The Forensic Chemistry Laboratory of Johannesburg (FCL JHB) is tasked with the chemical analysis of a variety of samples to assist in determining the cause of death where unnatural cause is suspected. Some of the samples submitted to the laboratory have a herbal or muti connotation, but a large portion of these cases turn out to have no herbal components present as only pharmaceutical or agricultural products are detected in these samples. This study combined, for the first time, forensic investigation, chemistry and botany to create a unique platform needed for the identification of poisonous plants and their components in forensic exhibits and viscera. The research was focussed on the poisonous plants previously detected at the laboratory, as well as the requests received for the analysis of muti/toxic plant components. The selection of plants included Nicotiana glauca, Datura stramonium / Datura ferox, Callilepis laureola, Boophone disticha / Ammocharis coranica, Abrus precatorius, Ricinus communis, Nerium oleander / Thevetia peruviana and Bowiea volubilis. All these species are known to have caused fatalities, hence their choice. Nicotiana glauca has been implicated in the deaths of at least 15 people since 2001. It was previously detected by GC-MS (EI) in plant exhibits, but could not be detected in a viscera matrix. A selective extraction method for alkaloids was used to extract botanical and viscera samples. Anabasine was successfully detected on the HPLC-MS (EI) system but this detection technique was not considered sensitive enough. A very sensitive HPLC-MS method was developed on the ZMD detector by using electrospray technology. This method outperformed both electron impact detectors (GC and HPLC) and could detect 1ng/ml anabasine with relative ease in full scan mode. Datura stramonium and D. ferox have not been previously positively linked to any human poisoning or death due to exposure to botanically derived products at the FCL JHB. Atropine and scopolamine were successfully ionised in ESI positive mode and could be detected at 10 pg/ml and 100 pg/ml level respectively. The identities of the compounds were confirmed by characteristic ISCID fragmentation patterns. The developed method was successfully applied to a suspected heart attack case. The results proved conclusively that the deceased was given D. ferox seeds as part of his meal and an overdose of atropine and scopolamine contributed to his death. Callilepis laureola is reputed to be one of the more commonly used medicinal plants in South Africa, and although its use has been indicated by the specific mention of a possible nephrotoxin and/or hepatotoxin as causative agent, it has not been detected in any of the forensic chemistry laboratories in South Africa. This was mainly due to the absence of a reliable method for the analysis of the main toxic component of C. laureola, atractyloside, by mass spectrometry. A sensitive and very selective HPLC-ESI-MS method was developed that could detect atractyloside, carboxyatractyloside and their monodesulfated analogues in botanical and viscera matrices. The method was successfully applied to a variety of forensic samples and proved that C. laureola may play an important role in herbal poisonings. In a selection of suspected herbal poisonings where the cause of poisoning was unknown, 30% of the samples tested positive for the presence of atractyloside, carboxyatractyloside or their monodesulfated analogues. The bulbs of Boophone disticha are rich in isoquinoline alkaloids and some of the alkaloids were detected by GC-EI-MS and LC-EI-MS, but the detection of these alkaloids in viscera samples was not successful. A routine method used for the screening for drugs of abuse in forensic samples, were successfully used for the analysis of the bulb extracts of B. disticha and the bulb scales of A. coranica. The chromatographic profile of these two plants appeared very similar at a first glance, but a closer evaluation of the mass spectra highlighted significant differences between the two plants. Six alkaloids from B. disticha were isolated and characterised by LC-MS and NMR and these compounds were detected in suspected herbal poisoning cases. It has been shown that B. disticha is one of the commonly used plants to “clean the system” but frequently results in the death of the patient. Abrus precatorius contains one of the most toxic compounds known to mankind, namely abrin that collectively refers to a group of glycoproteins. The seeds of A. precatorius also contain two indole alkaloids, abrine and hypaphorine. The two alkaloids were fractionated and characterised by LC-MS and NMR. Due to the fact that the instrumentation of the FCL JHB is not suited to the detection of proteins, an LC-ESI-MS method was developed for the detection of the two alkaloids in plant and viscera matrix as markers for A. precatorius. The presence of these two alkaloids was indicated on the TMD system (EI spectra) in a suspected herbal poisoning case. The LC-ESI-MS method was applied to the analysis of the samples and the absence of abrine and hypaphorine were proven in the samples. Ricinus communis is similar to A. precatorius in that it also contains a