Fetal and Infant Effects of Maternal Opioid Use during Pregnancy: A Literature Review including Clinical, Toxicological, Pharmacogenomic, and Epigenetic Aspects for Forensic Evaluation.

The two primary classes of opioid substances are morphine and its synthetic derivative, heroin. Opioids can cross the placental barrier, reaching fetal circulation. Therefore, at any gestational age, the fetus is highly exposed to pharmacologically active opioid metabolites and their associated adverse effects. This review aimed to investigate all the studies reported in a timeframe of forty years about prenatal and postnatal outcomes of opioid exposition during pregnancy. Clinical and toxicological aspects, as well as pharmacogenetic and epigenetic research focusing on fetal and infant effects of opioid use during pregnancy together with their medico-legal implications are exposed and discussed.

Validation and application of a method for the quantification of 137 drugs of abuse and new psychoactive substances in hair.

INTRODUCTION: In the dynamic universe of new psychoactive substances (NPS), the identification of multiple and chemically diverse compounds remains a challenge for forensic laboratories. Since hair analysis represents a gold-standard to assess the prevalence of NPS, which are commonly detected together with classical drugs of abuse (DoA), our study aimed at developing a wide-screen method to detect and quantify 127 NPS and 15 DoA on hair. MATERIALS AND METHODS: A multi-analyte ultra-high performance liquid chromatography mass spectrometry method for the identification and quantification of 127 NPS (phenethylamines, arylcyclohexylamines, synthetic opioids, tryptamines, synthetic cannabinoids, synthetic cathinones, designer benzodiazepines) and 15 DoA in hair samples was developed. A full validation was performed according to the European medicines Agency (EMA) guidelines, by assessing selectivity, linearity, accuracy, precision, limit of quantification (LOQ), limit of detection (LOD), matrix effect and recovery. As a proof of the applicability, the method was applied to 22 authentic hair samples collected for forensic purposes. RESULTS: Successful validation was achieved, by meeting the required technical parameters, for 137 compounds (122 NPS and 15 DoA), with LOQ set at 4 pg/mg for 129 compounds, at 10 pg/mg for 6 and at 40 pg/mg for 2. The method was not considered validated for 5 NPS, as LLOQ resulted too high for a forensic analysis (80 pg/mg). Among authentic forensic samples, 17 tested positive for DoA, and 10 to NPS, most samples showing positivity for both. Detected NPS were ketamine and norketamine, 5-MMPA, ritalinic acid, methoxyacetyl fentanyl, methylone and RCS-4. CONCLUSION: The present methodology represents an easy, low cost, wide-panel method for the quantification of 122 NPS and 15 DoA, for a total of 137 analytes, in hair samples. The method can be profitably applied by forensic laboratories. Similar multi-analyte methods on the hair matrix might be useful in the future to study the prevalence of NPS and the co-occurrence of NPS-DoA abuse.

External hair contamination from cannabis and "light cannabis" delivered by smoking and vaping: An in vitro study.

External contamination of hair by cannabis smoking requires a careful evaluation in forensic toxicology. Medical and recreational cannabis are increasingly consumed by e-cigarettes, which give rise to side-stream vapor. Moreover, products containing low Δ9-tetrahydrocannabinol (Δ9-THC) and rich in cannabidiol (CBD) started spreading legally. The goal of the present study was to assess whether hair analysis could allow to distinguish the type of delivered product, with low or high Δ9-THC, and the delivering mode, by smoking or vaping. Contamination of blank hair was mimicked by in vitro exposure to low- (0.4%) and high-Δ9-THC (9.7%) products delivered by smoking and vaping within a small confined system. Cannabis vaping extracts were prepared to deliver identical target Δ9-THC doses. Eighty samples were analyzed by ultrahigh-performance liquid chromatography mass spectrometry and quantified for Δ9-THC and CBD. After contamination by cannabis smoking, THC levels were in line with past in vitro and in vivo studies. Samples exposed to cannabis (169.30 ng/mg) showed significantly higher Δ9-THC than hair exposed to "light cannabis" (35.54 ng/mg), and the opposite was seen for the CBD/Δ9-THC ratio. Hair contaminated by vaping or smoking did not show a statistically different Δ9-THC content. Under our in vitro conditions, hair analysis might allow to discriminate whether external contamination is determined by products containing low or high Δ9-THC, but not the delivering mode. More research is needed in real-life conditions, to see whether the same also applies to the interpretation of forensic casework.

