New perspectives in the use of ink evidence in forensic science: Part I. Development of a quality assurance process for forensic ink analysis by HPTLC

The level of information provided by ink evidence to the criminal and civil justice system is limited. The limitations arise from the weakness of the interpretative framework currently used, as proposed in the ASTM 1422-05 and 1789-04 on ink analysis. It is proposed to use the likelihood ratio from the Bayes theorem to interpret ink evidence. Unfortunately, when considering the analytical practices, as defined in the ASTM standards on ink analysis, it appears that current ink analytical practices do not allow for the level of reproducibility and accuracy required by a probabilistic framework. Such framework relies on the evaluation of the statistics of the ink characteristics using an ink reference database and the objective measurement of similarities between ink samples. A complete research programme was designed to (a) develop a standard methodology for analysing ink samples in a more reproducible way, (b) comparing automatically and objectively ink samples and (c) evaluate the proposed methodology in a forensic context. This report focuses on the first of the three stages. A calibration process, based on a standard dye ladder, is proposed to improve the reproducibility of ink analysis by HPTLC, when these inks are analysed at different times and/or by different examiners. The impact of this process on the variability between the repetitive analyses of ink samples in various conditions is studied. The results show significant improvements in the reproducibility of ink analysis compared to traditional calibration methods.

Analysis of Passion Fruit rinds (Passiflora edulis): Isoorientin quantification by hptlc and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora Mulls fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P edulis (92.275 +/- 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 +/- 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P Mulls rinds as a natural source of flavonoids or as a possible functional food.

Analysis of passion fruit rinds (Passiflora edulis): isoorientin quantification by HPTLC and evaluation of antioxidant (radical scavenging) capacity

The content of isoorientin in passion fruit rinds (Passiflora edulis fo. flavicarpa O. Degener) was determined by HPTLC (high performance thin layer chromatography) with densitometric analysis. The results revealed a higher amount of isoorientin in healthy rinds of P. edulis (92.275 ± 0.610 mg L-1) than in rinds with typical symptoms of PWV (Passion fruit Woodiness Virus) infection (28.931 ± 0.346 mg L-1). The HPTLC data, allied to assays of radical scavenging activity, suggest the potential of P. edulis rinds as a natural source of flavonoids or as a possible functional food.

From traditional resource to global commodities: a comparison of Rhodiola species using NMR spectroscopy - metabolomics and HPTLC

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products has been overlooked in the debate about quality problems in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. We compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for Rhodiola authentication and the detection of potential adulterant compounds. Samples were collected from Europe and Asia and nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. A method was developed to quantify the amount of adulterant species contained within mixtures. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas crenulatin was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. Moreover, 7 % of the raw-material samples were not labelled satifactorily. The utilisation of both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences and novel identification method for R. rosea and R. crenulata. Using 1H-NMR spectroscopy it was possible to quantify the presence of R. crenulata in admixtures with R. rosea. This quantitative technique could be used in the future to assess a variety of herbal drugs and products. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Anticancer property of Bryophyllum pinnata (Lam.) Oken. leaf on human cervical cancer cells

Background: Bryophyllum pinnata (B. pinnata) is a common medicinal plant used in traditional medicine of India and of other countries for curing various infections, bowel diseases, healing wounds and other ailments. However, its anticancer properties are poorly defined. In view of broad spectrum therapeutic potential of B. pinnata we designed a study to examine anti-cancer and anti-Human Papillomavirus (HPV) activities in its leaf extracts and tried to isolate its active principle. Methods: A chloroform extract derived from a bulk of botanically well-characterized pulverized B. pinnata leaves was separated using column chromatography with step-gradient of petroleum ether and ethyl acetate. Fractions were characterized for phyto-chemical compounds by TLC, HPTLC and NMR and Biological activity of the fractions were examined by MTT-based cell viability assay, Electrophoretic Mobility Shift Assay, Northern blotting and assay of apoptosis related proteins by immunoblotting in human cervical cancer cells. Results: Results showed presence of growth inhibitory activity in the crude leaf extracts with IC50 at 552 mu g/ml which resolved to fraction F4 (Petroleum Ether: Ethyl Acetate:: 50: 50) and showed IC50 at 91 mu g/ml. Investigations of anti-viral activity of the extract and its fraction revealed a specific anti-HPV activity on cervical cancer cells as evidenced by downregulation of constitutively active AP1 specific DNA binding activity and suppression of oncogenic c-Fos and c-Jun expression which was accompanied by inhibition of HPV18 transcription. In addition to inhibiting growth, fraction F4 strongly induced apoptosis as evidenced by an increased expression of the pro-apoptotic protein Bax, suppression of the anti-apoptotic molecules Bcl-2, and activation of caspase-3 and cleavage of PARP-1. Phytochemical analysis of fraction F4 by HPTLC and NMR indicated presence of activity that resembled Bryophyllin A. Conclusions: Our study therefore demonstrates presence of anticancer and anti-HPV an activity in B. pinnata leaves that can be further exploited as a potential anticancer, anti-HPV therapeutic for treatment of HPV infection and cervical cancer.

