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The determination of carbon monoxide (CO) and carboxyhemoglobin (COHb) is of utmost importance in forensic toxicology to determine the cause of death in cases of CO poisoning, fire, and explosions. To this end, reliable and updated analytical methods are required. In this paper, four different methods for the determination of carbon monoxide in postmortem blood samples were compared: (i) the spectrophotometric determination of COHb applying the method proposed by Rodkey and modified by Beutler-West, (ii) the spectrophotometric determination of CO using a micro-diffusion-based method, (iii) the determination of CO by gas chromatography coupled to a TCD detector, and (iv) the determination of COHb by blood gas analysis. Three postmortem blood samples were analyzed with all methods, and the results were comparable. The applied methodologies showed different features depending on the sensitivity, sample preparation, and volume. The HS-GC/TCD method in our hand was the most appropriate, on postmortem samples, and versatile to apply. Unfortunately, only a limited number of postmortem blood samples were available for this study due to the rarity of that kind of intoxication in our jurisdiction.

The identification of cannabis chemotypes at an early stage of a plant's growth, which is long before anthesis, has been intensively pursued in order to control the on-target selection of the cultivar type at the beginning of cultivation, so as to avoid economic and legal drawbacks. However, this issue has been systematically addressed by only few and relatively recent studies of analytical chemistry, possibly because result validations require long-term monitoring of the content and ratio of cannabinoids and terpenes in a great number of plant specimens suitably selected and grown. Here, we review the procedures, the chromatographic techniques and the statistics used in topical investigations during the past thirteen years. Through heterogeneous and not easily comparable approaches, they prove the feasibility of chemotypes safe determination within the first month of a plant's life.

Cannabis (Cannabis sativa L.) is a highly promising medicinal plant with well-documented effectiveness and growing use in the treatment of various medical conditions. Cannabis oils are mostly used in galenic preparations, due to their easy adjustment of the administration dose, together with the enhanced bioavailability of its active compounds. As stated by the Italian Law (9/11/2015, 279 Official Gazette), “to ensure the quality of the oil-based cannabis preparation, the titration of the active substance(s) should be carried out.” This study aims to represent the Italian panorama of cannabis oils, which were analyzed (8,201) to determine their cannabinoids content from 2017 to 2019. After application of the exclusion criteria, 4,774 standardized cannabis oils were included, which belong to different medicinal cannabis varieties and prepared according to different extraction methods. The concentration of the principal cannabinoids was taken into account dividing samples on the basis of the main extraction procedures and cannabis varieties. According to this analysis, the most substantial variations should be attributed to different cannabis varieties rather than to their extraction protocols. This study may be the starting point of preparatory pharmacists to assess the correct implementation of the preparation procedures and the quality of the extracts.

Recently, an increasing number of pharmacists had to supply medicinal products based on Cannabis sativa L. (Cannabaceae), prescribed by physicians to individual patients. Cannabis olive oil preparation is the first choice as a concentrated extract of cannabinoids, even though standardized operative conditions for obtaining it are still not available. In this work, the impact of temperature and extraction time on the concentration of active principles was studied to harmonize the different compounding methods, optimize the extraction process, and reduce the variability among preparations. Moreover, starting from the cannabis inflorescence, the effect of temperature on tetrahydrocannabinolic acid decarboxylation was evaluated. For the analysis, a GC/MS method, as suggested by the Italian Ministry of Health, and a GC/flame ionization detection method were developed, validated, and compared.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

In this paper, we describe the approach to the characterization of an unusual material seized by the judicial authority, near Brescia City in Northern Italy. Usual analyses such as thin-layer chromatography, gas chromatography (GC)–flame ionization detection, and GC/mass spectrometry (MS) did not show the presence of drugs of abuse, in particular amphetamine-like compounds. The major solid component was identified as cornstarch; then taking into account the strong aromatic scent of the seized material; a preliminary experiment for volatile organic compounds was carried out by headspace (HS)-GC/MS. This analysis tentatively evidenced the presence of 1-phenyl-2-propanone (P2P), an amphetamine precursor. Therefore, we developed and optimized a new analytical method for determination of P2P in seized materials by HS-GC/MS. We also synthesized P2P, with the permission of the Ministry of Health, to have it as reference standard, because of its being illegal and the difficulty in obtaining it. This case had some analogies with the cases referred to as “wet amphetamine” by the judicial authority, in which amphetamines are sold mixed with P2P. The possible use of the material could be the production of tablets made of cornstarch with an aromatic scent similar to that of amphetamines to deceive consumers and to sell them as a drug of abuse.

