Sebastiano Arnoldi

Blood Cyanide Determination in Two Cases of Fatal Intoxication: Comparison Between Headspace Gas Chromatography and a Spectrophotometric Method*

Abstract:  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 μg/mL, respectively. Detection limits were 0.2 μg/mL for VIS and 0.05 μg/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.

Determination of the active principles of Catha Edulis: Quali–quantitative analysis of cathinone, cathine, and phenylpropanolamine

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.

Characterization of in vitro metabolites of JWH-018, JWH-073 and their 4-methyl derivatives, markers of the abuse of these synthetic cannabinoids.

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.

Capillary electrophoretic and extraction conditions for the analysis of Catha edulis FORKS active principles

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.

Improved GC method for the determination of the active principles of Catha edulis

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.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

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.

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®.