Blood ethanol is a widely accepted marker for recent ethanol intake (within the last 4-6 h). However, the rapid elimination of ethanol from the body nearly always makes it impossible to assess ethanol ingestion beyond the most recent 6-8h. But serum fatty acid ethyl ester (FAEE) is detectable in the blood both when ethanol is presented and long after ethanol has been removed from the circulation. For this FAEE should be better marker for prior ethanol intake.

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A matéria-prima na produção de biodiesel corresponde a mais que 70% do seu custo e o estudo de viabilidade tecnológica e econômica das diferentes matérias-primas se reveste de enorme importância. A extração do óleo de soja com solvente etanol resulta em duas miscelas, uma rica em óleo (miscela rica) e outra rica em etanol (miscela pobre). A miscela pobre pode ser reutilizada no processo de extração e a miscela rica pode ser utilizada diretamente sem a necessidade de dessolventização e de etapas de refino. A miscela rica em óleo foi esterificada por dois processos diferentes: químico e enzimático, com diferentes concentrações em razão molar (óleo:etanol), diferentes temperaturas e catalisadores básico (NaOH) ou enzimático (Novozym®435), buscando o maior rendimento em ésteres etilícos. O objetivo desse estudo foi avaliar o rendimento de ésteres etílicos aplicando enzimas imobilizadas Novozym®435 e um catalisador básico (NaOH) e analisar a viabilidade energética da produção de biodiesel a partir da transesterificação da miscela rica (óleo:etanol) em óleo de soja, sem necessidade de refino do óleo. Foi adotado o planejamento experimental e análise de superfícies de resposta para a seleção das melhores condições de processo, tendo como variáveis respostas o rendimento e a qualidade do biodiesel. Foram realizados ensaios de esterificação via enzimática e química. A reutilização da enzima foi estudada através da lavagem com diferentes solventes (etanol 96%, isopropanol e terc-butanol) e reações de transesterificação na presença do co-solvente terc-butanol. A produção de ésteres em miscelas permitiu a comparação dos custos entre o processo de catálise enzimática e catálise química com base na análise dos fluxos de materiais e energia. A miscela rica foi obtida após três banhos com miscela pobre e um último banho com etanol 99% (v/v), apresentando eficiência de 83% e um teor de óleo residual no farelo de 4,2%. Em sua composição, a miscela rica apresentou 90% em óleo de soja e até 7% de etanol. A transesterificação de miscela rica com catalisador NaOH foi otimizada e apresentou rendimento de ésteres etílicos (RE) 97,2% nas condições experimentais de: razão molar 1:12, concentração de catalisador 0,67% e temperatura de 30ºC. Na transesterificação enzimática, o rendimento máximo foi de 85% nas condições reacionais de razão molar 1:4,5, concentração de catalisador 9,5% e temperatura de 40ºC. A Novozym®435 não foi recuperada com sucessivas lavagens dos solventes. Entretanto, o terc-butanol como co-solvente aumentou o rendimento de ésteres para 94%. A análise dos fluxos de energia demonstrou que o a obtenção da matéria-prima (laminação e extração) foi a etapa que mais demandou energia. A produção de miscela rica em escala semi-piloto demandou mais energia que a de óleo refinado, porém, a etapa de transesterificação a partir de miscela rica, utilizando o catalisador químico, demandou menos energia comparada ao processo com catalisador enzimático e o convencional com metanol e etanol. A esterificação de miscela rica é energeticamente viável, entretanto, com um scale up adequado, a etapa de extração com etanol deve ser ajustada para viabilizar energeticamente a cadeia de produção de biodiesel por esta via alternativa. / The feedstock for biodiesel production represents more than 70% of the cost and technological and economic feasibility studies of different oil sources are of enormous importance. The extraction of soybean oil with ethanol solvent results in two miscella, one rich in oil (rich miscellae) and another rich in ethanol (poor miscellae). The poor miscellae can be reused in the extraction process and the rich miscellae can be used directly without dessolventizing and refining stages. The oil rich miscellae was esterified by two different processes: chemical and enzymatic, with different concentrations in the molar ratio (oil: ethanol), different temperatures and either basic catalyst (NaOH) or an enzyme (Novozym®435), searching for the highest production of ethyl esters. The study goal was to prove the feasibility of producing biodiesel from the transesterification of rich miscellae (oilethanol) in soybean oil, without oil refining and evaluating the performance of ethyl esters by applying immobilized enzymes Novozym®435 and a basic catalyst (NaOH). We adopted the experimental design and the surface response methodology for the best selection of process conditions, with the response variables the yield and the quality of biodiesel. Chemical and enzymatic esterification trials were conducted. The reuse of enzyme was studied by washing with different solvents (96% ethanol, isopropanol and tert-butanol) and the transesterification reaction in the presence of the co-solvent tert-butanol. The production of esters by enzymes in the miscellae allowed a comparison of the costs between the enzymatic and chemical catalysis process based on the energy flow analysis. The rich miscellae was obtained after three baths employing the poor miscellae and a last fourth bath with ethanol 99% (v/v), presenting efficiency of 83% and a residual meal oil content of 4.2%. In its composition, the rich miscellae showed 90% of soybean oil and up to 7% ethanol. The transesterification of the rich miscellae with NaOH catalyst was optimized and had a ethyl esters yield (RE) of 97.2% under the experimental conditions of: 1:12 molar ratio, catalyst concentration 0.67% and temperature 30° C. For the enzymatic transesterification, the maximum yield was 85% for the reaction conditions: molar ratio 1:4.5, catalyst concentration 9.5% and temperature 40° C. Novozym®435 was not recovered with successive washes of the solvents. However, the tertbutanol as a co-solvent increased the yield of esters to 94%. The energy flow analysis showed that obtaining the raw material (flaking and extraction) was the most energy demanding. The rich miscellae from the semi-pilot plant demanded more energy than the refined oil, however, the transesterification of the rich miscellae using chemical catalyst, required less energy compared to the enzymatic catalysis and the conventional process methanol and ethanol. The esterification of rich miscellae is feasible energetically, however, the extraction step with ethanol should be adjusted to enable energetically the chain of biodiesel production.

A matéria-prima na produção de biodiesel corresponde a mais que 70% do seu custo e o estudo de viabilidade tecnológica e econômica das diferentes matérias-primas se reveste de enorme importância. A extração do óleo de soja com solvente etanol resulta em duas miscelas, uma rica em óleo (miscela rica) e outra rica em etanol (miscela pobre). A miscela pobre pode ser reutilizada no processo de extração e a miscela rica pode ser utilizada diretamente sem a necessidade de dessolventização e de etapas de refino. A miscela rica em óleo foi esterificada por dois processos diferentes: químico e enzimático, com diferentes concentrações em razão molar (óleo:etanol), diferentes temperaturas e catalisadores básico (NaOH) ou enzimático (Novozym®435), buscando o maior rendimento em ésteres etilícos. O objetivo desse estudo foi avaliar o rendimento de ésteres etílicos aplicando enzimas imobilizadas Novozym®435 e um catalisador básico (NaOH) e analisar a viabilidade energética da produção de biodiesel a partir da transesterificação da miscela rica (óleo:etanol) em óleo de soja, sem necessidade de refino do óleo. Foi adotado o planejamento experimental e análise de superfícies de resposta para a seleção das melhores condições de processo, tendo como variáveis respostas o rendimento e a qualidade do biodiesel. Foram realizados ensaios de esterificação via enzimática e química. A reutilização da enzima foi estudada através da lavagem com diferentes solventes (etanol 96%, isopropanol e terc-butanol) e reações de transesterificação na presença do co-solvente terc-butanol. A produção de ésteres em miscelas permitiu a comparação dos custos entre o processo de catálise enzimática e catálise química com base na análise dos fluxos de materiais e energia. A miscela rica foi obtida após três banhos com miscela pobre e um último banho com etanol 99% (v/v), apresentando eficiência de 83% e um teor de óleo residual no farelo de 4,2%. Em sua