A special case of medicine in disguise: Tattoo inks containing anaesthetics.

This paper describes a case of medicine in disguise: seized tattoo inks containing lidocaine and tetracaine at high concentration. Identification of anaesthetics was performed by LC MS Q-TOF with ESI+ source, by accurate mass measurement and by comparing the fragmentation patterns of molecular ions, at 30 V and 10 V of collision-offset voltage, with reference standards. Quantification was also performed by LC MS Q-TOF on the chromatographic peaks in the extracted ion chromatograms, by calibration curves obtained at different standard concentrations and by standard additions approach. The measurement uncertainty was estimated from validation data. The paper gives also chromatographic parameters, MS and MS/MS data and a quantitation method, with a full validation, of other six "caines". Thus the paper intends to provide a tool for identification and quantitation of the most common local anaesthetics that could be fraudulently added to tattoo inks. The results here reported show that the seized samples of inks represent a serious health risk owing to the high anaesthetic content - therapeutic-like dosage - found.

A Q-TOF LC/MS method for identification and quantitation of Histamine in the antibiotic Gentamicin at ppm level: Validation and uncertainty evaluation.

Adverse reactions have been reported for antibiotics produced via fermentation with fish peptone due to Histamine contamination. Just few micrograms of Histamine can result in adverse reactions when administered intravenously. Thus in this paper a new method for identification and quantitation of Histamine at ppm levels in the antibiotic Gentamicin is described. The method is based on separation of Histamine from Gentamicin and other excipients present in the drug matrix, by hydrophilic interaction liquid chromatography (HILIC) coupled to a Q-TOF/MS detector; quantitation is based on the standard addition approach. The method was validated for repeatability, inter-day precision, specificity, accuracy (relative and absolute bias) linearity, limit of detection and quantitation. Uncertainty was estimated and evaluated by comparison with values expected according to the Horwitz theory. The method showed satisfactory performances and good sensitivity, reaching a limit of quantitation of approximately 1 ppm. The method proposed can be a starting point for the development of Histamine quantitation methods in other antibiotics or even in other medicinal products which active ingredient is produced via fermentation in presence of fish peptone.

Counterfeit Adderall Containing Aceclofenac from Internet Pharmacies.

A nontargeted approach based on liquid chromatography equipped with a quadrupole time-of-flight mass detector (LC-MS Q-TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti-inflammatory drug aceclofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic structures (M + H)+ and (M + Na)+ of a molecule M. The accurate mass data generated the molecular formula C16 H13 Cl2 NO4 . A screening of the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Aceclofenac structure was unambiguously confirmed by 1 H and 13 C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities.

Health risks related to illegal and on-line sale of drugs and food supplements: results of a survey on marketed products in Italy from 2011 to 2013.

OBJECTIVES: The increasing illegal and on-line market of medicines and food supplements is helping the widespread diffusion of harmful counterfeit and forbidden products among consumers of developed countries. The objectives of this survey were the description of the main frauds recognized by public officers and the detection of illegal or counterfeit drugs and food supplements. METHODS: Medicines and food supplements found by Police forces on the illegal market or resulting from seizures made by Italian Customs authorities were visually inspected and analysed to evaluate their quality and the presence of other undeclared substances. RESULTS: The visual inspection and the chemical analysis revealed unsuitable packaging (mostly lacking of adequate information for consumers), absence of the declared active substances and presence of undeclared active substances. Products containing doping agents, illegal substances and active ingredients requiring medical supervision were found. CONCLUSION: The present work confirmed the health risk associated with assumption of medicines purchased on the Internet and from the illegal supply chain and evidenced a new threat to consumer safety related to the presence of pharmaceutical active ingredients in food supplements claiming to contain only "natural ingredients".

Characterization of Sildenafil analogs by MS/MS and NMR: a guidance for detection and structure elucidation of phosphodiesterase-5 inhibitors.

Novel synthetic analogs of Sildenafil are constantly detected as adulterants in counterfeit drugs and dietary supplements. Their intake constitutes a serious health hazard as side effects are unknown. In this paper an investigation is carried out on NMR and MS/MS spectra of Sildenafil, Thiosildenafil, Acetildenafil and thirteen of their analogs: a list of key signals is reported and discussed with the intent to provide a tool that can help in detecting adulteration and in elucidating the structure of novel analogs. In this view extensive spectral data were reported, discussed and summarized in tables. A discussion on mass fragmentation and NMR chemical shifts is also provided to rationalize assignation. Moreover, a comprehensive information on the route of synthesis is provided for the benefit of those medicines control laboratories that need to synthesize analogs reference standards in-house.

A survey on illegal and counterfeit medicines for the treatment of erectile dysfunctions in Italy.