group of extremely poisonous glycoproteins, collectively refered to as ricin. The analysis of R. communis seeds encountered the same problems as the analysis of A. precatorius seeds, and the analysis was again focused on the detection of the minor piperidine alkaloid ricinine. The LC-ESI-MS method developed for abrine was modified to detect ricinine and functioned well in botanical and viscera matrices. This method will enable the forensic analyst to detect ricinine in very low levels when the presence of ricinoleic acid in samples indicates the use of a R. communis-based product. Nerium oleander is a common decorative garden plant that is used medicinally. The plant is rich in cardenolides with oleandrin the main compound. A reversed-phase chromatographic method with ESI mass spectral detection was developed to separate and detect 11 cardiac glycosides. The compounds were adequately separated to allow unambiguous identification, and displayed very stable cationisation with sodium. An extraction method was developed to extract the cardiac glycosides from the leaves of N. oleander and Thevetia peruviana and was also evaluated in a viscera matrix. The extraction method functioned well and extracted a variety of compounds that produced unique chromatographic fingerprints, allowing for the easy differentiation between the two plants. The method is ideally suited for the detection of oleandrin in high concentrations (full scan mode), low concentrations (selected masses) or trace levels (SIM analysis of ion clusters). The method is able to distinguish between extracts derived from N. oleander and T. peruviana and was able to detect and confirm neriifolin, odoroside and neritaloside in N. oleander leaf extracts. Analysis of forensic case exhibits were also successfully done with this method and performed well with liquid and solid matrices. With the new method oleandrin could be detected at trace levels in viscera samples that did not produce positive results in the past. Bowiea volubilis is widely used as a medicinal plant, but is also an extremely toxic plant. It is freely available at traditional healer markets, and is one of the most highly traded plants on the Durban market. Despite the high usage of the plant, it has not been detected by any of the forensic laboratories in South Africa. Bovoside A, a bufadienolide, is reported to be the main cardiac glycoside in the bulb of B. volubilis. The cardiac glycoside method was successfully applied to the analysis of the bulb extract of B. volubilis and bovoside A was identified as the main bufadienolide present in the bulb. Bovoside A was fractionated and characterised by LC-MS. Four extracts of botanical origin could be successfully distinguished from each other by monitoring the main masses of bovoside A, oleandrin and thevetin A and thevetin B. These marker compounds were well separated from each other and made the identification of the botanical extracts quite easy, and the identity of each extract was confirmed by the mass spectrum of each peak. / Prof. F.R van Heerden

Introduction: The macroporous Amberlite XAD resins were coated with LIX-64N and examined for the rate of uptake of copper . XAD-7 was by far the best support and gave a satisfactory rate of uptake up to loadings of 60% (w/w). The specific surface area of XAD-7 was measured by the adsorption of methylene blue from aqueous solution. The area of the wetted resin was five times less than that of the dry resin. LIX-65N was purified and the anti isomer characterised using spectroscopic techniques . The rate of uptake of copper was not improved by use of purified LIX-65N or by addition of LIX-63. XAD-7 coated with LIX-65N was used in columns. Elution curves for copper showed negligible tailing, and rapid separations of copper from iron (111), nickel, cobalt and magnesium by selective absorption were achieved. Copper was concentrated from very dilute solution at a flow rate of 50 ml min -1 ,and a 99% recovery was obtained. The method was applied to the rapid determination of copper in brass and bronze. A series of long-chain hydroxamic acids were synthesised and tested for suitability as stationary phase on XAD-7. Oleohydroxamic acid and naphthenohydroxamic acid were the most promising. The r ate of uptake of copper was reduced by the use of nonylphenol or amyl alcohol as a diluent. The capacities for copper of the hydroxamic acids were less when supported on XAD-7 than when used as liquid ionexchangers. The distribution coefficients of cobalt, nickel, zinc, lead, vanadium, uranium, iron (111) and copper were measured as a function of pH. XAD-7 coated with oleohydroxamic acid was used in columns for the rapid separation of iron (111) from copper and of copper from nickel, cobalt, lead and zinc. Copper was concentrated from very dilute solution at a flow rate of 45 ml min -1 and a 100,8% recovery was obtained. Copper was successfully separated from nickel by selective elution. The elution curves obtained show negligible tailing. The resin loaded with oleohydroxamic acid lost capacity due to chemical instability. Naphthenohydroxamic acid supported on XAD-7 was not suitable for use in columns, because it was physically unstable.

