Lucia Dell’Acqua

Determination of primary active constituents in Cannabis preparations by high-resolution gas chromatography/flame ionization detection and high-performance liquid chromatography/UV detection

Abstract 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.

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.

Identification of 1-butyl-3-(1-(4-methyl)naphtoyl)indole detected for the first time in “herbal high” products on the Italian market

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.

A case of fatal intoxication from metformin

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.

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.

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®.

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.