Cross-Species Transcriptomics Analysis Highlights Conserved Molecular Responses to Per- and Polyfluoroalkyl Substances.

In recent decades, per- and polyfluoroalkyl substances (PFASs) have garnered widespread public attention due to their persistence in the environment and detrimental effects on the health of living organisms, spurring the generation of several transcriptome-centered investigations to understand the biological basis of their mechanism. In this study, we collected 2144 publicly available samples from seven distinct animal species to examine the molecular responses to PFAS exposure and to determine if there are conserved responses. Our comparative transcriptional analysis revealed that exposure to PFAS is conserved across different tissues, molecules and species. We identified and reported several genes exhibiting consistent and evolutionarily conserved transcriptional response to PFASs, such as ESR1, HADHA and ID1, as well as several pathways including lipid metabolism, immune response and hormone pathways. This study provides the first evidence that distinct PFAS molecules induce comparable transcriptional changes and affect the same metabolic processes across inter-species borders. Our findings have significant implications for understanding the impact of PFAS exposure on living organisms and the environment. We believe that this study offers a novel perspective on the molecular responses to PFAS exposure and provides a foundation for future research into developing strategies for mitigating the detrimental effects of these substances in the ecosystem.

Infection Induced Fetal Inflammatory Response Syndrome (FIRS): State-of- the-Art and Medico-Legal Implications-A Narrative Review.

Fetal inflammatory response syndrome (FIRS) represents the fetal inflammatory reaction to intrauterine infection or injury, potentially leading to multiorgan impairment, neonatal mortality, and morbidity. Infections induce FIRS after chorioamnionitis (CA), defined as acute maternal inflammatory response to amniotic fluid infection, acute funisitis and chorionic vasculitis. FIRS involves many molecules, i.e., cytokines and/or chemokines, able to directly or indirectly damage fetal organs. Therefore, due to FIRS being a condition with a complex etiopathogenesis and multiple organ dysfunction, especially brain injury, medical liability is frequently claimed. In medical malpractice, reconstruction of the pathological pathways is paramount. However, in cases of FIRS, ideal medical conduct is hard to delineate, due to uncertainty in diagnosis, treatment, and prognosis of this highly complex condition. This narrative review revises the current knowledge of FIRS caused by infections, maternal and neonatal diagnosis and treatments, the main consequences of the disease and their prognoses, and discusses the medico-legal implications.

Routine Photography of Injuries: A Comparison Between Smartphone Cameras and Digital Single-Lens Camera-A Pilot Study.

ABSTRACT: Ten lesions were photographed with an entry-level (HUAWEI P smart 2019), a midrange (Samsung Galaxy S8) and a high range (Apple Iphone XR) smartphone camera and with a digital single-lens camera (DSLC). Images were independently rated by 3 pathologists, based on comparison to the real lesion and "visual impact." Difference of perceptual lightness coordinates between smartphones and the criterion standard (DSLC) was calculated.The highest ranking for adherence to reality was obtained with DSLC, while the highest ranking for visual impact was obtained with the Iphone. The color representation better reflecting the criterion standard (DSLC) was obtained for the entry-level smartphone.All the devices allow to assess the general features (ie, the color, the shape, and the main characteristics) of an injury during a forensic autopsy. However, results might be different when photos are obtained in suboptimal, such as low-light, conditions. Moreover, images acquired through a smartphone camera might be unsuitable for later image exploitation, such as enlargement of a portion of the image to magnification of a detail, which seemed not relevant when the photo was shot. Only a raw image, acquired using a dedicated camera and deactivating images manipulation software, might allow the preservation of the true data.

Development and validation of a fast UPLC-MS/MS screening method for the detection of 68 psychoactive drugs and metabolites in whole blood and application to post-mortem cases.