The aim of this study was to study the extraction of zinc from the mineral sphalerite, especially the leaching of concentrate recovered from the Rosh Pinah tailings by means of ferric chloride. To this end, the literature on zinc processing was surveyed and knowledgeable persons were consulted. The study also addressed the leaching kinetics of an upgrade Rosh Pinah tailings dam concentrate as well as those of a synthetic zinc sulphide in a ferric chloride medium. Valuable results were obtained, from the leaching of sphalerite concentrate in ferric chloride medium. An activation energy value of 45.82 kJ/mol was obtained, which compares well with what has been published in the literature. A chemical control model and a diffusion control model were applied to the data obtained. From neither of the models a straight-line relationship could be deduced over the leaching range. At t < 45 minutes it seems that the process is controlled by chemical reaction at the interface; at t > 45 minutes it seems that the process is controlled by diffusion through the product layer. If therefore seems that the rate-controlling step can be related to the process of diffusion through the product layer. The mixed control model proposed by Huang and Rowson, [1-(1-x)1/3+y/6[(1-x)1/3+1-2(1-x)2/3]=kMt, was applied to data obtained during this study. The resultant graphical fit was near perfect, indicating that sphalerite leached in ferric chloride follows a mixed control mechanism for the conditions reported in the study. An activation energy of 20.71 kJ/mol was determined for this model by using the following equation: / Dissertation (MSc)--University of Pretoria, 2006. / Chemistry / unrestricted

This thesis presents the development of chlorophyll, cholic acid, aflatoxin B1 molecularly imprinted polymer (MIP) particles and cholic acid MIP nanofibers for application as selective solid phase extraction (SPE) sorbents. The particles were prepared by bulk polymerization and the nanofibers by a novel approach combining molecular imprinting and electrospinning technology. The AFB1 MIP particles were compared with an aflatoxin specific immunoextraction sorbent in cleaning-up and pre-concentrating aflatoxins from nut extracts. They both recorded high extraction efficiencies (EEs) of > 97 % in selectively extracting the aflatoxins (AFB1, AFB2, AFG1 and AFG2). High reproducibility marked by the low %RSDs of < 1% and low LODs of ≤ 0.02 ng/g were calculated in all cases. The LODs were within the monitoring requirements of the European Commission. The results were validated with a peanut butter certified reference material. The chlorophyll MIP on the other hand selectively removed chlorophyll that would otherwise interfere during pesticide residue analysis (PRA) from > 0.6 to <0.09 Au in green plants extracts. The extracted chlorophyll was removed to far below the level of ≥ 0.399 Au that is usually associated with interference during PRA. Furthermore, the MIP demonstrated better selectivity by removing only chlorophyll (> 99%) in the presence of planar pesticides than the currently employed graphitized carbon black (GCB) that removed both the chlorophyll (> 88%) and planar pesticides (> 89%). For the interfering cholic acid during drug residue analysis, cholic acid MIP electrospun nanofibers demonstrated to be more sensitive and possessing higher loading capacity than the MIP particles. 100% cholic acid was removed by the nanofibers from standard solutions relative to 80% by the particles. This showed that the nanofibers have better performance than the micro particles and as such have potential to replace the particle based SPE sorbents that are currently in use. All the templates were optimally removed from the prepared MIPs by employing a novel pressurized hot water extraction template removal method that was used for the first time in this thesis. The method employed only water, an environmentally friendly solvent to remove templates to ≥ 99.6% with template residual bleeding of ≤ 0.02%.