Short and medium fatty acids derived from either dietary sources, gut microbiota, and liver production might play a role in the modulation of metabolism and inflammation. The outcome of different autoimmune or inflammatory diseases could be related to microbiota composition and consequently fatty acids production. Their analytical detection, historically completed by GC, was herein investigated using a sensitive approach of LC-MS/MS with straightforward chemical derivatization, using 3-NPH, to the respective acylhydrazines. An isopropanol protein precipitation coupled to LC-MS/MS analysis allowed to separate and quantify butyric, valeric, hexanoic acid and their branched forms. The serum physiological ranges of short and medium chain fatty acids were determined in a heterogeneous healthy population (n = 54) from 18 to 85 years finding a concentration of 935.6 ± 246.5 (butyric), 698.8 ± 204.7 (isobutyric), 62.9 ± 15.3 (valeric), 1155.0 ± 490.4 (isovaleric) and 468.7 ± 377.5 (hexanoic) ng/mL respectively (mean ± SD). As expected, the biological levels in human serum are reasonably wide-ranging depending on several factors such as body-weight, gut microbiome dysbiosis, gut permeability, cardiometabolic dysregulation, and diet.

In the last 10 years, the abuse of designer drugs has proliferated due to the ease of purchase on the Internet. Our research laboratory collaborates with the Financial Police (GdF) analyzing drugs of abuse on behalf of the Court (Procura della Repubblica––Tribunale di Busto Arsizio). Nine products sold on the Internet and suspected to contain illegal substances were brought to our laboratory after a seizure at the Malpensa Airport in Northern Italy. After first routine analyses carried out by thin-layer chromatography (TLC) and gas chromatography/flame ionization detection (GC/FID), we were able to exclude all “classical” drugs and common new psychoactive substances (NPSs). This prompted us to deeply analyze the unknown product to identify it by different analytical techniques. The structure of the unknown compound was elucidated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), accurate mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. This characterization allowed us to identify the unknown substance as a new designer drug thiothinone, [2-(methylamino)-1-(2-thienyl)-1-propanone]. The X-ray powder diffraction (XRPD) pattern and the analytical data presented herein can provide valid support to forensic chemists who may encounter this “legal high” in caseworks. To our knowledge, this is the first scientific disclosure of thiothinone from dubious products.

A man was found dead in a hotel located near Rome (Italy). The man was still holding a syringe attached to a butterfly needle inserted in his left forearm vein. The syringe contained a cloudy pinkish fluid. In the hotel room the Police found a broken propofol glass vial plus four sealed ones, an opened NaCl plastic vial and six more still sealed, and a number of packed smaller disposable syringes and needles. An opened plastic bottle containing a white crystalline powder labeled as potassium cyanide was also found. Systematic toxicological analysis (STA), carried out on blood, urine and bile, evidenced only the presence of propofol in blood and bile. So the validated L-L extraction protocol and the GC/MS-TOF method for the confirmation of propofol in the biological fluids optimized in our laboratory was applied to blood, urine and bile. The concentration of propofol resulted to be 0.432 μg/mL in blood and 0.786 μg/mL in bile. The quantitative determination of cyanide in blood was carried out by microdiffusion technique coupled to spectrophotometric detection obtaining a cyanide concentration of 5.3 μg/mL. The quantitative determination was then confirmed by GC/NPD and the concentration of cyanide resulted to be 5.5 μg/mL in blood and 1.7 μg/mL in bile. Data emerging from autopsy findings, histopathological exams and the concentrations of cyanide suggested that death might be due to poisoning caused by cyanide, however, respiratory depression caused by propofol could not be excluded.

Cannabis light preparations are products derived or containing dried female inflorescences of Cannabis sativa belonging to Chemotype III (THC/CBD ratio1); the total THC (THC+THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation. In Italy the most recent law for industrial hemp (242/2016) states that only for farmers this limit is extended to 0.6 %. On the other hand, the Ministry of the Interior published a note stating that the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5 % constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determining the total amount of THC. To this end a reliable LC-UV analytical method was developed and validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD= 0.002 % for all cannabinoids) and limit of quantification (LOQ= 0.005 % for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3 % and Er% between ± 6 % were obtained. The linearity was proven in the concentration range 0.005-1 % for THC, THCA and CBN and 0.005 %-50 % for CBD and CBDA. The analytical method was applied to more than nine hundred cannabis light samples. Based on the law 242/2016, only 18 % of the crops are to be considered legal for the market (total THC0.2 %). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24 %) would be considered illegal (total THC0.5 %). On the other hand, the most of the inflorescences (58 %) have a total THC content comprised between 0.2 % and 0.5 %, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.

