LC-MS/MS analysis of two new designer drugs (FLY serie) in rat plasma and its application to a pharmacokinetic study.

The widespread diffusion of new psychoactive substances, requires a continuous update and development of new methods able to identify and quantify these new molecules in biological matrices. In this study an analytical method for the determination of two new benzodifuranyl derivatives, 1-(2,3,6,7-tetrahydrofuro[2,3-f][1]benzofuran-4-yl)propan-2-amine and 2-(2,3,6,7-tetrahydrofuro[2,3-f][1]benzofuran-4-yl)ethanamine, in rat plasma was developed. A solid phase extraction using C18 cartridges was carried out obtaining good recoveries with low matrix effect. Quantification was performed by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Separation was carried out on a C18 reverse phase column with water/methanol containing 0.1% of formic acid as mobile phase. These conditions allowed to achieve adequate separation, resolution and signal-to-noise ratio for analytes and internal standard. Calibration curves were linear over the concentration range from 10 to 400 ng/ml with correlation coefficients that exceeded 0.995. Obtained precision, accuracy and recovery showed good reproducibility and selectivity. Finally, the validation method was successfully applied to an in vivo study in order to evaluate the pharmacokinetic profile of these new amphetamines.

Aceclofenac-Galactose Conjugate: Design, Synthesis, Characterization, and Pharmacological and Toxicological Evaluations.

Aceclofenac is a popular analgesic, antipyretic, and nonsteroidal anti-inflammatory drug (NSAID) used for prolonged treatment (at least three months) in musculoskeletal disorders. It is characterized by several limitations such as poor water solubility and low oral bioavailability. The main side-effect of aceclofenac, as well as all NSAIDs, is the gastrotoxicity; among other adverse effects, there is the risk of bleeding since aceclofenac reversibly inhibits platelet aggregation. With the aim to reduce these drawbacks, we have designed, synthesized, and characterized, both in vitro and in vivo, an orally administrable pro-drug of aceclofenac (ACEgal). ACEgal was obtained by conjugating carboxyl group with the 6-OH group of d-galactose; its structure was confirmed by X-ray powder diffractometry. The pro-drug was shown to be stable at 37 °C in simulated gastric fluid (SGF-without pepsin, pH = 1.2) and moderately stable in phosphate buffered saline (PBS, pH = 7.4). However, it hydrolyzed in human serum with a half-life ( t1/2) of 36 min, producing aceclofenac. Furthermore, if compared to its parent drug, ACEgal was four-times more soluble in SGF. To predict human intestinal absorption, cell permeability in a Caco-2 model of aceclofenac and ACEgal was determined. Anti-inflammatory, analgesic, and ulcerogenic activities have been investigated in vivo. In addition, oxidative stress parameters (thiobarbituric acid reactive substances, TBARS, and glutathione, GSH) and platelet antiaggregatory activity both of parent drug and pro-drug were evaluated. Results clearly showed that the conjugation of aceclofenac to a galactose molecule improves physicochemical, toxicological (at gastric and blood level), and pharmacological profile of aceclofenac itself without changing intestinal permeability and antiplatelet activity (in spite the new sugar moiety).

Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1ß). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1ß, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

Blood serum retinol levels in Asinara white donkeys reflect albinism-induced metabolic adaptation to photoperiod at Mediterranean latitudes.

Previous works on albinism form of Asinara white donkeys (Equus asinus) identified the mutation leading to the peculiar phenotype spread to all specimens of the breed. Inbreeding naturally occurred under geographic isolation, on Asinara Island, in the Mediterranean Sea. Albino individuals can be more susceptible to develop health problems when exposed to natural sun radiation. Alternative metabolic pathways involved in photoprotection were explored in this trial. Nutrition-related metabolites are believed to contribute to the conservation of Asinara donkeys, in which melanin, guaranteeing photoprotection, is lacking. Biochemical profiles with particular focus on blood serum ß-carotene and retinol levels were monitored. Identical natural grazing conditions for both Asinara (albino) and Sardo (pigmented) donkey breeds were assured on same natural pastures throughout the experimental period. A comparative metabolic screening, with emphasis on circulating retinol and nutrient-related metabolites between the two breeds, was carried out over one year. Potential intra- and interspecimen fluctuations of metabolites involved in photoprotection were monitored, both during negative and positive photoperiods. Differences (p = .064) between blood serum concentrations of retinol from Asinara versus Sardo breed donkeys (0.630 vs. 0.490 µg/ml, respectively) were found. Retinol levels of blood serum turned out to be similar in the two groups (0.523 vs. 0.493 µg/ml, respectively, p = .051) during the negative photoperiod, but markedly differed during the positive one (0.738 vs. 0.486, respectively, p = .016). Blood serum ß-carotene levels displayed to be constantly around the limit of sensitivity in all animals of both breeds. Variations in blood serum concentrations of retinol in Asinara white donkeys can reflect the need to cope with seasonal exposure to daylight at Mediterranean latitudes, as an alternative to the lack of melanin. These results may suggest that a pulsed mobilization of retinol from body stores occurs to increase circulating levels during positive photoperiod.