This thesis explores the robustness and the versatility of pressurized hot water extraction (PHWE) for a variety of analytes and matrices. Applications discussed include: selective extraction of alkaloids in goldenseal followed by their degradation studies; in-cell clean-up of pesticides in medicinal plants employing custom made molecularly imprinted polymers (MIPs) sorbents; in-cell pre-concentration followed by desorption of aflatoxins in plants with MIPs; desorption of pesticides from electrospun nanofiber sorbents; and removal of templates from MIPs sorbents. It was demonstrated that selective extractions could be achieved by just changing the temperature of water while adjusting the pressure. For instance, the alkaloids in goldenseal (hydrastine and berberine), were extracted at 140 °C, 50 bars, 1 mL min⁻¹ in 15 min; organochlorine pesticides from medicinal plants were extracted at 260 °C, 80 bars, 1 mL min-1 in 10 min; while aflatoxins AFG2, AFG1, AFB2 and AFB1 were extracted at 180 °C, 60 bars and a flow rate of 0.5 mL min⁻¹ in 10 min. The selectivity of PHWE was further enhanced by combining it with selective MIPs sorbents at higher temperatutes. In-cell clean-up of interfering chlorophyll was successfully removed from the medicinal plants during pesticides analysis while clean-up of aflatoxins AFG2, AFG1, AFB2 and AFB1 was achieved in two extraction cells connected in series. Ultrasound was also combined with PHWE for extraction of hydrastine and berberine at 80 °C and 40 bars in 30 min. PHWE was further evaluated for removal of templates from quercetin, phthalocynine and chlorophyll MIPs. The templates were thoroughly washed off their MIPs within 70 min with PHWE compared to over 8 h for Soxhlet and ultrasound assisted extraction. Pesticides were also desorbed from electrospun nanofibers at 260 °C, 80 bars in 10 min employing only water at 0.5 mL min⁻¹. In the light of green chemistry, the decrease in the usage of organic solvents was 100%, resulting in no organic solvent waste.

The thesis reports on fabricating alternative solid phase extraction (SPE) sorbents and colorimetric probes based on electrospun nanofibers for alkylphenols (APs). Hydroxyl methylated styrene [poly(co-styrene-CH₃OH)] and 3-oxobutanoate styrene [poly(co-styrene-OCOCH₃COCH₃)] copolymers were synthesized and fabricated into sorbent materials by electro-spinning/spraying. The fabricated morphologies consisting of bead free fibers, beaded fibers and particles were evaluated as SPE sorbents using batch experiments. Electropun fibers proved to be better sorbents as they exhibited extraction efficiency that exceeded 95% compared to 60% for beaded fibers and 40% for particles. In view to reduce sample and solvent volumes, smooth fibers were packed into pipette tips as SPE devices that yielded quantitative recoveries of APs from spiked wastewater samples. Recoveries ranged from 70% to 125% with LOD of 0.008, 0.01 and 0.1 μg mL⁻¹ for 4-tert octylphenol (4-t-OP), 4-octylphenol (4-OP) and 4-nonylphenol (4-NP) respectively, when using high performance liquid chromatography-fluorescence detector (HPLC-FLD). Furthermore, amino functionalised polydiacetylene polymers (PDAs), citrate capped gold (AuNPs) and silver nanoparticles (AgNPs) were evaluated as colorimetric probes for visual detection of APs. In colloidal studies, AuNPs probe showed a colour change from wine red to green upon introduction of analyte. UV-vis spectroscopy revealed the shifting of the surface plasmon resonance (SPR) peak from 525 nm to 729 nm induced by aggregation of AuNPs. For AgNPs probe, a colour change was observed from yellowish green to brown. Transmission electron microscopy (TEM) studies