In vitro incubation with human liver microsomes of JWH-018, JWH-073, JWH-122 and for the first time 1-butyl-3-(1-(4-methyl)naphthoyl)indole (the 4-methylnaphthoyl analogue of JWH-073) was investigated to identify the principal metabolites of alkylindole synthetic cannabinoids, thus helping the discovering of synthetic cannabinoids abusers. The results obtained showed that the most abundant metabolites were mono-hydroxylated derivatives either on the alkyl chain (ω or ω–1 position) or on the indole (presumably in position 5 or 6) and naphthalene moieties. Moreover the extraction conditions of these derivatives from biological fluids, mainly plasma and urine spiked with commercially available metabolite standards, and the incubation procedure were investigated to obtain a fast, reliable and suitable extraction protocol to detect either the parent drugs or their metabolites by means of GC/MS.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

Methadone (MTH) concentrations in those dying of MTH toxicity totally overlap concentrations where the presence of MTH is only an incidental finding, making it very difficult to make distinctions in actual cases. A biomarker, be it anatomical or biochemical for MTH toxicity is badly needed, particularly if that markers were known to disrupt effective ventilation. Because the brainstem houses the regulatory centers for cardiorespiratory-control enters, it would seem to be the most likely anatomical site to seek abnormalities in cardiorespiratory control. Objective To locate and describe the cells of nucleus of the solitary tract (TS)(NTS) in human brainstem and determine if neuronal cell death, either necrotic or apoptotic, within the TS of humans is more common in deaths due directly to MTH toxicity than with in the solitary tract itself. Design, setting, participants This was a single cohort study of MTH related decedents autopsied at a large university hospital. Each decedent had a recent history of non medical/illicit MTH use and had been pronounced dead in the field, prior to ever reaching the hospital. Complete autopsy and complete toxicology testing were performed on the formalin fixed brains of each individual. Multiple blocks were prepared of the area of interest, namely the tissue lying immediately between the inferior and the super colliculi. This volume, by definition, would have included the area of the Rostral Ventrolateral Medulla (RVLM), the location of the TS. Immunohistochemistry studies utilizing caspase-9 reaction (a protease enzyme involved in the process of preprogrammed death) were performed in order to estimate the degree and proportion of neuronal apoptosis, and also access the degree of classical necrosis within the NTS. Main outcomes and measures The primary outcome measure was the presence or absence of neuronal apoptosis and/or necrosis within the NTS. Results Cells displaying evidence of early apoptosis and advanced apoptosis, consisting primarily of nuclear fragmentation, admixed with other neurons displaying the features of classic necrosis were found. Evidence of classic necrosis was identifiable in most of the controls, though minor degrees of apoptosis were identifiable with Caspase staining and quantitative image analysis of immunohistochemical stains. Conclusions and Relevance: Our study shows that neurons, primarily along the TS, but occasionally in other cell nuclei (even controls) are vulnerable, both to direct MTH toxicity (via apoptosis) and indirectly (via hypoxia leading to classical cell necrosis). When MTH is found to be present in significant concentrations, but apoptotic lesions are absent, it would be reasonable to assume that MTH was not primarily the cause of cardiorespiratory arrest.

Two cases of suspected acute and lethal intoxication caused by propofol were delivered by the judicial authority to the Department of Sciences for Health Promotion and Mother-Child Care in Palermo, Sicily. In the first case a female nurse was found in a hotel room, where she lived with her mother; four 10 mg/mL vials and two 20 mg/mL vials of propofol were found near the decedent along with syringes and needles. In the second case a male nurse was found in the operating room of a hospital, along with a used syringe. In both cases a preliminary systematic and toxicological analysis indicated the presence of propofol in the blood and urine. As a result, a method for the quantitative determination of propofol in biological fluids was optimized and validated using a liquid-liquid extraction protocol followed by GC/MS and fast GC/MS-TOF. In the first case, the concentration of propofol in blood was determined to be 8.1 μg/mL while the concentration of propofol in the second case was calculated at 1.2 μg/mL. Additionally, the tissue distribution of propofol was determined for both cases. Brain and liver concentrations of propofol were, respectively, 31.1 and 52.2 μg/g in Case 1 and 4.7 and 49.1 μg/g in Case 2. Data emerging from the autopsy findings, histopathological exams as well as the toxicological results aided in establishing that the deaths were due to poisoning, however, the manner of death in each were different: homicide in Case 1 and suicide in Case 2.

Solid phase microextraction coupled to headspace sampling and GC/MS technique was applied to the characterization of the volatile components of several Cannabis preparations (hashish). Different parameters of the analytical method (fiber, coating thickness, sampling and exposition temperatures, sample preparation) were evaluated to optimize the characterization of the volatile components. a-Pinene, s-myrcene, limonene, 4-carene, trans-3(10) caren-2-ol, 4,7,7-trimethylbicyclo [4.1.0] heptan-3-ol, caryophyllene, s-humulene, azulene, gurjunene, ledene and caryophyllene oxide were identified among the volatile components of all hashish preparations. Moreover, a suitable internal standard (nonane) was chosen, the reproducibility and linearity of the method were evaluated in order to carry out the quantitative determination of caryphyllene, the most abundant volatile terpene. Its quantity ranged from 800 to 3000 µg/g.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

5-Substituted 3-(diethoxyphosphoryl)isoxazoles and -2-isoxazolines were synthesized regioselectively by 1,3-dipolar cycloaddition of(diethoxyphosphoryl)formonitrile oxide to monosubstituted alkynes and alkenes. By applying this methodology to an N-(tert-butoxycarbonyl)-substituted allylglycine methyl ester, we prepared the precursors of two diastereomeric 3-phosphono-2-isoxazolin-5-yl-substituted amino acids, which are bioisosteres of potent NMDA receptor antagonists.