Cultura de tecidos, análise fitoquímica e avaliação da atividade biológica de Arachis villosulicarpa Hoehne

O amendoim cultivado (Arachis hypogaea) possui várias substâncias com capacidade antioxidante que apresentam efeitos benéficos para a saúde humana. Entretanto, a produção dessas substâncias ainda não foi observada em outras espécies do gênero. O objetivo deste trabalho foi o estabelecimento de sistemas de cultura in vitro para Arachis villosulicarpa, visando o desenvolvimento de alternativas para a produção metabólitos especiais. Segmentos nodais, internodais e foliares foram excisados de plantas in vitro e inoculados em meio MS suplementado com BAP, ANA, PIC ou 2,4-D. Os explantes apresentaram respostas distintas de acordo com o regulador de crescimento utilizado. Na presença de PIC, foi observada a formação de calos friáveis levemente oxidados em todos os explantes. Por outro lado, quando cultivados na presença de BAP ou 2,4-D, os explantes apresentaram a formação de calos compactos, com formação de brotos em diferentes taxas. Na presença de ANA, segmentos nodais e internodais não apresentaram resposta, enquanto que segmentos foliares apresentaram a formação de raízes adventícias em todas as concentrações testadas. Neste trabalho foi realizado uma avaliação comparativa da produção de metabólitos especiais e da atividade biológica em extratos de calos oriundos de folíolos inoculados em BAP 8,8 M, plantas in vitro e plantas ex vitro . Nas análises por meio do HPTLC e do HPLC foi possível demonstrar a diferença na produção de algumas substâncias e indicar a presença de flavonóides e polifenóis nos materiais analisados. Além disso, a partir das atividades biológicas foi possível identificar tanto a atividade antioxidante quanto a não mutagênica dos extratos. Dessa forma, estudos visando à detecção de metabólitos especiais em A. villosulicarpa podem expandir o conhecimento sobre as possibilidades de utilização de espécies do gênero Arachis.

Validation of Thin Layer Chromatographic Methods

Thin-layer and high-performance thin-layer chromatography (TLC/HPTLC) methods for assaying compound(s) in a sample must be validated to ensure that they are fit for their intended purpose and, where applicable, meet the strict regulatory requirements for controlled products. Two validation approaches are identified in the literature, i.e. the classic and the alternative, which is using accuracy profiles.Detailed procedures of the two approaches are discussed based on the validation of methods for pharmaceutical analysis, which is an area considered having more strict requirements. Estimation of the measurement uncertainty from the validation approach using accuracy profiles is also described.Examples of HPTLC methods, developed and validated to assay sulfamethoxazole and trimethoprim on the one hand and lamivudine, stavudine, and nevirapine on the other, in their fixed-dose combination tablets, are further elaborated.

Adulteration and Poor Quality of Ginkgo biloba Supplements

Adulteration of Ginkgo products sold as unregistered supplements within the very large market of Ginkgo products (reputedly £650 million annually) through the post-extraction addition of cheaper (e.g. buckwheat derived) rutin is suspected to allow sub-standard products to appear satisfactory to third parties, e.g. secondary buyers along the value chain or any regulatory authorities. This study was therefore carried out to identify products that did not conform to their label specification and may have been actively adulterated to enable access to the global markets. 500 MHz Bruker NMR spectroscopy instrumentation combined with Topspin version 3.2 and a CAMAG HPTLC system (HPTLC Association for the analysis of Ginkgo biloba leaf) were used to generate NMR spectra (focusing on the 6–8 ppm region for analysis) and chromatograms, respectively. Out of the 35 samples of Ginkgo biloba analysed, 33 were found to contain elevated levels of rutin and/or quercetin, or low levels of Ginkgo metabolites when compared with the reference samples. Samples with disproportional levels of rutin or quercetin compared with other gingko metabolites are likely to be adulterated, either by accident or intentionally, and those samples with low or non-existent gingko metabolite content may have been produced using poor extraction techniques. Only two of the investigated samples were found to match with the High-Performance Thin-Layer Chromatography (HPTLC) fingerprint of the selected reference material. All others deviated significantly. One product contained a 5-hydroxytryptophan derivative, which is not a natural constituent of Ginkgo biloba. Overall, these examples either suggest a poor extraction technique or deliberate adulteration along the value chain. Investigating the ratio of different flavonoids e.g. quercetin and kaempferol using NMR spectroscopy and HPTLC will provide further evidence as to the degree and kind of adulteration of Gingko supplements. From a consumer perspective the equivalence in identity and overall quality of the products needs to be guaranteed for supplements too and not only for products produced according to a quality standard or pharmacopoeial monograph.