We report a rapid and sensitive LC-MS/MS method that allows the simultaneous detection of 68 commonly prescribed antidepressants, benzodiazepines, neuroleptics, and metabolites in whole blood with a small sample volume after a rapid protein precipitation. The method was also tested on post-mortem blood from 85 forensic autopsies. Three sets of commercial serum calibrators containing a mix of prescription drugs of increasing concentration were spiked with red blood cells (RBC) to obtain 6 calibrators (3 "serum calibrators" and 3 "blood calibrators"). Curves obtained from serum calibrators and from blood calibrators were compared using a Spearman correlation test and by analyzing slopes and intercepts, to assess if the points from six calibrators could be plotted together in a single calibration model. The validation plan included interference studies, calibration model, carry-over, bias, within-run and between-run precision, limit of detection (LOD), limit of quantification (LOQ), matrix effect and dilution integrity. Four deuterated Internal Standards (Nordiazepam-D5, Citalopram-D6, Ketamine-D4 and Amphetamine-D5) and two different dilutions were assessed. Analyses were performed using an Acquity UPLC® System coupled with triple quadrupole detector Xevo TQD®. The degree of agreement with a previously validated method was calculated on whole blood samples of 85 post-mortem cases, by performing a Spearman correlation test with a Bland-Altman plot. Percentage error between the two methods was evaluated. Slopes and intercepts of curves obtained from serum calibrators and from blood calibrators showed a good correlation, and the calibration model was built plotting all points together. No interferences were found. The calibration curve appeared to provide a better fit of the data using an unweighted linear model. Negligible carry-over was observed, and very good linearity, precision, bias, matrix effect and dilution integrity were achieved. The LOD and the LOQ were at the lower limits of the therapeutic range for the tested drugs. In a series of 85 forensic cases, 11 antidepressants, 11 benzodiazepines and 8 neuroleptics were detected. For all analytes, a very good agreement between the new method and the validated method was demonstrated. The innovation of our method consists in the use of commercial calibrators, readily available to most forensic toxicology laboratories, for the validation of a fast, inexpensive, wide-panel LC-MS/MS method that can be used as a reliable and accurate screening for psychotropic drug in postmortem samples. As observed in the implementation on real cases, this method could be profitably applied in forensic cases.

Insights in opiates toxicity: impairment of human vascular mesenchymal stromal cells.

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated ß-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.

Sudden cardiac death related to left coronary artery anomalies including hypoplasia and anomalous origin with retro-aortic course.

Congenital anomalies of the coronary arteries are a rare condition with an incidence of 0.3-1.3% in the general population. Clinically, sometimes these anomalies increase the risk of myocardial ischemia, which can present with a wide spectrum of symptoms, from angina to sudden cardiac death (SCD). This case report is about the SCD of an 8-year-old male, in apparent good health, during a football training. Although basic life support maneuvers were performed timely from bystanders and medical staff, the automated external defibrillator (AED) was not used. Autopsy revealed multiple left coronary artery (LCA) anomalies: origin from a separate ostium in the right sinus of Valsalva, slit-like shape of the ostium, acute angle take-off of the LCA from the aorta, retro-aortic course and focal coronary hypoplasia of some branches of the LCA. Microscopic examination revealed diffuse ischemic consequences at a different stage of tissue repair and mild multifocal lymphocytic infiltration. No other significant elements were detected at post-mortem examination. We discuss the forensic evaluation about the cause and the manner of death, considering also the modality of the resuscitation attempts and the claimed malpractice, as often occurs in case of sudden unexpected death in young athletes.

Importance of dashboard camera (Dash Cam) analysis in fatal vehicle-pedestrian crash reconstruction.

The reconstruction of dynamic of traffic injuries remains a challenge in forensic pathology and is often based on circumstantial data. Dash Cams are digital video recorders which can be located inside a vehicle and continuously record the view through the windscreen, thus providing objective evidence. Here we present the case of a traffic crash in which a pedestrian was hit by an articulated lorry. The analysis of a video recorded from a Dash Cam retrieved inside the vehicle during the death scene investigation (DSI) was crucial in the reconstruction of the manner of death. Indeed, the death, which was initially assumed to be accidental, was finally deemed as a suicide on the basis of the video recording, which showed an intentional and sudden rush of the victim to the middle of the roadway. Advantages and disadvantages of the use of Dash Cams will be discussed, focusing on the profound differences in the related national and international regulations. Based on the present case, in traffic crashes, the search for Dash Cams during the DSI may be recommended and the video recordings should be analyzed in the setting of a multidisciplinary and multimodal evaluation of the case, for a proper reconstruction of the facts.