The novel adamantane derivative APICA (N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide) was recently identified as a cannabinomimetic indole of abuse. Despite its novel structure, APICA recalls cannabinomimetic indoles, such as representative member JWH-018.In present study, the effects of APICA (1-3 mg/kg, i.p.) were tested in C57BL/6J mice, in the Tetrad task which includes the assessment of: body temperature; locomotor activity and behavioural reactivity; nociception; motor coordination; declarative memory. Furthermore, pre-treatment with the CB1 antagonist AM251 (3 mg/kg, i.p.) or the CB2 antagonist AM630 (3 mg/kg, i.p.) was carried out to characterize APICA activity.Our results show that APICA was able to dose-dependently decrease locomotor activity and behavioural reactivity in the open field, whereas only the highest dose was able to induce hypothermia, analgesia, motor incoordination and recognition memory impairment, with respect to vehicle (p < 0.01; p < 0.001).The pretreatment with the CB1 antagonist AM251 elicited an increase in body temperature, total distance travelled in the open field, latency to fall down in the Rotarod, and a decrease in tail flick latency (p < 0.05; p < 0.01). On the other hand, pretreatment with AM630 did not induced significant differences on APICA effects.This study supports preliminary reports on APICA cannabinomimetic properties, extending its detrimental effects on cognitive function. Moreover, these properties can be attributed to the CB1 receptor activity, indicating APICA as a selective CB1 receptor agonist.

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.

Opioid switching is often used to improve the opioid response in patients with cancer experiencing poor analgesia or adverse effects. When switching between drugs with delayed effect because of pharmacokinetics or type of delivery, concerns exist about the correct timing of introducing the second drug after stopping the previous one. The aim of this study was to assess plasmatic changes of fentanyl and methadone underlying the clinical events occurring during opioid switching. Eighteen patients with cancer receiving transdermal fentanyl with uncontrolled pain and/or moderate to severe opioid adverse effects, were switched to oral methadone using an initial fixed ratio of 1:20. Fentanyl patches were removed and the first of three daily doses of methadone was started concurrently. Blood samples were obtained at intervals after removing the fentanyl patch, and at 5-hour intervals for the first 25 hours. The intensity of pain and the adverse effects were assessed before switching, the day after, and then daily up to dose stabilization. A successful switch was considered when the intensity of pain and distress score decreased at least of 33% of the basal value recorded before the switch, within a reasonable period of time. Complete blood samples were obtained in 16 patients. Methadone plasma concentration increased from 2 to 245 ng/mL, and fentanyl plasma concentration decreased from 15 to 8 ng/mL, 25 hours after. A successful switch was determined the day after in 7 patients, while 4 patients did not respond favorably (effective switching, 63%). Five patients were considered too terminal for an appropriate evaluation. No differences in plasma concentration pattern of the two opioids were found between patients considered responders and nonresponders. Conversion ratios between opioids at time of stabilization did not significantly change in comparison with the initial conversion ratio chosen. Starting methadone soon after removing fentanyl patches results in a rapid increase of methadone concentration, while the half-life of transdermal fentanyl is reached after 25 hours. As a result, the rapid achievement of a clinical effect is obtained avoiding distressing therapeutic holes in patients with a clinical condition, mainly characterized by poor pain control and severe adverse effects, requiring an immediate intervention.

The taxonomic identification of the biological material contained in the hallucinogenic mushrooms culture media, was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are present as basidiospores mixed in culture media and spore-bearing fruiting body are not present. This approach is very useful as it allows the unequivocal identification of potentially illicit material before the cultivation and it enables to stop the material to the Customs and to destroy it due to its dangerousness without cultivating the "grow-kits" and without instructing a criminal case. In fact, even if psilocin and psilocybin and the whole mushrooms are illegal in many countries, there is no specific indication in the law about the so called "grow-kits", containing the spores. To confirm the data obtained by the taxonomic identification, a simple, reliable, efficient LC-UV method, using tryptamine as internal standard, suitable for the forensic quali-quantitative determination of psilocin and psilocybin in hallucinogenic mushroom was optimized, validated and applied to the mushrooms grown after the cultivation of the grow-kits seized by the judicial authority, with the authorization of the Ministry of Health. A cation exchange column was used in a gradient elution mode (Phase A: 50mMK2HPO4; 100mM NaCl pH=3 Phase B: methanol). The developed method was linear over the calibration range with a R(2)>0.9992 for both the analytes. The detection and quantification limits were respectively 0.01 and 0.1μg/mL for psilocybin and 0.05μg/mL and 0.1μg/mL for psilocin and the intra- and inter-day precision was satisfactory (coefficients of variation