composição, a miscela rica apresentou 90% em óleo de soja e até 7% de etanol. A transesterificação de miscela rica com catalisador NaOH foi otimizada e apresentou rendimento de ésteres etílicos (RE) 97,2% nas condições experimentais de: razão molar 1:12, concentração de catalisador 0,67% e temperatura de 30ºC. Na transesterificação enzimática, o rendimento máximo foi de 85% nas condições reacionais de razão molar 1:4,5, concentração de catalisador 9,5% e temperatura de 40ºC. A Novozym®435 não foi recuperada com sucessivas lavagens dos solventes. Entretanto, o terc-butanol como co-solvente aumentou o rendimento de ésteres para 94%. A análise dos fluxos de energia demonstrou que o a obtenção da matéria-prima (laminação e extração) foi a etapa que mais demandou energia. A produção de miscela rica em escala semi-piloto demandou mais energia que a de óleo refinado, porém, a etapa de transesterificação a partir de miscela rica, utilizando o catalisador químico, demandou menos energia comparada ao processo com catalisador enzimático e o convencional com metanol e etanol. A esterificação de miscela rica é energeticamente viável, entretanto, com um scale up adequado, a etapa de extração com etanol deve ser ajustada para viabilizar energeticamente a cadeia de produção de biodiesel por esta via alternativa. / The feedstock for biodiesel production represents more than 70% of the cost and technological and economic feasibility studies of different oil sources are of enormous importance. The extraction of soybean oil with ethanol solvent results in two miscella, one rich in oil (rich miscellae) and another rich in ethanol (poor miscellae). The poor miscellae can be reused in the extraction process and the rich miscellae can be used directly without dessolventizing and refining stages. The oil rich miscellae was esterified by two different processes: chemical and enzymatic, with different concentrations in the molar ratio (oil: ethanol), different temperatures and either basic catalyst (NaOH) or an enzyme (Novozym®435), searching for the highest production of ethyl esters. The study goal was to prove the feasibility of producing biodiesel from the transesterification of rich miscellae (oilethanol) in soybean oil, without oil refining and evaluating the performance of ethyl esters by applying immobilized enzymes Novozym®435 and a basic catalyst (NaOH). We adopted the experimental design and the surface response methodology for the best selection of process conditions, with the response variables the yield and the quality of biodiesel. Chemical and enzymatic esterification trials were conducted. The reuse of enzyme was studied by washing with different solvents (96% ethanol, isopropanol and tert-butanol) and the transesterification reaction in the presence of the co-solvent tert-butanol. The production of esters by enzymes in the miscellae allowed a comparison of the costs between the enzymatic and chemical catalysis process based on the energy flow analysis. The rich miscellae was obtained after three baths employing the poor miscellae and a last fourth bath with ethanol 99% (v/v), presenting efficiency of 83% and a residual meal oil content of 4.2%. In its composition, the rich miscellae showed 90% of soybean oil and up to 7% ethanol. The transesterification of the rich miscellae with NaOH catalyst was optimized and had a ethyl esters yield (RE) of 97.2% under the experimental conditions of: 1:12 molar ratio, catalyst concentration 0.67% and temperature 30° C. For the enzymatic transesterification, the maximum yield was 85% for the reaction conditions: molar ratio 1:4.5, catalyst concentration 9.5% and temperature 40° C. Novozym®435 was not recovered with successive washes of the solvents. However, the tertbutanol as a co-solvent increased the yield of esters to 94%. The energy flow analysis showed that obtaining the raw material (flaking and extraction) was the most energy demanding. The rich miscellae from the semi-pilot plant demanded more energy than the refined oil, however, the transesterification of the rich miscellae using chemical catalyst, required less energy compared to the enzymatic catalysis and the conventional process methanol and ethanol. The esterification of rich miscellae is feasible energetically, however, the extraction step with ethanol should be adjusted to enable energetically the chain of biodiesel production.