ELISA Detection of 30 New Amphetamine Designer Drugs in Whole Blood, Urine and Oral Fluid using Neogen® "Amphetamine" and "Methamphetamine/MDMA" Kits.

Amphetamine designer drugs are central nervous system stimulants that are widely disseminated in the illegal market. Generally, in forensic laboratories, immunoassay methods are the first line of screening for these types of drugs in a biological specimen (typically blood, urine or oral fluid). In this article, we describe the cross-reactivity profiles of 30 new amphetamine designer drugs, using the Neogen(®) [Amphetamine Specific and Methamphetamine/3,4-Methylenedioxymethamphetamine (MDMA) assays] drug tests. To assess the potential matrix influence on the response, each assay was tested on whole blood, urine and oral fluid. Concentrations of 10,000 ng/mL were not sufficient to produce a positive response for the majority of the analyzed amphetamines. This clearly demonstrates that, although these kits are extremely effective for the target drugs for which they are intended (amphetamine, methamphetamine and MDMA), they cannot be used to reliably identify the tested designer drugs in real cases, as these concentrations greatly exceed those expected to be found in forensic samples.

Validated LC-MS-MS Method for Multiresidual Analysis of 13 Illicit Phenethylamines in Amniotic Fluid.

A multi-residue analytical method was developed for the determination in amniotic fluid (AF) of 13 illicit phenethylamines, including 12 compounds never investigated in this matrix before. Samples were subject to solid-phase extraction using; hydrophilic-lipophilic balance cartridges which gave good recoveries and low matrix effects on analysis of the extracts. The quantification was performed by liquid chromatography electrospray tandem mass spectrometry. The water-acetonitrile mobile phase containing 0.1% formic acid, used with a C18 reversed phase column, provided adequate separation, resolution and signal-to-noise ratio for the analytes and the internal standard. The final optimized method was validated according to international guidelines. A monitoring campaign to assess fetal exposure to these 13 substances of abuse has been performed on AF test samples obtained from pregnant women. All mothers (n = 194) reported no use of drugs of abuse during pregnancy, and this was confirmed by the analytical data.

Simultaneous Determination of 11 Illicit Phenethylamines in Hair by LC-MS-MS: In Vivo Application.

Existing phenethylamines are a class of synthetic compounds that differ from each other only in small changes to a largely conserved chemical structure. The recreational and illicit use of phenethylamines is a widespread problem. A simple procedure for the simultaneous quantitative determination in hair of 11 phenethylamines that are officially recognized as illicit by Italian legislation (p-methoxyamphetamine; p-methoxymethamphetamine; 3,4,5-trimethoxyamphetamine; 2,5-dimethoxyamphetamine; 2,5-dimethoxy-4-methylamphetamine; 2,5-dimethoxy-4-ethylamphetamine; 2,5-dimethoxy-4-bromoamphetamine; 2,5-dimethoxy-4-bromophenethylamine; 2,5-dimethoxy-4-iodophenethylamine; 2,5-dimethoxy-4-ethylthiophenethylamine and 2,5-dimethoxy-4-n-propylthiophenethylamine) has been developed and validated. Extraction from the matrix was performed after incubation in methanolic HCl and filtered reconstituted extracts were injected into a liquid chromatography/tandem mass spectrometry system (LC-MS-MS) without any further purification steps. This validated LC-MS-MS method has been used to determine the in vivo accumulation/retention of the above target analytes in hair after repeat oral administration to rats. This experiment further permitted investigation of the effect of pigmentation on the uptake of these phenethylamines by hair and the effect of hair pigmentation. The developed method could potentially be used for forensic and toxicological purposes, in the detection and quantitation of these illicit substances in human hair in workplace drug testing; drug-facilitated crime investigation; driver re-licensing; determining drug abuse history and postmortem toxicology.

Identification and quantitation of 4-bromo-2,5-dimethoxyamphetamine in seized blotters.