Biogenic polyamines, such as spermidine (SPD, NH 2 -(CH 2 )4-NH-(CH 2 ) 3 -NH2), are ubiquitous polycationic molecules which play a definitive role in many biological processes such as nucleic acid metabolism, protein synthesis, and cell growth. SPD is commonly used as an ingredient in hair lotions, because it seems to promote hair growth. This work describes a capillary GC method for quantitative determination of SPD in hair lotions using 1,6-diaminohexane as internal standard, a methyl silicone capillary column, and a flame ionisation detector. Aliquots of hair lotion were treated with an alkaline aqueous solution and internal standard was added. The emulsion was extracted with diethyl ether containing ethyl chloroformate. Ether extracts, evaporated to dryness and reconstituted in ethyl acetate, were analysed by capillary GC with flame ionisation detection. Validation took into account the specificity, linearity, precision, and accuracy of the analytical method: these parameters were valid for the quantitative determination of SPD in hair lotion.

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.

Biogenic polyamines, among which is spermidine (SPD, NH2-(CH2)4-NH-(CH2)3-NH2), are ubiquitous polycationic molecules that have a definitive role in many biological processes, such as nucleic acid metabolism, protein synthesis and cell growth. SPD is present in diet integrators because it seems to favour the hair growth. This work describes a capillary gas chromatographic (CGC) method for the quantitative determination of SPD in diet integrators using cadaverine internal standard (IS), a methyl siliconic capillary column and flame-ionization detector (FID). Diet tablets, containing SPD, are pulverized; an aliquot of powder is treated with an alkaline aqueous solution and added with IS. The suspension is extracted with diethyl ether containing ethyl chloroformate (ECF). The ether extracts, evaporated to dryness and reconstituted in ethyl acetate were analyzed in CGC/FID. Derivatives of polyamines with ECF were characterized in CGC/MS too. Validation has considered specificity, linearity, precision and accuracy of analytical method; this parameters are valid for the quantitative determination of SPD in diet integrators.

This study reports the results of the detection and quantitation of cocaine and its metabolites in liver tissues fixed in formalin and in the formalin solutions in which the same tissues were fixed. Toxicological analyses were performed on formalin-fixed liver samples from four cases of death of cocaine abusers and on formalin solutions (10% buffered, pH 7) in which the samples were preserved. Analyses carried out at the time of autopsy on body fluids and tissues allowed identification of cocaine and the metabolite benzoylecgonine. Liver tissue samples were preserved in formalin solutions for four weeks before analysis. Results only showed the presence of benzoylecgonine in the studied materials. The mean levels of recovery of benzoylecgonine in fixed tissues were 12.31% in liver and 84.47% in formalin from liver. Results indicated that benzoylecgonine has good stability, even in biological specimens subjected to chemical fixation.

To evaluate CoQ10 concentration a UPLC/MS-MS method for its analysis in plasma samples was developed. Before validation, the choice of the anticoagulant, of the Internal Standard (CoQ9; IS) and the stability of CoQ10 in plasma were evaluated. These determinations were necessary to assess the effect of the oral intake of CoQ10 in nine healthy volunteers after 10 days of treatment with a food supplement containing it. The basal levels of CoQ10 were determined and the analyses were repeated after four days to take into account the variability of endogenous substances. After oral intake, the increase in plasma concentration of CoQ10 resulted statistically significant (P

Objective: Post-mortem brain samples from 15 deceased patients whose death was heroin related, were analyzed to determine 6-monoacetyl-morphine (6-MAM) concentrations. The samples belonged to people died between 2008 and 2014. The first eight samples were also analyzed in 2012 to determine only morphine and codeine levels. Method: A GC/MS method was studied in order to enhance sensitivity, thus helping the determination of 6-MAM whose detection is in most cases difficult because of the complexity of the biological matrix. The analytical method was validated using deuterated internal standards (IS-D3, morphine-D3 and codeine-D3) and it showed adequate specificity, linearity, LOD, LOQ precision and accuracy for the determination of the analyte of interest. Results: 6-MAM was evidenced only in the more recent samples, thus pointing out its low stability. Its concentration ranged from 15.6 to 28.9 ng/g. Morphine and codeine was also determined and a comparison was carried out between the blood and the brain levels of the three analytes. Moreover a parallel was established between the concentrations of morphine and codeine found in the brain in 2012 and 2015. Conclusion: 6-MAM determination in the brain is particularly important when discriminating between morphine assumption and heroin abuse. In fact in the cases in which it is not detectable in the blood it can be present in the brain. It was noticed that the concentrations of morphine found in the brain in 2015 are higher respect to the levels of 2012; a possible explanation could be that 6-MAM originally present in the brain has hydrolyzed to morphine, thus increasing its levels.

