Gamma-hydroxybutyrate (GHB) is an endogenous compound in the human body, found in regions of the mammalian brain and believed to be a cerebral neurotransmitter.1,2 GHB also acts as a powerful central nervous system depressant commonly used as a “date rape” drug due to its hypnotic and sedative properties.1 The drug has also been used medicinally to treat alcohol withdrawal, opiate-withdrawal syndrome, and narcolepsy.3–5 Toxicological analysis of GHB in drug facilitated sexual assault (DFSA) cases is typically performed using blood and urine specimens.1 However, due to the endogenous nature of GHB, toxicological interpretation of these biological specimens can be complex and challenging.1,4 Additionally, urine and blood analysis of GHB can be impacted by sample collection, sample analysis times, and sample storage conditions.6,7 Due to the challenges and limitations associated with blood and urine analysis of GHB along with the prominence of GHB in DFSA cases, it would be beneficial to determine the possibility of GHB analysis using alternative biological matrices. The primary goal of this research was to develop a sample preparation method that could accurately and reliably identify and quantify GHB in oral fluid, as an alternative biological matrix. Additionally, this research was carried out to compare the identification and quantitation capabilities of GHB in oral fluid to that of traditional biological matrices, specifically urine and blood. The methods employed in this study utilized gas chromatography – mass spectrometry (GC-MS) instrumentation in order to correctly identify GHB. A deuterated internal standard, GHB-d6, was used to quantify all samples. The methods were assessed using the parameters set forth by the Scientific Working Group of Forensic Toxicologists (SWGTOX) for quantitative analysis methods. The following factors were considered: calibration model, bias, precision, limit of detection and quantitation, carryover, and interferences. Urine and blood samples were prepared using 200 uL of urine (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) or blood (Equitech-Bio Inc., Kerrville, TX, U.S.A.), varying amounts of the 200 mg/L working calibrator and control solution prepared using certified reference standards (Cerilliant, Round Rock, TX, U.S.A.), and 50 uL of 100 mg/L working internal standard solution resulting in an internal standard concentration of 25 mg/L in each sample. Solid phase extraction (SPE) was performed using United Chemical Technologies (UCT), Inc. (Bristol, PA, U.S.A) Clean Screen GHB columns (ZSGHB020) on all samples.1 Samples were reconstituted, derivatized, and analyzed using GC-MS. Oral fluid samples were prepared using 1.0 uL of drug-free oral fluid, and 1.0 mg/mL (as salt) in methanol GHB received from Cerilliant. The samples were spiked with 1 uL of 1.0 mg/mL (as salt) in methanol GHB-d6 received from Cerilliant. Each sample had an internal standard concentration of 10 mg/L. Samples were fortified with 100 uL of ethyl acetate, and derivatized with 100 uL of bis(trimethyl)trifluoroacetamide (BSTFA) with 1% Trimethylchlorosilane (TMCS) received from Cerilliant. The samples were incubated, and analyzed using GC-MS.8 All analyses were conducted using an Agilent 7890A GC system, Agilent 5975C Mass Detector System (MSD), and an Agilent 7683B Autosampler (Agilent Technologies Inc. Santa Clara, CA). The chromatographic component was carried out using an Agilent HP5-MS 30m x 250um x 0.25um capillary column and an Agilent HP- 5MS 15m x 250um x 0.25um capillary column. All data was analyzed using Agilent MSD ChemStation software (version E.02.02.1431). The method has a total length of 12.75 minutes. Selective ion monitoring (SIM) was used to monitor the ions of interest for each analyte. GHB-d6 was monitored using the ions 239, 240, and 241. GHB was monitored using the ions 233, 234, 235.1 Results revealed that GHB and GHB-d6 could be identified and differentiated due to their fragmentation patterns. All calibration curves for the three matrices exhibited R2 values > 0.98 using a linear dynamic range of 5-100 mg/L with a minimum of four calibration points. The limit of detection for the three matrices was determined to be 1 mg/L, and the limit of quantitation for the three matrices was determined to be 5 mg/L. Bias and precision were analyzed at concentrations of 8 mg/L, 45 mg/L, and 90 mg/L for each matrix. All urine and blood samples were calculated to be within the acceptance range of +20% bias and +20% coefficient of variation. Oral fluid samples were outside of the +20% acceptance range for both bias and coefficient of variation. The highest concentration analyzed that did not produce carryover into subsequent matrix blanks was found to be 350 mg/L for each matrix. Significant interferences were found to be present in urine and blood samples, but negligible for all oral fluid samples. This research illustrates that the developed sample preparation method can be used to accurately and reliably identify GHB in oral fluid. Additionally, this research suggests that the quantitation capabilities of GHB in oral fluid are not as accurate and precise as those of urine and blood. Therefore, the developed method has better qualitative analysis capabilities, while the urine and blood methods have better quantitative analysis capabilities for forensic toxicology casework.