Blotters are usually impregnated with hallucinogens such as lysergic acid diethylamide (LSD); only rarely other psychoactive substances are detected. In this work we identified 4-bromo-2,5-dimethoxyamphetamine (DOB) and 2,5-dimethoxyamphetamine (DMA) in illicit blotters seized in Italy. This report describes a rapid method for the simultaneous identification and quantitation of DOB and its precursor (DMA) by liquid chromatography tandem mass spectrometry (LC-MS-MS), using 2,3-dimethoxyphenethylamine-d3 as internal standard. Regression equations were linear over the tested concentration range with good correlation coefficients. The achieved levels of sensitivity may be suitable to confirm the possible presence of DOB and DMA also in low concentration or in traces in seized material for forensic analysis. The developed method showed good reproducibility and sensitivity, and could be used for similar routine analysis. To our knowledge, this is the first report describing the detection of DOB and DMA from blotters.

Effects of homeopathic mother tinctures on breath alcohol testing.

In some countries, it is illegal to drive with any detectable amount of alcohol in blood; in others, the legal limit is 0.5 g/L or lower. Recently, some defendants charged with driving under the influence of alcohol and have claimed that positive breath alcohol test results were due to the ingestion of homeopathic mother tinctures. These preparations are obtained by maceration, digestion, infusion, or decoction of herbal material in hydroalcoholic solvent. A series of tests were conducted to evaluate the alcoholic content of three homeopathic mother tinctures and their ability to produce inaccurate breath alcohol results. Nine of 30 subjects gave positive results (0.11-0.82 g/L) when tests were taken within 1 min after drinking mother tincture. All tests taken at least 15 min after the mother tincture consumption and resulted in alcohol-free readings. An observation period of 15-20 min prior to breath alcohol testing eliminates the possibility of false-positive results.

Investigation on gabapentin residues in eggs from free-range hens exposed to saline slags from pharmaceutical industry.

A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to monitor the presence of gabapentin as environmental contaminant in albumen and yolk of eggs from grazing flocks exposed to open air stored saline wastes from pharmaceutical industry. The method involved a simple liquid extraction followed by a gradient elution with formic acid 0.2 % and acetonitrile in reverse phase. ESI ionization was performed in positive ion mode. The tandem mass spectrometer was operated in multiple reaction monitoring mode. The calibration curves were linear in the concentration range from 5 to 400 ng/g for the two matrices with correlation coefficients that exceeded 0.990. The limits of quantitation were 12.0 and 14.8 ng/g in albumen and yolk, respectively. Results are discussed in light of the pharmacokinetics of gabapentin in experimentally exposed hens, accounting for the top soil intake in such free grazing animals.

Evaluation of commercial multi-drug oral fluid devices to identify 39 new amphetamine-designer drugs.

Recently, the diffusion on the black market of new psychoactive substances not controlled and often sold as 'legal highs', is exponentially increasing in Europe. Generally, the first analysis for these drugs involves an immunoassay screening in urine or plasma. Actually, there is growing interest in the use of oral fluid (OF) as alternative specimen over conventional biological fluids for drug testing, because of the significant advantages, as a non-invasive collection under direct observation without undue embarrassment or invasion of privacy, and a good correlation with plasma analytical data. Few assays have been developed for detection of new psychoactive compounds in biological samples, so it is important to investigate how they may or may not react in pre-existing commercial immunoassays. In this paper, two different multi-drugs oral fluid screen devices (OFDs) (Screen® Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD) were evaluated to determine the cross-reactivity of thirty-nine new amphetamine designer drugs, including twelve substances officially recognized as illicit by italian legislation. Cross-reactivity towards most drugs analyzed was <1 in assays targeting amphetamine (AMP) or methamphetamine (MET). Only two (p-methoxyamphetamine and p-methoxymethamphetamine) of all tested amphetamines gave a positive result.

Enantiomeric separation of 13 new amphetamine-like designer drugs by capillary electrophoresis, using modified-B-cyclodextrins.

An easy-to-prepare chiral CE method for the enantiomeric separation of 13 new amphetamine-like designer drugs, using CDs as chiral selectors, was developed. Sulfated-ß-CD was found to be the best chiral selector among the three used (sulfated-ß-CD, caroboxymethyl-ß-CD, dimethyl-ß-CD). The separation of the analytes was achieved in a fused-silica gel capillary at 20 °C using an applied voltage of +25 kV. The optimized background electrolyte consisted of 63.5 mM H3 PO4 and 46.9 mM NaOH in water. Several electrophoretic parameters such as CD type, CD concentration (1 - 40 mg/mL), buffer pH (2.6, 3.6, 5.0, 6.0), length of the capillary (70 - 40 cm total length), amount of the organic solvent (methanol and acetonitrile) were investigated and optimized.