The quali-quantitative determination of the principal components of “uva di Troia canosina” seed extracts by LC/ DAD analysis and the optimization of the extraction and purification processes for the development of an industrial nutraceutical product, are described. Two different fractions of seeds collected at different stages of fermentation were compared: “Tesi” 2 when there is a spontaneous stratification of the seeds at the bottom of the recipient and “Tesi” 4 at the end of fermentation. Percolation was applied and compared to maceration and the purification step carefully evaluated to obtain extracts free of contaminant species endowed with polyphenolic content comparable to commercial preparations such as Leucoselect® (Indena, SpA, Italy), Vitis Vinifera extract 95% (seeds), Vitis Vinifera dry extract 95%, Biovin grape seed and vinasse extract. (Farmalabor, Italy). In particular, “Tesi” 2 extract obtained by percolation and purified with a LLE extraction with ethyl acetate showed a polyphenolic content similar to Leucoselect®. From the quantitative analyses it was evident, as expected, that “Tesi”2 has a higher polyphenolic content compared to “Tesi” 4, because during vinification the must extracts polyphenols from the seeds. On the other hand, “Tesi” 4 is particularly convenient since it is easily obtained and very economical, being a waste product. The residual content of organic solvents (ethanol and ethyl acetate) and water was assessed in the grape extracts according to ICH rules by means respectively of HS/GC and Karl Fisher titration in order to meet the requirements for commercialization. Furthermore, the high molecular weight polyphenolic fraction of our extracts was investigated through gel permeation chromatography (GPC) and compared to that of Leucoselect®.

The taxonomic identification of mushrooms suspected to contain hallucinogenic active principles was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are difficult to identify because the morphologic identification is prevented, due to the bad conservation of the vegetable material. To confirm the presence of the illegal active principles, the optimization of a LC/MS-MS method for the qualitativequantitative analysis of psilocin and psilocybin in mushroom samples seized by the judicial authority is described. For the quantitative determination it was necessary to identify and synthesize a proper internal standard (IS, i.e., 5-hydroxy-N,N-diethyltryptamine), endowed with chromatographic features suitable for the analysis of the active principles. LC/MS-MS analysis evidenced that the amount of psilocybin ranged from 0.5 to 1.4% while that of psilocin from 1.3 to 2.5% (w/w), confirming literature data. The concentration of psilocin was higher in the cap and in the distal part of the stem (near to the soil) than in the part of the stem proximal to the cap. On the other hand the concentration of psilocybin was higher in the cap and in the proximal part, being lower in the distal part of the stem.

Objectives: The aim of this work was the quantitative determination of N,N-Dimethylacetamide (DMA) as crystallization solvent in samples of Diacerein. DMA is commonly used as a solvent in the chemical, agricultural and pharmaceutical industries. However, in order to ensure product quality and to protect patients from the potentially toxic properties, the substances used as active ingredients in therapeutic drugs should not contain high levels of residual solvents. Methods: LC is commonly used in the pharmaceutical industry to check DMA in pharmaceutical products, but in this work we were interested in validating and comparing LC/UV and GC/FID techniques for determining the presence of DMA in Diacerein Results: Both methods showed good linearity, precision and accuracy with comparable LOD and LOQ. Conclusion: The GC method, however, since it uses DMSO as an internal standard, has higher analytical versatility, thus allowing the qualitative and quantitative determination of DMA at lower levels than those obtained with LC.

Objectives: Acid-base dissociations constants of a series of poorly water-soluble nicotinic ligands designed as anti-inflammatory agents were determined in order to characterize the pharmacokinetic profile of this kind of ligands. Methods: pKas values were assessed by means of potentiometric and electrophoretic methods and investigated by computational protocols. Results: Both electrophoretic and potentiometric measurements produced reliable results. However, with the electrophoretic technique only an average value for close pKas was found, whereas the potentiometric method allowed determination of each pKa value in water-cosolvent mixtures. A theoretical treatment with various prediction programs - i.e. ADME Boxes v. 4.1, ACD/pKa DB and ACD/pKa GALAS - led in most cases to values which were not in accordance with the experimental ones. Conclusion: Electrophoretic and potentiometric techniques showed complementary features. Indeed, with capillary electrophoresis, the problem associated with the low water solubility of the studied samples could be easily overcome, although this technique did not allow to measuring all dissociation constants. In contrast, application of the potentiometric method afforded all the theoretical pKa values, although we had to perform the titrations in watercosolvent mixtures, a less precise, more laborious and time-consuming approach.

For a complete quantitative analysis of primary active constituents in Cannabis preparations Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN), we have compared two different chromatographic techniques, high-resolution gas chromatography (HRGC)/flame ionization detection (FID) and high-performance liquid chromatography (HPLC)/UV. The two different methods have been validated using crude drug (hashish) with methyloleate and tetraphenylethylene as internal standard for HRGC/FID and HPLC/UV, respectively.