Master of Public Health - MPH / Background: The identification of undiagnosed HIV positive individuals through HIV testing services is one of the key measures in ensuring that the UNAIDS/ WHO 90 90 90 targets are achieved by the year 2020. The identification and subsequent initiation of HIV positive individuals on ART helps to minimise or prevent HIV transmission following viral suppression. Around 25% of people living with HIV globally are unaware of their positive status. In many countries, critical gaps exist in HIV services including prevention, testing and treatment. In Africa, the East and Southern region has a high burden of HIV/ AIDS where 50% of people are living with HIV, with 800,000 new infections in 2017. Despite concerted efforts in rolling out HIV testing services in Zimbabwe, a significant number of the population, especially men and young people, remain with an unknown HIV status, which is hindering progress towards the control and prevention of the HIV pandemic. A recent study confirmed that HIV testing in men is lower as compared to women. Consequently, progress is slower in trying to achieve the year 2020 milestone. The identification of suitable options for reaching out to these groups and the general population is important to achieve a reduction HIV prevalence. HIV self-testing oral fluid sample is thought to have the capacity to increase uptake of HIV testing. However, it still requires evidence on its acceptability and feasibility for those with undiagnosed HIV. Aim: The aim of this study was to explore the perceptions and experiences of the accessibility and feasibility HIV self-testing kits for individuals who received and used this method in Neta ward in Mberengwa District, Zimbabwe. The study identified some key factors that led to individuals accepting the kits and using them. The findings of this study will hopefully be able to inform policy makers’ decision-making about the importance of rolling out HST in the country.

Cannabidiol (CBD) is one of over 80 active cannabinoids found in Cannabis Sativa and is the second most abundant cannabinoid derived from the plant following d(9)-Tetrahydrocannabinol (THC). As opposed to THC, CBD does not appear to have any psychotropic effects, rather CBD is often utilized for its therapeutic properties, which include effects such as antinociception, anti-convulsion, and anti-inflammation. During the extraction of CBD from plant material, THC may be co-extracted. Therefore, screening and quantitating potential THC levels in individuals using CBD products is important in instances where the legality of use of THC does not match that of CBD. In recent years, oral fluid has gained recognition as a non-invasive and expedient matrix for both drug testing and forensic casework. Due to its relevance, oral fluid was selected for analysis. This project evaluated the detection and quantitation of CBD, THC, and two primary metabolites in oral fluid samples of a therapeutic-use cannabidiol donor using Biotage Supported Liquid Extraction (SLE) and subsequent testing by PerkinElmer QSight 220 CR LC-MS/MS in positive ionization mode All calibrators and quality controls were prepared by fortifying synthetic oral fluid with certified reference standards. Standards and samples were prepared in a 1:3 dilution with extraction buffer. Calibrators were prepared at 0.25, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, and 500 ng/mL, with quality controls analyzed at 0.75, 70, and 425 ng/mL. Internal standard was added to the appropriate samples to account for any variation produced by sample preparation. SLE was performed using ISOLUTE SLE+ 1 mL columns with elution in hexane:ethyl acetate:methyl tert-butyl ether (80:10:10), followed by evaporation using an Organomation Multivap Nitrogen Evaporator (Berlin, Massachusetts). All samples were reconstituted in 100 uL of 0.1% formic acid in deionized water:0.1% formic acid in acetonitrile (70:30). Validation parameters were assessed using Academy Standards Board (ASB) Standard 036-Standard Practices for Method Validation in Forensic Toxicology, including linear dynamic range (LDR), limit of detection (LOD), limit of quantitation (LOQ), analyte recovery, ion suppression/enhancement, and carryover. Following reconstitution, samples were placed onto the autosampler for injection and subsequent chromatographic separation using a PerkinElmer Brownlee C18 2.1x50 millimeter (mm), 2.7 micrometer (um) column. Analysis of the samples by mass spectrometry was performed in positive mode with multiple reaction monitoring (MRM). Total run time including equilibration was 10.5 minutes. All compounds were quantified using