showed growth of AgNPs. A presumed oxidation of the analyte, forming an absorbing compound at 279 nm in both AgNPs and PDAs probes was also observed. For PDAs probe the colour change was from purple to pink. Concentrations as low as 30 μg mL⁻¹ were detectable in all colloidal based probes. Further colorimetric investigations were conducted with electrospun AuNPs-nylon 6 fiber mat. A colour change from purplish red to navy blue at concentrations of 1000 μg mL⁻¹ was observed. Electrospun AgNPs –nylon 6 fiber mat did not show a distinct colour change. High resolution scanning electron microscopy (HRSEM) revealed the analyte inducing the assembly of AuNPs and AgNPs as they covered the surface of the nanofiber mat. Electrospun nanofibers are a platform for analysis and thus tuning their chemistry will lead to sensitive and selective methods

L'efficacité des antibiotiques actuellement utilisés dans le monde entier est en baisse à un rythme inquiétant. La majorité des produits actifs naturels sont isolés des ascomycètes ou des Actinobactéries. Parmi les 10 000 antibiotiques connus, plus de la moitié sont produits par des bactéries du genre Streptomyces. Il est donc intéressant de rechercher de nouvelles molécules actives dans des niches encore sous explorés, tels que les symbioses microbiennes mutualistes. Ainsi, les lichens sont des organismes complexes abritant des communautés bactériennes à la surface et, plus rarement, à l'intérieur de leurs thalles et constituent un modèle d’étude pour la découverte de nouvelles molécules d’intérêts. Une optimisation des conditions d'extraction des lichens a été développée. Le profilage chimique par LC / MS de neuf lichens (2 à algues vertes : Roccella fuciformis et R. phycopsis et de 7 cyanolichens: Lichina confinis, L. pygmaea, Leptogium lichenoides, Synalissa symphorea, Collema auriforme, C. cristatum et C. fuscovirens) ont été effectués et comparés avec des approches de «molecular network". Cela a permis de souligner la similitude chimique entre tous les cyanolichens d’une part et les espèces lichéniques à algues vertes d’autre part. Une étude chimique plus approfondie de R. fuciformis et R. phycopsis a été par la suite effectuée et dix composés différents ont été isolés et identifiés. Neuf d'entre eux ont été isolés et identifiés par RMN et des voies de fragmentation ont été proposés pour cinq d'entre eux. Une étude de localisation in situ de leurs métabolites majeurs respectifs (érythrine et acide roccellique pour R. phycopsis et érythrine, acide léprarique et acetylportentol pour R. fuciformis) a été réalisée et a démontré leur emplacement spécifique au sein des thalles lichéniques. Les communautés bactériennes cultivables associées à trois lichens de la côte bretonne (France) (Roccella fuciformis, Lichina confinis, L. pygmaea) et un lichen terrestre récolté en Autriche (Collema auriforme) ont été étudiées afin de trouver de nouveaux métabolites d'intérêts. L'abondance et la diversité des communautés bactériennes associées à ces lichens a été montré: 247 souches ont été isolées et identifiées par l’étude du gène de l'ARNr 16S. Ainis, plus de 30% de toutes les souches expriment des gènes permettant la production potentielle des composés bioactifs et 12% appartiennent probablement à de nouvelles espèces bactériennes. Les métabolites secondaires de deux bactéries cultivables associées ont été étudiés (MOLA1488, Streptomyces sp. et MOLA1416, Hoeflea sp.) et certains métabolites spécialisés actifs ont été isolés (des dicétopipérazines, des alcaloïdes, des dérivés phénoxazine par exemple ...) présentant des propriétés biologiques intéressantes. Enfin, pour mettre en évidence les interactions possibles entre