Integrated multidisciplinary approach in a case of occupation related planned complex suicide-peticide.

The combined event of a suicide and the killing of a pet has been hardly explored in forensic literature, but it is not rare at all. In the reported case the dead corpse of a 60-year old mentally ill woman and the carcass of a dog were found on the bed of a private apartment. In light of death scene investigation, necroscopic examination and toxicological analyses death was attributed to licit drug intoxication and self-strangulation after lethal poisoning of the dog. Due to the presence of two lethal means (cervical noose and drugs), acting in chronological order, the event was classified as a secondary complex suicide. Moreover, the woman, although mentally ill, was a psychiatrist. Thus, her professional background, namely the easy access to psychiatric drugs together with her knowledge of drug composition and properties lead to suppose an occupation related suicide. The comprehensive analysis of all the available information, including death scene investigation, occupational, necroscopic and toxicological data, resulted of the utmost importance for a proper reconstruction of the events and are recommended in complex cases such as occupation related planned complex suicides combined to the killing of pets.

Ethyl Glucuronide Concentration in Hair of Detainees: A Preliminary Study.

Through the measurement of ethyl glucuronide in hair (hETG), it is possible to assess chronic alcohol abuse over time. In this paper, we present a study on hETG in Italian prison inmates. Analyses were performed by LC-MS according to a previously published method. Results were evaluated using the cut-offs established by the Society of Hair Testing. Positives samples (ETG > 30 pg/mg) accounted for 6% of all subjects, with concentrations ranging from 42 pg/mg up to 270 pg/mg, abstinent subjects (ETG < 7 pg/mg) accounted for 88%, and moderate alcohol consumption (7 < ETG < 30 pg/mg) for 6% of the subjects. No females displayed ETG values above 30 pg/mg. Among positive samples, only two subjects did not declare heavy alcohol consumption and were found strongly positive at 210 and 270 pg/mg. To the best of our knowledge, this represents the first study on ETG hair concentration on prison inmates.

Possible fatal hyperthermia involving drug abuse in a vehicle: case series.

Major interplaying causes of heat stroke (HS) and fatal hyperthermia are climate, physical activity, artificial extreme ambient temperatures, confinement in a small compartment, and effects of drugs and chemicals, combined with predispositions and complications. A common and unfortunate cause of HS is vehicular hyperthermia (VH) death. Hyperthermia in a vehicle is a type of environmental thermal disorder, involving victim's susceptibility, confinement with restraint, and possible influences of drugs and poisons, including exhaust gas containing complex fumes, carbon dioxide and monoxide. Whereas VH typically occurs when babies or young children are being left unattended in parked vehicles in direct sunlight, it has been reported only anecdotally for adult subjects. Three cases of adult fatal VH will be herein presented. In each presented case the corpse was found enclosed in a vehicle on spring/summer days. During crime scene investigation (CSI) psychoactive substances were found near to the corpses leading to the suspect of a suicidal fatal drug intoxication. Basing on this misleading suspect in Case 2 and 3 a forensic expert was not charged for the CSI and a fatal VH was not suspected nor properly investigated. Later, a comprehensive autopsy, including biochemical and toxicological analyses, excluded a death related to natural causes, fatal intoxications, ketoacidosis and traumas. On the other hand, the reconstruction of the temperature, the humidity, the heat index and the related risk of HS allowed the diagnosis of fatal VH in all the reported cases. In particular, death occurred because of the long-lasting stay into a hot parked vehicle which was facilitated from self-administration of psychoactive drugs with related neuro-depression. This case series confirms that a comprehensive CSI followed by an autopsy including histology, biochemical and toxicological analysis remains mandatory in cases of forensic interest, as well as when a corpse is found enclosed in a vehicle. Anyway, sometimes the diagnosis of heat-related fatalities remains a medley of investigative and medicolegal observations.

"Tampering to Death": A Fatal Codeine Intoxication Due to a Homemade Purification of a Medical Formulation.