linear calibration models with 1/X weighting (1/concentration) and measured values were normalized by their respective internal standards. The LDR was determined to be 0.25 to 500 ng/mL. For all analytes, LOD was assessed and determined to be 0.25 ng/mL with an LOQ of 1 ng/mL. Carryover was assessed by running a double blank following a sample spiked at 500 ng/mL with no analytes observed. The donor samples were collected at several timepoints around the oral administration of an 8 mg dose of CBD. These timepoints included: prior to administration, at the time of administration, 30 minutes post-administration, 45 minutes post-administration, 60 minutes post-administration, 90 minutes post-administration, and 120 minutes post-administration. CBD was quantified within the diluted oral fluid samples from below LOQ to 325.75 ng/mL. THC was detected above LOD but below LOQ, concentrations which lie below typical cut-offs used in both workplace drug testing and forensic casework. The two metabolites were not detected above the LOD. As such, the CBD product can be concluded to be of reasonable purity as it relates to legal implications. Overall, the use of laminar flow mass spectrometry was effective in detecting various cannabinoids in oral fluid samples following SLE sample extraction.

Drug screening is widespread in contexts such as the criminal justice system, employment, and substance abuse treatment centers. Traditionally, drug testing methods have targeted urine matrices and extensive research is available regarding urine drug screening. Due to the nature of sample collection, urine specimen may be tampered or adulterated in efforts to invalidate or pass a drug test. Examples of this include substituting a sample with synthetic urine, diluting a sample with water, or adding a substance to the sample. The addition of a substance is referred to as adulteration and is done in an effort to mask drugs in the sample or to invalidate the test results. For various reasons, including the effects of adulteration, time, and costs associated with urine drug tests, oral fluid (OF) has become an increasingly important alternative matrix for screening drugs of abuse. It offers distinct advantages since tests can be administered noninvasively, quickly, and under observation, thus reducing the risk of tampering. Studies have also shown that drugs of abuse detected in OF may correlate better to user impairment at the time of collection, as compared to other matrices. In 2019, the Substance Abuse and Mental Health Service Administration (SAMHSA) published mandatory guidelines for oral fluid testing. Although these guidelines only directly impact federal spaces, they also serve as a blueprint for developing drug testing laws and policies in other organizations. Despite requirements and procedures controlling for specimen adulteration, it is recognized that manufacturers will continue to develop and market new products to avoid drug detection, just as with urine drug tests. The design of this experiment was to investigate the effects of the commercially available drug testing subversion products High Voltage Saliva Cleanse Mouthwash (High Voltage Detox, Las Vegas, Nevada, USA) and Stinger Detox Mouthwash (Stinger Detox, Phoenix, Arizona, USA) on immunoassay testing for tetrahydrocannabinol (THC) and methamphetamine (MET) in OF. The High Voltage Saliva Cleanse was designated adulterant “A”, and the Stinger Detox was designated adulterant “B”. Two separate immunoassay kits, Discover™ (American Screening Corporation, Inc., Shreveport, Louisiana, USA) and Orawell® (Jiangsu Well Biotech Co., Ltd, Changzhou, Jiangsu, China), were assessed to investigate the differences in performance of the current available test devices in addition to the effects of the subversion products. Using Discover™, samples were spiked according to 0.5 times (x), 1x, and 2x the cutoff concentrations of 50 ng/mL THC and 50 ng/mL MET without adulterant, with Adulterant A, and with Adulterant B. Testing with Orawell® devices was initially conducted at 1x and 2x the cutoff concentrations of 40 ng/mL THC and 50 ng/mL of MET. Based on the lack of response, testing at 0.5x was not conducted. Additional testing was conducted at 1.5x and 3x the cutoff concentrations without adulterant, with Adulterant A, and with Adulterant B. It was concluded that the adulterants affected the test results in the Orawell® device, by producing false positives for drugs of abuse not present in the sample for 17 (56.7%) of the 30 tests containing adulterants. Additionally, it was concluded that both immunoassay tests assessed were lacking in analytical sensitivity and reproducibility.