les lichens et leurs bactéries associées, une approche de culture (extraits lichéniques et bactéries associées) a été réalisée à partir de 4 souches bactériennes les plus abondantes associées à Roccella fuciformis pour (1) évaluer l'impact de ces métabolites sur la croissance de ces quatre souches et également, (2) à évaluer la capacité de bioconversion de l'acide leprarique et de l’érythrine. Ces bactéries ont montré la capacité de bio-converser l’érythrine en acide orsellinique, mais aucun des quatre métabolites testés n’a affecté leur croissance. / Efficiency of currently used antibiotics is worldwide decreasing at a worrying rate, while we are faced with new and emerging pathogens. The majority of active natural products are isolated from the Ascomycetes or from the Actinobacteria. Among the 10000 known antibiotics, more than half are produced by bacteria of one single genus, Streptomyces. It is therefore most interesting to search for novel active molecules in yet under explored niches, such as mutualistic microbial symbioses. Lichens are complex organisms harboring bacterial communities on the surface and, more rarely, inside their thalli and present a model to discover new biomolecules. Optimization of extraction conditions of lichens has been developed. Chemical profiling by LC / MS of nine lichens (2 green algae Roccella fuciformis and R. phycopsis and 7 cyanolichens: Lichina confinis, L. pygmaea, Leptogium lichenoides, Synalissa symphorea, Collema auriforme, C. cristatum and C. fuscovirens) were made and compared with a "Molecular network" approach. This has allowed to identify the chemical similarity between all cyanolichens and between two lichen species containing green algae. On the other hand, further chemical study on R. fuciformis and R. phycopsis was conducted and ten different compounds were isolated. Nine of them have been isolated and identified by NMR and mass fragmentation pathways have been highlighted for five of them. In situ localization of their major respective metabolites (erythrin and roccellic acid for R. phycopsis and erythrin, lepraric acid and acetylportentol for R. fuciformis) was performed and showed a specific location in the lichen thallus. We focused also on the cultivable bacterial communities associated to three lichens from Brittany coast (France) (Roccella fuciformis, Lichina confinis, L. pygmaea) and one inland lichen from Austria (Collema auriforme) to find new secondary metabolites of interest. The abundance and the diversity of the bacterial communities associated to these lichens were showed: 247 strains were isolated and identified by 16S rRNA gene analysis. More than 30% of all strains express potential bioactive compounds and 12% represent probably new species. The secondary metabolites patterns of their cultivable associated bacteria were studied (MOLA1488, Streptomyces sp. and MOLA1416, Hoeflea sp.) and some active secondary metabolites were isolated (e.g. dicetopiperazines, pyrrole alkaloïds, phenoxazine derivatives …) showing biological properties. Finally, to highlight potential interactions between lichens and their associated bacteria, an approach of culture (lichen extracts and bacteria) was performed from 4 most abundant bacterial strains associated with Roccella fuciformis to (1) assess the impact of major metabolites (compounds 4) of this lichen on the growth of these four strains by a an optimized method of viability using MTT; and also to evaluate (2) the ability to bioconversion of these four strains of lepraric acid and erythrin. These bacteria have shown the ability to metabolize erythrin in orsellinic acid, but none of the four tested metabolites has affected their growth.