Within a project aimed to reintroduce non-drug hemp cultivars in the Italian Po valley, for fibre but also high added-value nutraceutical production, investigation on locally grown plants has been performed, in order to assess their oil and metabolic content. This study provides useful information regarding three different hemp cultivars, from two sites, in view of their potential industrial application. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of ?-3 and ?-6 fatty acids, as requested for healthy foods. The alcoholic extracts, for which a high content of amino acids and phenolic compounds has been highlighted, could provide dietary supplements to help in preventing oxidative stress. By investigating the Carmagnola cultivar, six known and four new lignanamides have been identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants. © 2014 © 2014 Taylor & Francis.

Seeds and skins derived from the small berries of “uva di Troia canosina” grape, a Vitis vinifera variety autochthonous of Apulia region (South Italy), collected at four different stages (“Tesi” 1–4) of the fermentation process were extracted by means of a maceration. The extracts were purified and analyzed, in order to study the influence of fermentation over grape seed and skin polyphenolic content. Seed extraction was performed by a multistep maceration with two solvents: ethanol and acetone, the former giving the best results; moreover, the extracts were purified with pure ethyl acetate in order to enrich their polyphenolic content. On the other hand, skin extraction was achieved by a single-step maceration in methanol and a purification with a brominated synthetic adsorbent resin. The evaluation of the extraction yield and polyphenolic content was carried out by TLC, UV/VIS, and LC/DAD analyses. In the seed extracts, the characteristic polyphenols (catechin, epicatechin, and procyanidin B1 and B2) useful for the development of a nutraceutical product, endowed with antioxidant properties, were present, while no resveratrol was detected in “uva di Troia canosina” grape skin extracts, even in an LC/MS-MS analysis.

A capillary electrophoretic method, which allowed the detection and separation of the active principles of Catha edulis, i.e. cathinone, cathine and phenylpropanolamine, was developed. A suitable internal standard (nicotinamide), which permitted the quantification of the analytes reducing the variability of the migration times due to EOF changes, was identified. The analytical method was validated, assessing linearity, sensitivity and repeatability, showing optimal features for the analysis of the vegetable material. Moreover extraction conditions were investigated to achieve the exhaustion of the plant material in the fastest and most efficient way to meet the requirements of the Court.

The GC method previously reported by our research group for the analysis of the active principles of Catha edulis, i.e. cathine, cathinone and phenylpropanolamine, was considerably improved. N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) as derivatizing agent was employed, thus allowing an accurate determination of the analytes and a suitable internal standard for quantitative analyses (nicotinamide) was introduced. Moreover the chromatographic conditions were carefully studied to improve the separation of the alkaloids and sensitivity. To this end different chromatographic capillary columns and temperature gradients were investigated. The optimized GC method was validated and resulted adequate for the application in forensic analysis. Finally on behalf of the Tribunal, C. edulis vegetable material seized by the police in northern Italy was analyzed, the quantity of cathine ranging from 0.095 to 0.29%, the quantity of PPA from 0.010 to 0.21% and the quantity of cathinone from 0.025 to 0.374% of the weight of the vegetable material.

The results of the analysis of some herbal products and powders adulterated with alkylindoles recovered on the Italian market between 2010 and 2011 are reported. Besides the well-known alkyindoles JWH-018 and JWH-073, other derivatives such as JWH-250 and AM-694 have been detected and for the first time in Italy 1-butyl-3-(1-(4-methyl)naphthoyl)indole (compound 1), the 4-methylnaphthoyl analogue of JWH-073. This compound as well as the other alkylindoles has been synthesized and characterized by (1)H NMR, (13)C NMR, DSC, GC/MS, and elemental analysis. The quantitative analyses of the samples have been carried out by means of the GC/FID method developed in our laboratory for the analysis of herbal high products containing naphthoylindoles; the quantity of the cannabimimetic substances ranged from 6 mg/g to 47 mg/g.

Brain distribution pattern of “street” heroin metabolites (morphine and codeine) was investigated in two fatalities due to “acute narcotism”. A suitable sample pretreatment prior to solid-phase-extraction was developed to achieve a good recovery of the analytes and to eliminate the interfering species. After derivatization with MSTFA, samples were analyzed by GC/MS. Specificity, accuracy, precision and linearity of the method were evaluated; LOD and LOQ were, respectively, 10 ng/25 ng for morphine and 5 ng/10 ng for codeine. This method was applied to the analysis of six brain areas (hippocampus, frontal lobe, occipital lobe, nuclei, bulb and pons) coming from two cases of heroin-related deaths. No evidence of accumulation of metabolites in a specific brain region was found.