No description available.

A elevada incidência de acidentes de trânsito está fortemente relacionada ao consumo de bebidas alcoólicas. No Brasil a verificação do uso de álcool entre os condutores é realizada através dos etilômetros e da confirmação do etanol presente no sangue pela técnica de headspace (HS) associada à cromatografia em fase gasosa com detector de ionização de chama (CG/DIC). Não foram encontrados na literatura métodos para determinação de etanol em fluido oral (FO), utilizando a cromatografia em fase gasosa com detector de massas (CG/EM). Objetivos: Realizar a otimização da extração do etanol do fluido oral pela técnica de HS através de desenho experimental e posterior validação de metodologia analítica para a determinação de etanol em FO através de CG/EM e CG/DIC, utilizando o Quantisal® como dispositivo de coleta. Métodos: O desenho experimental foi desenvolvido através do Box–Behnken Design (BBD), onde foram avaliados a temperatura, o tempo de agitação e o volume injetado. O método foi validado de acordo com as recomendações do FDA e ANVISA considerando os parâmetros de seletividade, efeito residual, efeito matriz, linearidade, precisão, exatidão, limite de detecção e quantificação, estabilidade e recuperação. Resultados: As melhores condições do HS obtidas pelo desenho foram: temperatura de 90ºC, volume de injeção de 1000 μL e tempo de extração de 7 min. O método mostrou-se linear na faixa de 0,05-2 g/L (0,5-20 dg/L). Os valores de exatidão situaram-se na faixa de 101,56 e 111,29% e os valores obtidos para a precisão intra e interdia foram inferiores a 12%. Os limites de quantificação e de detecção encontrados foram iguais a 0,0125 g/L e 0,005 g/L para a CG/EM e 0,05 g/L e 0,0129 g/L para a CG/DIC, respectivamente. Conclusões: O método desenvolvido mostrou-se eficaz na determinação inequívoca de etanol em fluido oral através da técnica de HS-CG/EM e utilizando o dispositivo de coleta Quantisal®, atingindo limites de detecção inferiores ao encontrados pelas análises em CG/DIC, sem a necessidade de confirmação por outros sistemas cromatográficos e podendo ser facilmente aplicado na rotina laboratorial. / The high incidence of traffic accidents is strongly related to alcohol consumption. In Brazil the verification of alcohol use among drivers is performed through the breath alcohol analyzers and confirmation of ethanol in blood by the headspace technique (HS) associated to gas chromatography with flame ionization detector (GC/FID). To the best of our knowledge there are no methods for the determination of ethanol in oral fluid (OF), using gas chromatography with mass detection (GC/MS) in the literature. Propose: Perform the optimization of the extraction of ethanol from OF by headspace technique (HS) through experimental design and subsequent validation of analytical method for the determination of ethanol in OF by GC/MS and GC/DIC, using Quantisal® as a collection device. Methods: The experimental design was performed using the Box-Behnken Design (BBD) and the evaluated parameters were temperature, stirring time and sample volume injected. The methods were validated according to FDA and ANVISA recommendations considering the parameters of selectivity, residual effect, matrix effect, linearity, precision, accuracy, limit of detection and quantification, stability and recovery. Results: The best conditions of HS obtained by design were: temperature 90°C, injection volume 1000 μL and extraction time of 7 min. The method was linear in the range of 0.05-2 g/L (or 0.5-20 dg/L). The values of accuracy stay in the range of 101.56 and 111.29% and values for intra and inter-day precision were less than 12%. The limits of detection and quantification were found equal to 0.0125 g/L and 0.005 g/L for GC/MS and 0.05 g/L and 0.0129 g/L for GC/FID, respectively. Conclusions: The method was effective in unequivocal determination of ethanol in oral fluid by HS-GC/MS and using the collection device Quantisal®, reaching detection limits lower than that found by analysis on HS-GC/FID, without the need confirmation by other chromatographic systems and can be easily applied for routine monitoring.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.