INTRODUCTION: In developed countries the phenomenon of pharmaceutical counterfeiting is steadily increasing through the illegal and the Internet market. Medicines for the treatment of erectile dysfunctions containing phosphodiesterase type 5 inhibitors (PDE5) are especially prone to falsification. AIMS: To obtain evidence of the health risks for patients taking these products and to provide useful information to general practitioners and specialists in sexual medicine. METHODS: First the samples were visually inspected and then analyzed to get information about their identity and quality. MAIN OUTCOME MEASURES: A survey on the PDE5 medicines analyzed by the Italian official medicines control laboratory between 2005 and 2011 was performed. All the analyzed medicines were gathered from the Italian illegal market (seizures by police forces) or were bought from illegal online pharmacies. Results. The study revealed that 24% of the analyzed samples were counterfeit and 54% were illegal medicines. In 12% of the cases an intermediate classification (illegal/counterfeit) was assigned. Only 7% of the samples were original. Moreover, the examination of the packaging evidenced potential risks: outer and immediate packaging missing; inconsistency between the carton box and the blister as regards the expiry date and/or the batch number; expiry date or manufacturer's name or country missing. CONCLUSIONS: In 19% of the samples a potential health risk for patients was identified due to either the presence in the sample of more than one undeclared PDE5(s) or an amount of the active ingredient higher than that declared (up to 190% of the maximum dose) or to the presence of potentially dangerous excipients of non-pharmaceutical origin or quality (e.g., gypsum or non-purified talc).

Counterfeit drugs detection by measurement of tablets and secondary packaging colour.

The growth of pharmaceutical counterfeiting is a major public health problem. This growth is resulting in a proportional increase in the number of samples that medicines control laboratories have to test. Thus the need for simple and affordable preliminary screening methods to be used by inspectors to decide in the field whether to collect a sample for further laboratory analysis or not. This paper intends to evaluate the possibility to employ for preliminary examinations of suspicious samples an optical spectrophotometer (colorimeter) used in the graphic industry, capable of measuring the reflectance visible spectrum of solid materials. The colorimeter was tested on original and counterfeited Viagra, Cialis and Levitra by measuring the colour of tablets' surface and of a specific spot of the packages. Various batches of the original drugs were employed both to investigate precision and robustness of the technique and to build spectral libraries. These libraries were used to compare suspicious samples to the corresponding original by means of a wavelength distance pattern recognition method. The method was eventually tested on suspicious samples sized by police authorities in order to evaluate its effectiveness. The device resulted precise and robust toward ambient conditions changes, although some limits emerged: the libraries of original samples need a frequent update and a lower precision is to be expected for tablets which surface is extremely convex.

Medicines informal market in Congo, Burundi and Angola: counterfeit and sub-standard antimalarials.

BACKGROUND: The presence of counterfeits and sub-standards in African medicines market is a dramatic problem that causes many deaths each year. The increase of the phenomenon of pharmaceutical counterfeiting is due to the rise of the illegal market and to the impossibility to purchase branded high cost medicines. METHODS: In this paper the results of a quality control on antimalarial tablet samples purchased in the informal market in Congo, Burundi and Angola are reported. The quality control consisted in the assay of active substance by means of validated liquid chromatographic methods, uniformity of mass determination, disintegration and dissolution tests. Moreover, a general evaluation on label and packaging characteristics was performed. RESULTS: The results obtained on thirty antimalarial tablet samples containing chloroquine, quinine, mefloquine, sulphadoxine and pyrimethamine showed the presence of different kinds of problems: a general problem concerning the packaging (loose tablets, packaging without Producer name, Producer Country and sometimes without expiration date); low content of active substance (in one sample); different, non-declared, active substance (in one sample); sub-standard technological properties and very low dissolution profiles (in about 50% of samples). This last property could affect the bioavailability and bioequivalence in comparison with branded products and could be related to the use of different excipients in formulation or bad storage conditions. CONCLUSION: This paper evidences that the most common quality problem in the analysed samples appears to be the low dissolution profile. Here it is remarked that the presence of the right active substance in the right quantity is not a sufficient condition for a good quality drug. Dissolution test is not less important in a quality control and often evidences in vitro possible differences in therapeutic efficacy among drugs with the same active content. Dissolution profile can be dramatically affected by the choice of excipients in the oral solid formulation and, in many cases, is out of specifications due to the absence of formulation studies by producers of developing countries.

Calcitonin forms oligomeric pore-like structures in lipid membranes.

Calcitonin is a polypeptidic hormone involved in calcium metabolism in the bone. It belongs to the amyloid protein family, which is characterized by the common propensity to aggregate acquiring a beta-sheet conformation and include proteins associated with important neurodegenerative diseases. Here we show for the first time, to our knowledge, by transmission electron microscopy (TEM) that salmon-calcitonin (sCT) forms annular oligomers similar to those observed for beta-amyloid and alpha-sinuclein (Alzheimer's and Parkinson's diseases). We also investigated the interaction between sCT and model membranes, such as liposomes, with particular attention to the effect induced by lipid "rafts" made of cholesterol and G(M1). We observed, by TEM immunogold labeling of sCT, that protein binding is favored by the presence of rafts. In addition, we found by TEM that sCT oligomers inserted in the membrane have the characteristic pore-like morphology of the amyloid proteins. Circular dichroism experiments revealed an increase in beta-content in sCT secondary structure when the protein was reconstituted in rafts mimicking liposomes. Finally, we showed, by spectrofluorimetry experiments, that the presence of sCT allowed Ca(2+) entry in rafts mimicking liposomes loaded with the Ca(2+)-specific fluorophore Fluo-4. This demonstrates that sCT oligomers have ion-channel activity. Our results are in good agreement with recent electrophysiological studies reporting that sCT forms Ca(2+)-permeable ion channels in planar model membranes. It has been proposed that, beyond the well-known interaction of the monomer with the specific receptor, the formation of Ca(2+) channels due to sCT oligomers could represent an extra source of Ca(2+) entry in osteoblasts. Structural and functional data reported here support this hypothesis.