In the last years, all the vegetable material supposed to belong to the Catha Edulis species, seized at the Malpensa and Orio al Serio airports, were analyzed in our laboratory on behalf of the Tribunals of Busto Arsizio and Bergamo, respectively. After a preliminary botanic examination, the quali-quantitative determination of the active principles cathinone, cathine and phenylpropanolamine (PPA) was carried out by means of GC/MS and GC/FID techniques, which meet the requirements of the forensic analyses. We developed a fast, effective and reliable derivatization procedure which allowed to simultaneously detect cathine and PPA, whose discrimination is mandatory since PPA is not a psychoactive agent. Cathine was distributed in the various parts of the plant (leaves and stems) and its quantity ranged from 0.03% to 0.17% of the weight of the vegetable material; PPA was not detected in the twigs and its quantity in the leaves ranged from 0.07% to 0.16%. The quantitative determination of cathinone was carried out directly on the methanol solution after maceration of the vegetable material, its quantity ranging from 0.02% to 0.10%. No significant difference in the content of the two active principles was found between the fresh and the dried material.

A case of fatal intoxication from metformin is presented. The decedent was an obese 58-year-old-woman with type II diabetes, in whom severe lactic acidosis secondary to metformin accumulation was precipitated by acute renal failure. She had been on metformin 500 mg twice a day. Postmortem blood was deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected into high-performance liquid chromatography system. Separation was performed on a analytical 125 × 4 mm i.d. RP-8 column. The wavelength was set at 235 nm. The mobile phase was acetonitrile (40%), sodium lauryl sulfate, and sodium dihydrogen phosphate adjusted to pH 5.1 (60%) at a flow rate of 1.0 mL/min. The concentration of metformin in postmortem blood was 77.3 μg/mL. The qualitative result was also confirmed by LC/APCI/MS/MS analysis.

Maternal smoking during pregnancy has been associated with a reduced content of n-3 long-chain PUFA (LC-PUFA) in breast milk, thereby reducing the intake of key nutrients by the infants. We postulated that the mammary gland is affected by maternal smoking in the process of n-3 LC-PUFA secretion into milk. This prompted us to investigate the effects of cigarette smoke on the synthesis of n-3 LC-PUFA in vitro by using a line of healthy epithelial cells from the human mammary gland, MCF-10A. Cells were exposed to cigarette smoke under controlled conditions by adding to the medium aliquots of horse serum containing smoke components, as analyzed by GC-MS. The major findings concern the inhibition of both the conversion of the precursor 14C-ALA (alpha-linolenic acid) to n-3 LC-PUFA and of the A5 desaturation step (assessed by HPLC analysis with radiodetection of n-3 FAME) following exposure to minimal doses of smoke-enriched serum, and the dose-dependent relationship of these effects. The data indicate that exposure to cigarette smoke negatively affects the synthesis of n-3 LC-PUFA from the precursor in mammary gland cells.

Pinacidil (CAS 85371-64-8) is an antihypertensive drug of the class of agents called “potassium channel openers”. The pharmacokinetics of were studied after repeated oral administration of a new slow release tablet formulation (Pindac™) as compared with the standard slow release capsule formulation in healthy volunteers. Eighteen healthy volunteers (3 males and 15 females), aged from 22 to 48 years, and 49 to 95 kg in weight, were given a 12.5 mg dose of each formulation every 12 h for 7 days on two occasions, in a randomized, cross-over trial with at least two weeks interval between trials. Blood samples were drawn immediately before drug administration on the morning of days 1, 2, 3, 4, 5, 6 and 7 and 0.5, 1, 2, 3, 4, 6, 8, 12, 24 and 36 h after the last drug administration. Blood pressure, heart rate, and respiratory function were assessed at adminission and on the morning of day 0 (baseline), 2 and 7, before drug administration, and at 4, 24 and 36 h after the final administration. Pinacidil plasma levels were determined by an HPLC method. Both formulations elicited similar raductions of systolic and diastolic pressures from 4 h after administration, but did not change heart rate. The main model-independent pharmacokinetic parameters of pinacidil (Cmax, Tmax, AUC, MRT), as well as the absorption and elimination half-lives were similar with the two formulations. During the study there were no complaints of side-effects with either of the formulations. One advantage of the new formulation as compared to the capsules is that the tablets can be cut easily and the dosage adapted to a patient’s needs.

The gas chromatographic identification of barbiturates isolated from biological material is not optimal, and due to their polar nature, it is difficult to obtain good resolution on most columns. To improve resolution, flash alkylation appeared to be a reasonable alternative. Methyl, ethyl, and butyl derivatives were prepared for various drugs found in the "acid-neutral" extract and the Kovats retention indices (Ir) were determined on SE-30, OV-17, and SP-2250. The Ir of the alkylated derivatives of the barbiturates are highly reproducible. One column and two alkylated derivatives can be used for the qualitative analysis of barbiturates in extracts of biological material, even when the Stas-Otto extract is used.