O Papilomavírus Humano é uma das infecções sexualmente transmissíveis mais comuns no mundo. A história natural da infecção oral pelo HPV não é bem esclarecida e seus fatores de risco não são bem explorados. Pessoas imunocomprometidas, como pacientes infectados pelo HIV, apresentam maior risco para a infecção pelo HPV.Objetivo: determinar a prevalência do HPV no fluído oral de homens infectados pelo HIV e sua relação com os fatores de risco. Casuística: Um total de 283 amostras de lavado oral foram analisadas. Todas as amostras passaram por processos de lavagem, extração de DNA, amplificação do DNA por PCR convencional (MY09/11), eletroforese em gel de agarose e as amostras positivas para HPV DNA foram submetidas à genotipagem do HPV por meio de hibridização. Resultados: A genotipagem do HPV identificou a presença dos tipos 06, 16, 44, 51, 56, 58, 62, 66, 67, 69, 72, 83 e 84. O tipo mais prevalente no grupo de alto risco foi HPV-66 e no grupo de baixo risco HPV-6 e 83. Em relação aos fatores de risco, o tabagismo foi o único fator considerado significativo [OR (CI) = 10,04 (1,98 - 50,92), p>0,01] para a infecção do HPV oral em homens infectados pelo HIV em São Paulo, Brasil Discussão: A prevalência do HPV oral é altamente variável em decorrência de fatores como método de coleta e análises das amostras. Apesar de dados sobre a infecção pelo HPV em homens serem escassos, estudos mostram que grande parte dos homens brasileiros são infectados pelo vírus. O fator de risco principal é a exposição sexual de risco, porém, outros fatores são considerados como possíveis para aquisição do HPV como o tabagismo, consumo excessivo de álcool e a infecção pelo HIV. Conclusão:. A prevalência do HPV oral nos indivíduos estudados foi de 3,5%, e dentre os tipos encontrados, a maioria não são encontrados na vacina do HPV comercialmente disponível. / Human Papillomavirus is one of the most common sexually transmitted infection worlwide. The natural history of oral HPV infection is unclear and its risk factors have not been explored. Immunocompromised people, as exemplified by HIV patients, are at high risk for HPV-infection. Objectives: To determine the prevalence of HPV in the oral tract of HIV-1-positive male subjects and its association with risk factors. Case series: A total of 283 oral wash samples were analyzed. All samples were processed by washing processes, DNA extraction, DNA amplification by conventional PCR (MY 09/11), agarose gel electrophoresis and HPV DNA positive samples were submitted to HPV genotyping by hybridization. Results: HPV genotyping revealed of types 06, 16, 44, 51, 56, 58, 62, 66, 67, 72, 83 and 84; major high risk HPV type identified was HPV-66 and low risk types were HPV-6 and 83. Regarding risk factors, smoking was the only risk factor considered significant [OR (CI) = 10,04 (1,98 - 50,92), p>0,01] for the oral HPVinfection in HIV-1-infected subjects in São Paulo, Brazil. Discussion: The prevalence of oral HPV is highly variable due to factors such as method of collection and analysis. Although data on HPV infection in men are scarce, studies show that most Brazilian men are infected by the virus. The main risk factors are unprotected sexual intercourse, but other factors for this infection have been described elsewhere including smoking, alcohol consumption and HIV-positive serostatus. Conclusion: the prevalence of oral HPV in the individuals studied was 3.5% and among the types analyzed, most are not found in HPV vaccine commercially available.

