtG and CDT-HPLC, respectively; sensitivity: 1.00 vs. 0.47 for CDT-immunonephelometry; 0.98 vs. 0.51 for CDT-HPLC). Our results indicate that HEtG, as compared to CDT measured using different methods, is a selective marker of ethanol heavy chronic use providing considerably higher sensitivity.

Testing ethylglucuronide in maternal hair and nails for the assessment of fetal exposure to alcohol: comparison with meconium testing.

Background: The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioral, and/or learning disabilities that are included in the term fetal alcohol spectrum disorder. The measurement of ethylglucuronide (EtG) in alternative biological matrices, including neonatal and maternal hair, neonatal meconium, and maternal nails, is receiving increasing interest for the accurate evaluation of the in utero exposure to alcohol. Objective: To evaluate the correlation between EtG in maternal hair and nails with EtG in neonatal meconium to further explore the suitability of these biomarkers in disclosing prenatal exposure to ethanol. Methods: A total of 151 maternal hair strands (0–6 cm), nail clips (2–6 mm), and corresponding neonatal meconium and nails samples were obtained from neonatal wards of 4 Mediterranean public hospitals: Rome, Florence, and Belluno in Italy and Barcelona in Spain. Hair, nails, and meconium were analyzed for the presence of EtG by validated liquid chromatography mass spectrometry assay. Meconium was also analyzed for the presence of fatty acid ethyl esters (FAEEs) as a complementary biomarker of potential in utero exposure to alcohol. Results: Eighteen newborns resulted in utero exposed to maternal alcohol consumption by FAEE testing in meconium with EtG values between 0.5 and 1.5 nmol/g. Unfortunately, none of these cases were confirmed by the presence of EtG in maternal hair and nails samples, which resulted all negative to this biomarker. Discussion and Conclusions: The results confirm that FAEEs and EtG in meconium are the best biomarkers to assess in utero exposure to maternal alcohol. EtG in hair and nails are not good biomarkers to disclose alcohol consumption lower than on daily basis and lower than 1–2 alcoholic units per day.

Liquid chromatography with tandem mass spectrometric detection for the measurement of ethyl glucuronide and ethyl sulfate in meconium: new biomarkers of gestational ethanol exposure?

A liquid chromatography tandem mass spectrometric (LC-MS/MS) method with postcolumn addition of acetonitrile for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium was developed and validated using pentadeuterated EtG and pentadeuterated EtS as internal standards. The analytes were extracted from the matrix by acetonitrile, concentrated by solid phase extraction, separated using a reversed-phase chromatographic column, and quantified within 9 minutes. Lower limits of quantification were 5 and 1 ng/g meconium for EtG and EtS, respectively. Calibration curves were linear from lower limits of quantifications to 500 ng/g, with a minimum r2 > 0.999. At 3 concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 78.7% and 96.8% for EtG and between 72.1% and 95.6% for EtS. Inaccuracy was better than 8.1%, with intra-assay and interassay imprecision better than 7.2% and 10.5%, respectively. Matrix effects (ion suppression/enhancement) were found to be negligible. The analytes of interest were stable at room temperature, at 4°C, when exposed to 3 freeze-thaw cycles, and when stored at −20°C for up to 6 months. This sensitive and specific method was used to assess the presence of these alcohol biomarkers in meconium samples from 2 different city cohorts.

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography-mass spectrometry

A rapid and reliable gas chromatographic-mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether-n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.

Population Baseline of Meconium Ethyl Glucuronide and Ethyl Sulfate Concentrations in Newborns of Nondrinking Women in 2 Mediterranean Cohorts.