Development and validation of a reversed-phase LC method for analysing potentially counterfeit antimalarial medicines.

Pharmaceutical counterfeiting is more and more a public health problem, especially in developing countries where the most counterfeit drugs are antibiotics, antimalarials and other life-saving drugs. The evaluation of the phenomenon extent is of great concern to the World Health Organization for carrying out a global strategy to combat the phenomenon. To this purpose, a reversed-phase liquid chromatographic method to perform the separation and simultaneous determination of three different kinds of antimalarial drugs (chloroquine, quinine and mefloquine) was developed. The method was validated by using both commercial and in-laboratory produced tablets and was then verified on various in-laboratory produced formulations differing in excipient composition. Finally, the method was successfully applied to the analysis of medicinal samples purchased from the informal market in Congo, Burundi and Angola.

Early stages of salmon calcitonin aggregation: effect induced by ageing and oxidation processes in water and in the presence of model membranes.

The natural ageing- and hydrogen peroxide-induced aggregation of salmon calcitonin were studied in water and in the presence of dipalmitoylphosphatidylcholine (DPPC) liposomes. The early stages of the aggregation process at low protein concentration were investigated by means of Circular Dichroism spectroscopy (CD) and conventional and immunogold labelling Transmission Electron Microscopy (TEM). In buffered water solution, salmon calcitonin showed a two-stage conformational variation related to fibril formation and phase-separation of larger aggregates. A first stage, characterised by small conformational changes but a decrease in dichroic band intensity, was followed by a second stage, 6 days after, leading to higher conformational variations and aggregations. Salmon calcitonin showed a distinct modification in the secondary structure and aggregate morphology in the presence of hydrogen peroxide with respect to natural ageing, indicating that the two aggregation processes (natural and chemical-induced) followed a distinct mechanism. The oxidised forms of the peptide were separated by liquid chromatography. The same study was performed in the presence of DPPC liposomes. The results obtained by conventional and immunogold labelling TEM evidenced that salmon calcitonin in buffered water solution essentially does not enter the liposomes but forms around them a fibril network characterised by the same conformational changes after 6 days. The oxidised sample in the presence of liposomes showed a "fibrils hank", separated from liposomes. The presence of liposomes did not affect either the aggregation or the conformational modifications yet observed by TEM and CD in water solution.

Diclofenac sodium multisource prolonged release tablets--a comparative study on the dissolution profiles.

The aim of this work was to compare the dissolution behaviour of six diclofenac sodium prolonged release tablets of different brands obtained from the national market. The formulations contain the same amount of drug substance but different types and/or amount of excipients. The influence of these differences in formulation on the release characteristics of the dosage forms was evaluated on the European Pharmacopoeia apparatus 2 (paddle) employing eight different dissolution media in the pH range 1.2-8. Friability and hardness were tested too according to the European Pharmacopoeia. Dissolution profiles obtained from the studied formulations showed that the release characteristics vary considerably among different manufacturers and that even identical formulations show rather dissimilar release profiles in all the studied media. Use of both SIF without pancreatin and SIF without pancreatin containing 1% (w/v) Tween 20 resulted in strong discrimination among products. A correlation between friability and hardness and in vitro dissolution was evidenced for two formulations having identical excipient composition.

Effects induced by hydroxyl radicals on salmon calcitonin: a RP-HPLC, CD and TEM study.

The effects of hydroxyl radical attack on a peptidic drug were studied in vitro. Different chemico-physical techniques were used to investigate structural damage induced by oxidative stress conditions in salmon calcitonin (sCT), a peptide hormone used in treating osteoporosis. Reversed-phase liquid chromatography (RP-HPLC), circular dichroism (CD) and transmission electron microscopy (TEM) were applied to measure formation of oxidation/degradation products and to reveal the conformational and ultrastructural modifications in the presence of OH. free radicals. Hydroxyl radicals were obtained from ferrous sulfate and ascorbic acid mixtures. The RP-HPLC results revealed the formation of new chromatographic peaks indicating a number of degradation/oxidation products formed in the presence of OH. free radicals. CD spectra showed slight protein conformational modifications as well as aggregation. TEM confirmed sCT aggregation and suggested the formation of fibrillar aggregates.

Simultaneous determination of zidovudine and nevirapine in human plasma by RP-LC.

A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid-liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250 x 4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.