Hair testing applied to the assessment of in utero exposure to drugs: Critical analysis of 51 cases of the University Hospital of Verona.

The work discusses the results of hair and urine testing performed in 51 cases of suspected in utero drug exposure handled at the University Hospital of Verona from 2016 to 2022. On the day of birth or the day after birth, urine from mother and newborn (UM and UN) and hair from mother (HM), newborn (HN) and father (HF), if possible, were collected. Urine underwent immunoassay and GC-MS analysis, whereas hair underwent LC-MS/MS and GC-MS/MS analysis. In 50 out of 51 cases, HM and/or HN were available. In 92% of them, hair testing resulted in a positive, often (>50% cases) for more than one class of substance. The most detected substances were cocaine, opiates, methadone and cannabinoids. Maternal segmental analysis showed a prevalent decreasing concentration trend during pregnancy in case of positivity for one class of substances, whereas, as expected, a neatly prevalent increasing trend in the case of positivity for more than one class of substances. In nine cases, HF was also available, resulting in all being positive, usually for the same classes of substances identified in HM, thus questioning parental responsibility. In 33 cases, urine samples from the mother or newborn were also collected. Of them, 27 cases (82%) tested positive, showing peri-partum drug consumption and then confirming the severity of the addiction. Hair testing showed to be a reliable diagnostic tool to investigate in utero drug exposure because of the possibility of obtaining a complete picture of maternal addictive behaviour and family background, thanks to segmental maternal hair analysis and father hair testing.

Drug screening by using the Toxtyper™ 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™ 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.

Lactate determination in human vitreous humour by capillary electrophoresis and time of death investigation.

Forensic inquests, particularly, in assessing time since death currently recognize the importance of the analysis of vitreous humour (VH) biomarkers. Present research, studies, and validates the determination of lactate (La) in VH by CZE with indirect UV detection. The BGE (pH 8.9) consisted of Tris buffer (37 mM) containing 4-methoxybenzoic acid (4 mM) and alkyl-trimethyl-ammonium bromide (1.2 mM). Each VH specimen was diluted with a butyric acid solution (internal standard 0.057 mM) and La and butyrate were separated within 3-5 min (30 kV). The La LOQ and LOD were 4 and 2 mM, respectively. The calibration curve linearity ranged from 4 to 80 mM; intra- and interruns precisions were less than 10% for standard as well as for VH specimen, respectively. To investigate postmortem interval (PMI) and VH lactate level correlation, human VH specimens were collected during autopsy (n = 40) and stored at -20°C until assay. La levels ranged from 16 to 42 mM; PMI values ranged from 10 to 141 h. La (mM) and PMI (h) correlation was statistically significant (r2 = 0.527; p < 0.05). In conclusion, the present CZE analysis is efficacious to determine VH La as a biomarker for PMI investigation.

Vitreous humor endogenous compounds analysis for post-mortem forensic investigation.

The chemical and biochemical analysis of bodily fluids after death is an important thanatochemical approach to assess the cause and time since death. Vitreous humor (VH) has been used as a biofluid for forensic purposes since the 1960s. Due to its established relevance in toxicology, a literature review highlighting the use of VH with an emphasis on endogenous compounds has not yet been undertaken. VH is a chemically complex aqueous solution of carbohydrates, proteins, electrolytes and other small molecules present in living organisms; this biofluid is useful tool for its isolated environment, preserved from bacterial contamination, decomposition, autolysis, and metabolic reactions. The post-mortem analysis of VH provides an important tool for the estimation of the post-mortem interval (PMI), which can be helpful in determining the cause of death. Consequently, the present review evaluates the recent chemical and biochemical advances with particular importance on the endogenous compounds present at the time of death and their modification over time, which are valuable for the PMI prediction and to identify the cause of death.

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.

Use of finger-prick dried blood spots (fpDBS) and capillary electrophoresis for carbohydrate deficient transferrin (CDT) screening in forensic toxicology.

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger-prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger-prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 µL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening.

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×50µm (ID) uncoated fused silica capillaries, using a background electrolyte composed of 150mM sodium phosphate pH 4.4 added with 5mM 2-(hydroxypropyl)-ß-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.

Micellar electrokinetic chromatography: a new simple tool for the analysis of synthetic cannabinoids in herbal blends and for the rapid estimation of their logP values.