Differenzierung von blauen Kugelschreibertinten mit LDI-MS: ein Vergleich gegenüber Routinemethoden

Die Differenzierung von Tinten erweist sich oft als wichtig in der Echtheitsprüfung von Dokumenten. Sie wird üblicherweise durch optische Vergleiche und Dünnschicht Chromatographie durchgeführt (TLC). Laser Desorption Ionisation Massenspektrometrie (LDI-MS) ist auch als nützlich gefunden worden und besonders leistungsfähig um Farbstoffe aus Kugelschreibertinte zu analysieren. Diese analytische Methode ist mit Hochleistungs Dünnschichtchromatografie TLC (HPTLC) verglichen worden, mit dem Ziel deren Tinten-Differenzierungskapazität zu testen. Tinteneinträge von 31 blauen Kugelschreibern sind analysiert worden und gemäß deren Farbstoffzusammensetzung klassifiziert worden. Typische Farbstoffe sind durch beide Methoden identifiziert worden und mehrere sind in vielen Tinten-Zusammensetzungen gefunden worden. LDI-MS ist leistungsfähiger als HPTLC um Tinten zu differenzieren, weil es Informationen über Farbstoffstrukturen (Molekular Gewicht) enthält und eine präzise relative Quantifizierung (Signalfläche) erlaubt. Dazu ist für LDI-MS Proben die Vorbereitung minimal und die Analysezeit kurz im Vergleich zu HPTLC mehr komplexen Schritte, wie Extraktionen, Spots Applikationen und Lösungsmittelelution. Allerdings sind mit LDI-MS zwei Analysen nötig um kationische und anionische Farbstoffe zu analysieren, während mit HPTLC nur eine Analyse nötig ist.

Estudo das rotas de síntese dos gangliosídios nos dois fenótipos da célula estrelada hepática

Glicoesfingolipídios (GSL) são constituintes da membrana plasmática e possuem bases de cadeia longa (bases esfingóides) como componente estrutural lipídico. Açúcares podem ser adicionados à ceramida sintetizada de novo (rota 1), sintetizada pela reciclagem da esfingosina (rota 2) e em GSL reciclados através do Golgi (rota 3). A serina palmitoiltransferase (SPT) é a enzima marca-passo e catalisa o primeiro passo na biossíntese de novo destes componentes. A linhagem celular GRX, representativa das células estreladas hepáticas, expressa o fenótipo miofibroblástico e pode ser induzida in vitro a adquirir o fenótipo lipocítico. Ambos fenótipos possuem gangliosídios da série-a (GM2, GM1 e GD1a) bem como o seu precursor GM3, que são expressos como doublets em HPTLC (bandas 1 e 2, respectivamente). Para o estudo da biossíntese dos GSL neste modelo biológico, foram determinadas as condições ideais para a atividade da SPT, e foi avaliada sua atividade na fração microssomal nos dois fenótipos. Também foi determinada a contribuição de cada rota de biossíntese para as duplas bandas. As células foram pré-incubadas com 5mM de -cloroalanina (inibidor da SPT) ou com 25M de fumonisina B1 (inibidor da ceramida sintase) e então, [U-14C]galactose foi adicionada no meio de cultura na presença contínua dos inibidores. Culturas controles (sem inibidores) foram realizadas simultaneamente. Os lipídios foram extraídos, os gangliosídios purificados em colunas Sep-Pack C18 e analisados por HPTLC, a qual foi revelada por auto-radiografia e após, submetida à análise densitométrica. Em ambos fenótipos, a síntese de novo, a reciclagem da esfingosina e a reciclagem pelo Golgi contribuem com a biossíntese dos GSL No miofibroblasto, os doublets dos gangliosídios complexos (GD1a e GM1) são, principalmente, sintetizados pelas rotas de reciclagem; enquanto as bandas do GM2 e do GM3 têm uma participação importante da síntese de novo. No lipócito, as rotas de reciclagem são as mais importantes. Contudo, no lipócito, ambas bandas do GM2 e a banda 2 do GM3 apresentam considerável síntese pela rota de novo. Esta rota tem uma contribuição menor no lipócito do que no miofibroblasto, o que está de acordo com os níveis da atividade da SPT detectados nestes fenótipos. Isto sugere que os fenótipos miofibroblasto e lipócito utilizam pools de ceramida distintos para a síntese de seus GSL e que apresentam importantes diferenças entre as rotas biossintéticas, o que pode se refletir no seu comportamento celular.