Many homemade tamper processes of medical codeine formulations are available on selected "forums" on the Internet, where recreational codeine users claim to be able to purify codeine by removing additives, such as acetaminophen, to avoid or limit adverse effects. In this work, it is reported and discussed a fatal case of codeine intoxication. The findings of objects such as jars, filters, and tablets, and amounts of unknown liquid material at the death scene investigation suggested a fatal codeine intoxication after the tampering procedure called "cold water extraction." Toxicological results obtained from the analysis of both the nonbiological material and the body fluids of the decedent integrated with the information collected at the death scene investigation confirmed the above-mentioned hypothesis. This report underlines the importance of a tight interconnection between criminalistics and legal medicine to strengthen the identification of the cause of death and the reconstruction of the event.

Recent advances in the application of CE to forensic sciences, an update over years 2009-2011.

The present article reviews and comments the applications of capillary electrophoresis in the different areas of forensic sciences covering the time from the second half of 2009 until the first part of 2011, being the latest update of previous reviews covering the years from 2001 to 2009. Numerous articles reporting applications of capillary electrophoresis to analytical problems of potential interest for the forensic researchers and scientists can be found in the most qualified journals of analytical chemistry, analytical biochemistry, pharmacology, toxicology, laboratory medicine, human genetics, etc. However, the present review has been focused on discussing only the most relevant examples of analytical applications of capillary electrophoretic and electrokinetic techniques published in the following fields: (i) illicit and abused drugs, (ii) ions and small molecules of forensic interest, (iii) proteins and peptides of forensic interest, (iv) dyes and inks, (v) forensic DNA. The present review collects and comments on 60 references.

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.

Capillary zone electrophoresis (CZE) coupled to time-of-flight mass spectrometry (TOF-MS) applied to the analysis of illicit and controlled drugs in blood.

A new method for the determination of illicit and abused drugs in blood by capillary zone electrophoresis-electrospray ionization-time-of-flight mass spectrometry is proposed, in view of its application in clinical and forensic toxicology. The analytes (methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine, 6-acethylmorphine, benzoylecgonine) were separated with capillary zone electrophoresis by applying 15 kV within 25 min, in an uncoated fused-silica capillary (75 microm x 100 cm) using a 25 mM ammonium formate electrolyte solution (pH 9.5). The capillary electropherograph was coupled to time-of-flight mass spectrometry through an orthogonal electrospray ionization source, with a coaxial sheath liquid interface. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 0.5% formic acid delivered at 4 microL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically < or =5 ppm) and by matching of the isotopic pattern. Under optimized conditions, linearity was assessed in the range 10-2000 ng/mL, with correlation coefficients between 0.9744 and 0.9982 for all the analytes. LODs were in the range of 2-10 ng/mL (S/N > or =3) and LOQs of 10-30 ng/mL. The CVs (tested at 40 and 800 ng/mL in biological matrix) were below 2.97% for migration times and below 14.61% for peak area ratios (analyte/internal standard). Blood samples were extracted by using a liquid-liquid extraction procedure and injected under field-amplified sample stacking conditions. The method was successfully applied to real cases.

Implementation and performance evaluation of a database of chemical formulas for the screening of pharmaco/toxicologically relevant compounds in biological samples using electrospray ionization-time-of-flight mass spectrometry.

Electrospray ionization (ESI)-time-of-flight (TOF) MS enables searching a wide number of pharmaco/toxicologically relevant compounds (PTRC) in biosamples. However, the number of identifiable PTRC depends on extension of reference database of chemical formulas/compound names. Previous approaches proposed in-house or commercial databases with limitations either in PTRC number or content (e.g., few metabolites, presence of non-PTRC). In the frame of development of a ESI-TOF PTRC screening procedure, a subset of PubChem Compound as reference database is proposed. Features of this database (approximately 50,500 compounds) are illustrated, and its performance evaluated through analysis by capillary electrophoresis (CE)-ESI-TOF of hair/blood/urine collected from subjects under treatment with known drugs or by comparison with reference standards. The database is rich in parent compounds of pharmaceutical and illicit drugs, pesticides, and poisons and contains many metabolites (including about 6000 phase I metabolites and 180 glucuronides) and related substances (e.g., impurities, esters). The average number of hits with identical chemical formula is 1.82 +/- 2.27 (median = 1, range 1-39). Minor deficiencies, redundancies, and errors have been detected that do not limit the potential of the database in identifying unknown PTRC. The database allows a much broader search for PTRC than other commercial/in-house databases of chemical formulas/compound names previously proposed. However, the probability that a search retrieves different PTRC having identical chemical formula is higher than with smaller databases, and additional information (anamnestic/circumstantial data, concomitant presence of parent drug and metabolite, selective sample preparation, liquid chromatographic retention, and CE migration behavior) must be used in order to focus the search more tightly.