A própolis vermelha é uma resina natural produzida por abelhas do gênero Apis a partir de exsudados vegetais e tem atraído a atenção dos pesquisadores de todo o mundo devido à sua potente atividade em diversos modelos biológicos. Essa resina natural é rica em polifenóis e compostos voláteis, sendo que mais 300 compostos químicos já foram identificados em amostras de diferentes regiões. Técnicas de identificação química de alta precisão são imprescindíveis para a correta elucidação dos compostos químicos presentes nas amostras e para o entendimento dos efeitos biológicos. Neste estudo, buscou-se utilizar técnicas de fracionamento como uma metodologia capaz de ser explorada em estudos envolvendo amostras de própolis vermelha, buscando-se a identificação e caracterização de frações ativas, bem como o uso de modelos celulares de câncer de cólon para o screening de moléculas bioativas. As frações ativas foram identificadas por meio HPLC-MS e ESI-MS/MS, evidenciando uma composição química complexa baseada em compostos fenólicos e flavonoides. Entre os resultados obtidos, destacam-se as frações 05 e 06, as quais foram capazes de produzir inibição significativa sobre as linhagens tumorais HT-29 e HCT-116, respectivamente, com menores efeitos sobre as células não-tumorais. As frações obtidas no estudo apresentaram menor diversidade química do que a encontrada no extrato total da própolis, favorecendo a sua bioatividade in vitro, e, como resultado, esta técnica pode favorecer a descoberta e o desenvolvimento de novos fármacos utilizando a própolis como fonte de potencial de moléculas contra o câncer. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES. / Red propolis is a natural resin produced by bees of the genus Apis from plant exudates and has attracted the attention of researchers worldwide due to its potent activity in several biological models. This natural resin is rich in polyphenols and volatile compounds and more than 300 chemical compounds have been identified in samples from different regions. High precision chemical identification techniques are essential for the correct elucidation of the chemical compounds present in the samples and for the understanding of the biological effects. In this study, we sought to use fractionation techniques as a methodology able to be explored in studies involving samples of red propolis, seeking the identification and characterization of active fractions, as well as the use of cellular models of colon cancer for screening of bioactive molecules. The active fractions were identified by techniques of HPLC-MS and ESI-MS/MS, evidencing a complex chemical composition based on phenolic compounds an flavonoids. Among the obtained results, we highlight the fractions 05 and 06, which are able to produce significant inhibition on the HT-29 and HCT-116 tumor lines.The fractions obtained in this study showed lower chemical diversity than that found in the total extract of propolis, favoring its bioactivity, and as a result, this technique may favor the discovery and development of new drugs using propolis as a potential source of molecules against cancer.

No description available.

Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the DNA found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the DNA originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mRNA profiling to be utilized in routine forensic casework, RNA of sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current DNA analysis methodologies. Several DNA/RNA co-extraction methods were evaluated based on the quantity and quality of DNA and RNA recovered and were also compared to standard non-co-extraction methods. The two most promising methods, the in-house developed NCFS co-extraction and the commercially available AllPrep DNA/RNA Mini kit, were then optimized by improving nucleic acid recovery and consistency of CE (capillary electrophoresis) detection results. The sensitivity of the two methods was also evaluated, and DNA and RNA profiles could be obtained for the lowest amount of blood (0.2 µL) and saliva and semen (1 µL) tested. Both extraction methods were found to be acceptable for use with forensic samples, and the ability to obtain full DNA profiles was not hindered by the co-extraction of RNA. It is generally believed that RNA is less stable than DNA which may prevent its use in forensic casework. However, the degradation rates of DNA and RNA in the same biological fluid stain have not been directly compared. To determine the relative stability of DNA and RNA, the optimized NCFS co-extraction protocol was used to isolate DNA and RNA from iv environmentally compromised stains. Dried blood, saliva, and semen stains and vaginal secretions swabs were incubated at set temperatures and outside for up to 1 year. Even at 56°C, DNA and RNA were both stable out to 1 year in the blood and semen stains, out to 3 months (DNA) and 1 year (RNA) in the saliva stains, and out to 6 months (DNA) and 3 months (RNA) in the vaginal secretions swabs. The recoverability of both nucleic acids was reduced when the samples were exposed to increased humidity, sunlight, and rain. In general, DNA and RNA stability was found to be similar with a loss in ability to obtain a DNA or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no DNA profile was obtained, indicating that RNA in dried stains is sufficiently stable for mRNA body fluid typing to be used in forensic casework.