Caracterização lipídica de duas cepas de Leishmania (Viannia) braziliensis causadoras da leishmaniose tegumentar americana

A leishmaniose tegumentar americana (LTA) constitui uma doença infecciosa causada por protozoários do gênero Leishmania com elevada incidência na região Amazônica. Uma variedade de espécies de leishmania é responsável por esta patologia. Desta forma, dependendo da espécie e da resposta imunológica do hospedeiro vertebrado, a doença pode apresentar diferentes formas clínicas, como a leishmaniose cutânea localizada (LCL) e a leishmaniose mucocutânea (LMC). A principal espécie responsável pela LTA é a Leishmania (Viannia) braziliensis. Contudo, devido à existência de uma multiplicidade de cepas desta espécie e ao reduzido número de estudos relacionados, torna-se importante o conhecimento dos aspectos metabólicos básicos do protozoário, como o metabolismo lipídico, na tentativa de caracterizar vias ou componentes fundamentais para seu desenvolvimento e infectividade. Desta forma, este trabalho teve como objetivo analisar distribuição de corpos lipídicos (CLs) e o perfil lipídico de duas cepas de L. (V.) braziliensis, isolada de diferentes casos clínicos, em diferentes períodos da fase estacionária do crescimento celular. As formas promastigotas das cepas M17593 (LCL) e M17323 (LMC) de L. (V.) braziliensis foram utilizadas na fase estacionária inicial (EST-I) e estacionária tardia (EST-T) de crescimento. Inicialmente, foi realizada análise ultraestrutural das formas promastigotas por microscopia eletrônica de transmissão (MET) e foram observadas estruturas sugestivas de CLs distribuídos no citoplasma do parasito, confirmados pela técnica citoquímica ósmio-imidazol, organelas necessárias para o metabolismo energético do parasito. Para quantificar a distribuição de CLs entre os dias de cultivo e entre as cepas, foi realizada análise por citometria de fluxo com Bodipy® 493/503. Os resultados indicaram que a cepa responsável pela LMC apresentou maior quantidade de CLs durante a fase estacionária tardia. Na cepa LCL não foi observado diferença significativa entre as fases estudadas. Assim, pode ser sugerido que a exacerbada resposta inflamatória que ocorre em pacientes com LMC, esteja relacionada com o acúmulo de CLs no parasito, fonte de energia e eicosanoides, como prostaglandinas. Outra hipótese é a possível correlação de CLs com a baixa exposição do fosfolipídio fosfatidilserina para a superfície externa da membrana, importante para a infectividade do parasito. Para análise dos lipídios totais, os parasitos foram submetidos à extração lipídica, seguido da técnica de HPTLC, onde foram encontrados predominantemente fosfolipídios, esterol esterificado, esteróis, triglicerídeos e ácidos graxos compondo o parasito, com variações entre as cepas e entre as fases estudadas. A cepa LCL na fase estacionária tardia possui maior quantidade de lipídios totais, que pode ser justificado por já ser conhecida como a cepa mais infectiva e possivelmente apresentar maior quantidade de glicoconjugados associados com subdomínios lipídicos importantes para o reconhecimento de fagócitos. É importante ressaltar que a maior infectividade da cepa LCL quando comparada à cepa LMC, resulta em um menor processo inflamatório. Estes resultados indicam que há uma variação no perfil lipídico e na distribuição de CLs entre as diferentes cepas de L. (V.) braziliensis, que pode estar relacionado com a infectividade do parasito e com a manifestação clinica da doença.

Caracterização por espectrometria de massas e investigação das atividades anti-inflamatória e gastroprotetora do extrato etanólico e diterpenos clerodânicos de folhas de Casearia sylvestris Swartz

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização por espectrometria de massas e investigação das atividades anti-inflamatória e gastroprotetora do extrato etanólico e diterpenos clerodânicos de folhas de Casearia sylvestris Swartz

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^