In der vorliegenden Arbeit wurden Einsatzmöglichkeiten der Headspace-Festpahsenmikroextraktion (HS-SPME) in Kombination mit der GC-MS für forensische und toxikologische Fragestellungen, insbesondere in der Haaranalytik, untersucht. Für basenstabile Drogen- und Medikamentenwirkstoffe wurden Nachweismethoden nach alkalischer Hydrolyse der Haare mit Natriumhydroxid und anschließender HS-SPME entwickelt. Für das Lokalanaesthetikum Lidocain und für das Heroinsubstitutionsmittel Methadon wurden optimierte Verfahren auf Haarproben von Drogentodesfällen angewandt. Um die Einsatzmöglichkeiten der HS-SPME gerade für Wirkstoffmetaboliten zu erweitern, wurden drei Derivatisierungsmöglichkeiten überprüft: (i) Umsetzung von primären und sekundären Aminen im alkalischen Haarhydrolysat mit Alkylchloroformiaten zu Carbamaten ("in sample" Derivatisierung), (ii) "On Fiber" Derivatisierung primärer und sekundärer Amine nach erfolgter HS-SPME mit N-Methyl-bis-trifluoracetamid (MBTFA) sowie (iii) Reaktion von Monofluoressigsäure mit 1-Pyrenyldiazomethan (PDAM) an einer vorher mit dem Derivatisierungsreagenz benetzten SPME-Faser. Fettsäureethylester konnten nach einer Flüssigextraktion der Haare von Alkoholikern mit einem DMSO/n-Hexan-Gemisch extrahiert und nach optimierter HS-SPME nachgewiesen werden. Im Vergleich zu Haarproben von Abstinenzlern und Gelegenheitstrinkern ergaben sich deutliche Unterschiede in den Konzentrationen. Für die schnelle Bestimmung von Valproinsäure und Trichlorethanol wurde eine Quantifizierungsmethode aus Blut- und Urinproben erarbeitet. Die Anwendung der HS-SPME ermöglichte eine schnelle, einfache und lösungsmittelfreie Extraktion bei geringem Probenverbrauch. Sie führte zu sauberen Extrakten und für dieses Verfahren hohen absoluten Extraktionsausbeuten. Bessere Reproduzierbarkeiten, ein hoher Probendurchsatz und eine verlängerte Einsetzbarkeit der SPME-Faser wurden durch die Verwendung eines automatischen Probengebers erreicht. / In the presented thesis possible fields of applications of the headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) for forensic and toxicological problems were investigated with special focus on hair analysis. Detection methods after alkaline hydrolysis of hair with sodium hydroxid and following HS-SPME were developed for basic stable drugs. For the local anaesthetic lidocaine and for the heroine substitute methadone optimized methods were applied to hair of drug fatalities. In order to enhance the fields of application of the HS-SPME especially for drug metabolites three derivatization procedures were tested: (i) conversion of primary and secondary amines in the alkaline hair extract with alkylchloroformates to carbamates ("in sample" derivatization), (ii) "on fibre" derivatization of primary and secondary amines after HS-SPME with N-methyl-bis-trifluoroacetamide (MBTFA) and (iii) reaction of monofluoroacetic acid (compound 1080) with 1-pyrenyldiazomethane (PDAM) on a SPME-fibre previously occupied with the derivatization reagent. Fatty acid ethyl ester (FAEE) could detected after liquid extraction from hair of alcoholics with a mixture of DMSO/n-hexane and optimized HS-SPME. In comparison to hair samples of teetotalers and social drinkers significant differences in the concentrations of the FAEE were found. A quantification method were developed for the fast determination of valproic acid and trichloroethanol from blood and urine. The application of HS-SPME allows a fast, simple and solvent free extraction from a small sample amount. HS-SPME lead to clean extracts and high absolute extraction yields for this kind of extraction. Better reproducibilities, a high sample throughput and extended lifetime of the SPME-fibre were obtained using an automatic sampler.