For the first time a capillary separation based on micellar electrokinetic chromatography (MEKC) with diode array detection (DAD) was developed and validated for the rapid determination of synthetic cannabinoids in herbal blends. Separations were carried out on a 30 µm(ID) × 40 cm uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 25 mM sodium tetraborate pH 8.0, 30 mM SDS and n-propanol 20% (v/v). Separations were performed at 30 kV. Sample injection conditions were 0.5 psi, 10s. Diazepam and JWH-015 were used as internal standards. The determination of the analytes was based on the UV signal recorded at 220 nm, corresponding to the maximum wavelength of absorbance of the molecules, whereas peak identification and purity check were also performed on the basis of the acquisition of UV spectra between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 25 min without any significant interference from the matrix. Linearity was assessed within a concentration range from 5 to 100 µg/mL. The intra-day and inter-day imprecision values were below 2.45% for relative migration times and below 10.75% for relative peak areas. The present method was successfully applied to the direct determination of synthetic cannabinoids in 15 different herbal blend samples requiring only sample dilution. In addition, the developed MEKC separation was also applied to estimate the octanol/water partition coefficients (logP) of these new and poorly known molecules.

[Assessment of fitness to drive in correlation with narcotic and psychotropic drug use. Epidemiologic study in Verona]. / La valutazione dell'idoneità alla guida in relazione all'uso di sostanze stupefacenti e psicotrope. Studio epidemiologico della casistica veronese.

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.

Screening for pharmaco-toxicologically relevant compounds in biosamples using high-resolution mass spectrometry: a 'metabolomic' approach to the discrimination between isomers.

High-resolution mass spectrometry (HRMS) enables the identification of a chemical formula of small molecules through the accurate measurement of mass and isotopic pattern. However, the identification of an unknown compound starting from the chemical formula requires additional tools: (1) a database associating chemical formulas to compound names and (2) a way to discriminate between isomers. The aim of this present study is to evaluate the ability of a novel 'metabolomic' approach to reduce the list of candidates with identical chemical formula. Urine/blood/hair samples collected from real positive cases were submitted to a screening procedure using ESI-MS-TOF (positive-ion mode) combined with either capillary electrophoresis or reversed phase liquid chromatography (LC). Detected peaks were searched against a Pharmaco/Toxicologically Relevant Compounds database (ca 50,500 compounds and phase I and phase II metabolites) consisting of a subset of PubChem compounds and a list of candidates was retrieved. Then, starting from the mass of unknown, mass shifts corresponding to pre-defined biotransformations (e.g. demethylation, glucuronidation, etc.) were calculated and corresponding mass chromatograms were extracted from the total ion current (TIC) in order to search for metabolite peaks. For each candidate, the number of different functional groups in the molecule was automatically calculated using E-Dragon software (Talete srl, Milan, Italy). Then, the presence of metabolites in the TIC was matched with functional groups data in order to exclude candidates with structures not compatible with observed biotransformations (e.g. loss of methyl from a structure not bearing methyls). The procedure was tested on 108 pharmaco-toxicologically relevant compounds (PTRC) and their phase I metabolites were detected in real positive samples. The mean list length (MLL) of candidates retrieved from the database was 7.01 +/- 4.77 (median, 7; range, 1-28) before the application of the 'metabolomic' approach, and after the application it was reduced to 4.08 +/- 3.11 (median 3, range 1-17). HRMS allows a much broader screening for PTRC than other screening approaches (e.g. library search on mass spectra databases). The 'metabolomic' approach enables the reduction of the list of candidate isomers.

Rapid and direct determination of creatinine in urine using capillary zone electrophoresis.

BACKGROUND: In clinical medicine creatinine determination is used for the diagnosis of renal diseases and muscular dysfunctions. Also, in forensic toxicology creatinine concentration is used as a standardization tool for the quantitative measurement of therapeutic or illicit drugs and xenobiotics in urine. The present work was aimed at developing a robust and reliable CE determination of creatinine in urine, meeting the needs of simplicity, rapidity and low cost required by routine toxicological screening. METHODS: The optimized buffer electrolyte was composed of 200 mM phosphate and 200 mM acetic acid (pH 3.8). The separation capillary (50 microm x 10 cm of effective length) was made of naked fused silica. Separations were carried out under 25 kV potential. Urine samples were diluted 20 fold with water and directly injected. Direct UV absorption detection at 200 nm was employed. RESULTS: Linearity was assessed in the range 0.2-32 mM. Precision tests resulted in CV's % below 0.56% for migration times and below 3.78% for peak area ratios (analyte/I.S.). CONCLUSIONS: The described CE creatinine assay meets the strict requirements of forensic analysis and looks particularly useful to test the possible adulteration or dilution of urine samples undergoing toxicological screening.