Broad-spectrum toxicological analysis of hair based on capillary zone electrophoresis-time-of-flight mass spectrometry.

The coupling of capillary electrophoresis-electrospray ionization and time-of-flight mass spectrometry, combining efficiency and speed of separation with high mass accuracy and fast scanning capability, was for the first time applied to the determination of drugs of abuse (amphetamine, methamphetamine, MDA, MDMA, ephedrine, cocaine, morphine, codeine) and their metabolites in hair (6-MAM, benzoylecgonine). Experimental conditions were as follows. Separation: voltage 15 kV, uncoated fused-silica capillary (75 microm ID, 100 cm total length), running electrolyte 25 mM ammonium formate, pH 9.5, field-amplified sample stacking injection. Forensic drugs could be identified by exact mass determination (mass accuracy typically < or = 5 ppm) and by match of the isotopic pattern. The method was fully validated, showing limit of detections (LODs) suitable for the determination of all the compounds below the cut-off usually adopted for hair analysis (0.1 ng/mg). Analytical precision in real matrices (tested at 0.1 and 1.0 ng/mg) was typically characterized by CV's < or = 24% in both intra-day and day-to-day experiments. Quantitative determination was also tested by using a single internal standard (folcodine). Results, although with a moderate accuracy, conceivably depending on the lack of deuterated internal standards, proved useful for diagnostic use of the results from hair analysis. A single liquid-liquid extraction procedure was applied for all analytes, allowing the detection of a broad spectrum of basic drugs and their major metabolites.

Hair analysis for illicit drugs by using capillary zone electrophoresis-electrospray ionization-ion trap mass spectrometry.

In forensic toxicology, hair analysis has become a well established analytical strategy to investigate retrospectively drug abuse histories. In this field, gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry are currently used, often after preliminary screening with immunoassays. However, on the basis of previous applications to pharmaceutical analysis, capillary zone electrophoresis coupled to ion trap mass spectrometry looks also highly promising. The purpose of the present work was the development of a simple and rapid CZE-MS method for sensitive and quantitative determination of the main drugs of abuse and their metabolites (namely, 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethampthetamine (MDMA), benzoylecgonine, ephedrine and cocaine) in human hair. Hair samples (100 mg) were washed, cut and incubated overnight in 0.1 M HCl at 45 degrees C, then neutralized with NaOH and extracted by a liquid-liquid extraction method. CZE separations were carried out in a 100 cm x 75 microm (I.D.) uncoated fused silica capillary. The separation buffer was composed of 25 mM ammonium formate, pH 9.5; the separation voltage was 15 kV. Electrokinetic injections were performed at 7 kV for 30 s under field amplified sample stacking conditions. ESI-ion trap MS detection was performed in the ESI positive ionization mode using the following conditions: capillary voltage 4 kV, nebulizer gas (nitrogen) pressure 3psi, source temperature 150 degrees C and drying gas (nitrogen) flow rate 8l/min. A sheath liquid, composed of isopropanol-water (50:50, v/v) with 0.5% formic acid, was delivered at a flow rate of 4 microl/min. The ion trap MS operated in a selected ion monitoring mode (SIM) of positive molecular ions for each drug/metabolite. Collision induced fragmentation was also possible. Nalorphine was used as internal standard. Under the described conditions, the separation of all compounds, except amphetamine/methamphetamine, MDA/MDMA and morphine/6-MAM was achieved in 20 min, with limits of detection lower than the most severe cut-offs adopted in hair analysis (i.e. 0.1 ng/mg). Linearity was assessed within drug concentration ranges from 0.025 to 5 ng of each analyte/mg of hair. Analytical precision was fairly acceptable with RSD's < or = 3.06% for migration times and < or = 22.47% for areas in real samples, in both intra-day and day-to-day experiments. On these grounds, the described method can be proposed for rapid, selective and accurate toxicological hair analysis for both clinical and forensic purposes.