In der vorliegenden Arbeit wurde ein analytisches Verfahren zur quantitativen Bestimmung von Fettsäureethylestern (FSEE) im Haar und in Hautoberflächenlipiden mittels Headspace-Festphasenmikroextraktion (HS-SPME) und Gaschromatographie-Massenspektrometrie (GC-MS) sowie eine auf Hochleistungs-Flüssigchromatographie mit Photodiodenarray-Detektion (HPLC-DAD) basierende Methode zur Bestimmung der Squalenkonzentrationen in Lipidextrakten entwickelt. Die bei Untersuchung von Proben verschiedener Konsumentengruppen erhaltenen Konzentrationswerte wurden hinsichtlich ihrer Eignung als Marker für chronisch exzessiven Alkoholkonsum untersucht. Aus den Ergebnissen lässt sich schließen, dass Fettsäureethylester im Haar als Alkoholmarker den bisher üblicherweise genutzten Markern wie GGT, CDT oder MCV bezüglich Sensitivität und Spezifität mindestens ebenbürtig sind. Es wurden die folgenden vorläufige Cut-off-Werte festgelegt: wenn sich im Haar für die Summenkonzentration der vier in der höchsten Konzentration vorkommenden FSEE (Ethylmyristat, Ethylpalmitat, Ethyloleat und Ethylstearat) ein Wert > 1 ng/mg ergibt, kann mit hoher Sicherheit von chronisch exzessivem Alkoholkonsum ausgegangen werden, für Abstinenzler werden typischerweise Werte < 0,4 ng/mg gefunden. Durch Bildung des Quotienten der FSEE-Konzentrationen und der Squalenkonzentrationen wurden relative FSEE-Konzentrationen erhalten, die im Falle der Haaranalyse zu einer Verbesserung der Zuordnungssicherheit zu den entsprechenden Konsumentengruppen führten bzw. bei der Analyse von Hautoberflächenlipiden einen sinnvollen Vergleich der Werte erst ermöglichten. Als vorläufiger Cut-off-Wert für die relativen FSEE-Konzentrationen wurde ein Wert von 2 ng/µg vorgeschlagen. Als weiteres wichtiges Ergebnis der Arbeit wurde der Einlagerungsmechanismus der FSEE ins Haar aufgeklärt. Es konnte gezeigt werden, dass Fettsäureethylester in erster Linie über das Sebum ins Haar gelangen. / The current doctoral thesis presents the development of an analytical procedure for the quantitative analysis of fatty acid ethyl esters (FAEE) in hair and in skin surface lipids using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) as well as a method based on high-performance liquid chromatography with photodiode array detection (HPLC-DAD) to determine squalene concentrations in lipid extracts. The results obtained from analysis of samples from different alcohol consuming groups showed that FAEE are suitable markers for long-term alcohol misuse. Concerning sensitivity and specifity they are at least as good as other commonly used markers like GGT, CDT or MCV. The following provisional cut-off values were established: for chronically excessive alcohol consumption, the sum of the four FAEE (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) found in the highest mean concentrations should be > 1 ng/mg in hair; for non-drinkers, concentrations < 0,4 ng/mg are typical. The quotient obtained by dividing the FAEE concentration by the squalene concentration was defined as the relative FAEE concentration, which provides a better classification of the samples regarding the consumer groups through hair analysis. Relative FAEE values also allow a reasonable comparison in the case of skin surface lipid concentrations for the first time. 2 ng/µg is suggested as a preliminary cut-off value. As a further important result of the current work, the mechanism of incorporation of FAEE into hair was clarified. It was shown that fatty acid ethyl esters are incorporated into hair mainly through sebum.