Giacomo Luca Visconti

Capillary gas chromatographic determination of spermidine in diet integrators.

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.

Validation Study of Analysis of 1-Phenyl-2-Propanone in Illicit Methamphetamine Samples by Dynamic Headspace Gas Chromatography Mass Spectrometry

A new method based on dynamic headspace sampling (DHS) coupled to GC/MS analysis was developed, optimized and validated for the analysis of 1-phenyl-2-propanone (P2P) in illicit methamphetamine (MAMP) samples. The DHS parameters were investigated to reach the sensitivity suitable for this kind of analysis. The method showed of a good specificity, linearity, accuracy, precision and robustness. The analysis of ten MAMP samples seized by the judicial authority was carried out. P2P was found in all the seizure, confirming that P2P is the starting compound of the synthesis of amphetamines.

Determination of 6-Monoacetyl-Morphine (6-MAM) in Brain Samples from Heroin Fatalities

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.

Determination of 1-phenyl-2-propanone (P2P) by HS-GC/MS in a material sold as “wet amphetamine”

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.

Quali-Quantitative Analysis by LC/DAD and GPC of the Polyphenols ofUva Di Troia Canosina Grape Seeds for the Development of an IndustrialNutraceutical Product

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®.

New spirocyclic Δ2-isoxazoline derivatives related to selective agonists of α7 neuronal nicotinic acetylcholine receptors

A set of structural analogues of spirocyclic quinuclidinyl-Δ(2)-isoxazolines, characterized as potent and selective α7 nicotinic agonists, was prepared and assayed for binding affinity at α7 and α4β2 neuronal nicotinic acetylcholine receptors (nAChRs). The investigated derivatives (3a-3c, 4a-4c, 5a-5c, 6a-6c, and 7a-7c), synthesized via the 1,3-dipolar cycloaddition of nitrile oxides to suitable dipolarophiles, showed an overall reduced affinity at the α7 subtype when compared with their model compounds. Solely Δ(2)-isoxazolines 3a, 3b, and 6c maintained a binding affinity in the nanomolar range at the α7 nAChRs (K(i) = 230, 420 and 700 nM, respectively). The quaternary ammonium salt 6c retained also a noteworthy α7 vs. α4β2 subtype selectivity, whereas 3a and 3b showed a sharp reduction in selectivity compared with 1a and 1b, their quinuclidinyl higher homologues.

Characterization of the Volatile Components of Cannabis Preparations bySolid-Phase Microextraction Coupled to Headspace-Gas Chromatographywith Mass Detector (SPME-HSGC/MS)

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.

Determination of Propofol by GC/MS and Fast GC/MS-TOF in Two Cases of Poisoning

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.

Taxonomic Identification of Hallucinogenic Mushrooms Seized on the IllegalMarket Using a DNA-Based Approach and LC/MS-MS Determination ofPsilocybin and Psilocin

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.

Methods for the Evaluation of Polyphenolic Content in ''Uva Di Troia Canosina'' Grape and Seeds at the Different Maceration Stages

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.