The detection of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium has been investigated recently as an alternative to meconium fatty acid ethyl esters (FAEEs) measurement as an objective estimate of prenatal alcohol exposure, independent of maternal self-reporting. We report the results of the first study conducted to investigate the concentrations of EtG and EtS in meconium from newborns with and without intrauterine exposure to ethanol, defined by questionnaire and meconium FAEEs concentration. FAEEs, EtG, and EtS were quantified by liquid chromatography tandem mass spectrometry in meconium samples obtained from the Arcispedale Santa Maria Nuova, Reggio Emilia, Italy (n = 80) and from the Hospital del Mar in Barcelona, Spain (n = 105). Median EtG and EtS values in meconium from newborns without intrauterine exposure to ethanol varied between 0.100 and 0.140 nmol/g and 0.010 and 0.020 nmol/g in Reggio Emilia and Barcelona samples, respectively. In meconium from newborns with uncertain prenatal ethanol exposure, the EtG median value was 0.160 nmol/g in the Italian cohort and 0.250 nmol/g in the Spanish one. The median EtS concentration was 0.020 in both cohorts. EtG and EtS median values in 5 meconium samples from newborns of heavily drinking mothers were 7.240 nmol/g and 0.033 nmol/g, respectively. A positive cutoff of 2.0 nmol/g for EtG yielded the best sensitivity and specificity (100%) to discriminate for true prenatal exposure to ethanol. It was not possible to establish a proper cutoff for EtS because of the low number of positive samples. Based on our results, meconium EtG can be proposed as an alternate biomarker for intrauterine alcohol exposure. In contrast to the 7 FAEEs, EtG is just one molecule that could be screened in meconium samples from all newborns by a simple, low-cost, easy-to-perform immunoassay, which can be routinely applied in neonatology wards for the early diagnosis of prenatal exposure to ethanol.

Ethyl-glucuronide and ethyl-sulfate in placental and fetal tissues by liquid chromatography coupled with tandem mass spectrometry.

The aim of this study was to develop a method for the determination of ethyl-glucuronide (EtG) and ethyl-sulfate (EtS), two direct ethanol metabolites, in early placental and fetal human tissues, as potential biomarkers of transplacental ethanol transfer from the mother to the fetus. Placental and fetal tissue samples were obtained from women undergoing voluntary termination of pregnancy at 12 weeks of gestation. Samples were deproteinized and directly injected into a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) system. Limits of detection of 13.0 and 23.0 pmol/g and lower limits of quantification of 22.0 and 40.0 pmol/g were reached for EtG and EtS, respectively. Inter- and intraday imprecision and accuracy were always lower than 15%. The method was applied to 70 samples (35 placentas and 35 fetal tissues). Of 35 samples, 4 samples collected from 4 women tested positive for EtG and EtS, always showing higher concentrations for EtG. The placenta/fetal tissue ratio for EtG was 2.9 ± 0.9, whereas EtS showed a ratio of 1.7 ± 0.7. Preliminary results suggest that these metabolites are present in both tissues. Further studies should now corroborate the hypothesis, not yet confirmed, that transplacental transfer of ethanol takes place not only for the parent compound but also for EtG and EtS.

Comparison of extraction procedures for benzodiazepines determination in hair by LC-MS/MS.

INTRODUCTION The use of a LC-MS/MS system for benzodiazepines detection remarkably increased the analytical sensitivity of these drugs in biological matrices, in particular in non-conventional ones such as hair. Since the amount of hair sample available for the analysis is frequently limited and, moreover, it needs to be checked for many other drugs and compounds of forensic interest, it is important to develop a sample preparation procedure able to detect either benzodiazepines and as many as possible other substances. The aim of this study was to compare the sensitivity of two different hair sample preparation procedures for benzodiazepines detection in hair. METHODS About 20mg hair, previously washed with organic solvent and cut into small pieces, were ultrasonicated with a phosphate buffer (pH 8.4) up to 1h and then extracted with dichloromethane/diethyl ether. The organic solvent was then dried under nitrogen flow and samples were reconstituted with 60μl methanol. Finally a 5μl aliquot was injected in the LC-MS/MS system. The second procedure consisted of an ultrasonication of hair samples in 700μl of methanol. Samples were then directly analyzed. Both the methods were fully validated. RESULTS Thirty-five compounds among benzodiazepines and their metabolites were screened using both the procedures. The methods fulfilled all the validation parameters and were applied on either spiked blank hair and real positive samples. While phosphate extraction allowed to reach a LOQ for almost all the substances ranging from 0.1 to 5pg/mg, thus guaranteeing to evaluate even a single dose administration (as confirmed by real positive cases) the sensitivity of the methanol extraction showed a LOQ ranging from 1 to 20pg/mg, still enough to assess a therapeutic use of almost all the benzodiazepines; yet the methanolic incubation allows a simple and rapid analytical procedure due to the direct injection of the extraction solvent. CONCLUSION Even though a methanol extraction procedure for benzodiazepines determination is useful for forensic toxicological purposes also when a wider range of substances is needed and in case of a small amount of hair available, it is advisable to prefer a phosphate extraction when detection of a single dose administration is required.