O Brasil destaca-se no cenário mundial em relação ao consumo de estimulantes anfetamínicos (ATS), e estudos nacionais têm evidenciado grande prevalência na utilização destas substâncias por motoristas profissionais. O fluido oral apresenta uma série de vantagens sobre as matrizes tradicionais para a monitorização do consumo de ATS no trânsito, e vem sendo empregada em diversos países. Este trabalho objetivou avaliar a especificidade de imunoensaios comerciais na detecção preliminar dos ATS comercializados no Brasil em amostras de fluído oral e desenvolver e validar um método para a confirmação e quantificação de anfetamina (AMP), metanfetamina (MET), anfepramona (DIE), fenproporex (FEN) e metilfenidato (MPH) no fluído oral por microextração em fase sólida (SPME) e cromatografia a gás com detector de massas (CG/EM). A especificidade analítica dos imunoensaios foi avaliada por meio do estudo das informações técnicas de diversos produtos e da realização de ensaios experimentais com três testes. O método confirmatório por SPME-CG/EM foi desenvolvido a partir da técnica de imersão (DI-SPME), sob agitação magnética, à temperatura ambiente, utilizando fibras revestida por polidimetilsiloxano (30 μm), propilcloroformato como agente derivatizante e adição de Na2CO3 e de Na2SO4 para aumentar o pH e a força iônica do meio, respectivamente. Os testes imunológicos atualmente disponíveis para a triagem de ATS em fluido oral são importados e não detectam, mesmo em altas concentrações, os principais ATS consumidos no Brasil: FEN, DIE e MPH. O método por SPME-CG/EM foi linear para os ATS estudados no intervalo de 2-256 ng.mL-1, exceto para o FEN cujo intervalo foi de 4-256 ng.mL-1. Os limites de detecção foram 0,5 ng.mL-1 (MET), 1 ng.mL-1 (MPH) e 2 ng.mL-1 (DIE, AMP, FEN). A exatidão do método situou-se entre 98,2 – 111,9% e a precisão não excedeu 15% de desvio padrão relativo. O método foi aplicado com sucesso na estimação do perfil farmacocinético do FEN e da AMP no fluído oral de seis indivíduos do sexo masculino, após a administração de dose única de especialidade farmacêutica nacional contendo 25 mg de cloridrato de FEN. / The Brazil stands out on the world as a major consumer of amphetamine-type stimulants (ATS), and several national studies have shown high prevalence in the consumption of these substances by professional drivers. Oral fluid has many advantages over the conventional biological fluids for monitoring ATS use on roads, and has been employed with this purpose in several countries. The aim of this study is to assess the specificity/cross-reactivity of commercial oral fluid immunoassays in detecting the prescription ATS marketed in Brazil and to develop and validate a method for confirmation and quantification of amphetamine (AMP), methamphetamine (MET), amfepramone (DIE), fenproporex (FEN) and methylphenidate (MPH) in oral fluid by solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). Analytical specificity of immunoassays was evaluated through the study of the technical information of commercial products and through experimental testing of three kits. The confirmatory SPME-GC-MS method employed SPME immersion technique (DI-SPME), under magnetic stirring, at room temperature, using polydimethylsiloxane (30 μm) fibers, in-matrix propylchloroformate derivatization, Na2CO3 and Na2SO4 to increase both pH and ionic strength. Immunological tests currently available for ATS screening in oral fluid are imported and do not detect, even at high concentrations, the main ATS consumed in Brazil: FEN, MPH and DIE. The SPME-GC-MS method was linear for the studied ATS over the range of 2-256 ng.mL-1, except for FEN where the linear range was 4-256 ng.mL-1. The detection limits were 0.5 ng.mL-1 (MET), 1 ng.mL-1 (MPH) and 2 ng.mL-1 (DIE, AMP, FEN). Accuracy was within 98.2 – 111.9% of the target concentrations and precision did not exceed 15% of relative standard deviation. The method was successfully applied to estimate the pharmacokinetic profile of FEN and AMP in oral fluid of six male subjects after administration of a single dose of 25 mg FEN hydrochloride.

A elevada incidência de acidentes de trânsito está fortemente relacionada ao consumo de bebidas alcoólicas. No Brasil a verificação do uso de álcool entre os condutores é realizada através dos etilômetros e da confirmação do etanol presente no sangue pela técnica de headspace (HS) associada à cromatografia em fase gasosa com detector de ionização de chama (CG/DIC). Não foram encontrados na literatura métodos para determinação de etanol em fluido oral (FO), utilizando a cromatografia em fase gasosa com detector de massas (CG/EM). Objetivos: Realizar a otimização da extração do etanol do fluido oral pela técnica de HS através de desenho experimental e posterior validação de metodologia analítica para a determinação de etanol em FO através de CG/EM e CG/DIC, utilizando o Quantisal® como dispositivo de coleta. Métodos: O desenho experimental foi desenvolvido através do Box–Behnken Design (BBD), onde foram avaliados a temperatura, o tempo de agitação e o volume injetado. O método foi validado de acordo com as recomendações do FDA e ANVISA considerando os parâmetros de seletividade, efeito residual, efeito matriz, linearidade, precisão, exatidão, limite de detecção e quantificação, estabilidade e recuperação. Resultados: As melhores condições do HS obtidas pelo desenho foram: temperatura de 90ºC, volume de injeção de 1000 μL e tempo de extração de 7 min. O método mostrou-se linear na faixa de 0,05-2 g/L (0,5-20 dg/L). Os valores de exatidão situaram-se na faixa de 101,56 e 111,29% e os valores obtidos para a precisão intra e interdia foram inferiores a 12%. Os limites de quantificação e de detecção encontrados foram iguais a 0,0125 g/L e 0,005 g/L para a CG/EM e 0,05 g/L e 0,0129 g/L para a CG/DIC, respectivamente. Conclusões: O método desenvolvido mostrou-se eficaz na determinação inequívoca de etanol em fluido oral através da técnica de HS-CG/EM e utilizando o dispositivo de coleta Quantisal®, atingindo limites de detecção inferiores ao encontrados pelas análises em CG/DIC, sem a necessidade de confirmação por outros sistemas cromatográficos e podendo ser facilmente aplicado na rotina laboratorial. / The high incidence of traffic accidents is strongly related to alcohol consumption. In Brazil the verification of alcohol use among drivers is performed through the breath alcohol analyzers and confirmation of ethanol in blood by the headspace technique (HS) associated to gas chromatography with flame ionization detector (GC/FID). To the best of our knowledge there are no methods for the determination of ethanol in oral fluid (OF), using gas chromatography with mass detection (GC/MS) in the literature. Propose: Perform the optimization of the extraction of ethanol from OF by headspace technique (HS) through experimental design and subsequent validation of analytical method for the determination of ethanol in OF by GC/MS and GC/DIC, using Quantisal® as a collection device. Methods: The experimental design was performed using the Box-Behnken Design (BBD) and the evaluated parameters were temperature, stirring time and sample volume injected. The methods were validated according to FDA and ANVISA recommendations considering the parameters of selectivity, residual effect, matrix effect, linearity, precision, accuracy, limit of detection and quantification, stability and recovery. Results: The best conditions of HS obtained by design were: temperature 90°C, injection volume 1000 μL and extraction time of 7 min. The method was linear in the range of 0.05-2 g/L (or 0.5-20 dg/L). The values of accuracy stay in the range of 101.56 and 111.29% and values for intra and inter-day precision were less than 12%. The limits of detection and quantification were found equal to 0.0125 g/L and 0.005 g/L for GC/MS and 0.05 g/L and 0.0129 g/L for GC/FID, respectively. Conclusions: The method was effective in unequivocal determination of ethanol in oral fluid by HS-GC/MS and using the collection device Quantisal®, reaching detection limits lower than that found by analysis on HS-GC/FID, without the need confirmation by other chromatographic systems and can be easily applied for routine monitoring.