Rafael Menck de Almeida

Determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (LPME) and gas chromatography–mass spectrometry (GC–MS)

In this study, it is shown a method for the determination of benzodiazepines and their main metabolites in urine samples by hollow-fiber liquid-phase microextraction (LPME) in the three-phase mode. Initially, the hydrolysis step was performed using 100 μL of sodium acetate 2.0 mol/L buffer solution (pH 4.5), 25 μL of β-glucuronidase enzyme and incubation for 90 min at 55 °C. In parallel with hydrolysis, the LPME fiber (9 cm) was prepared. Its pores were filled with a mixture of dihexyl ether: 1-nonanol (9:1). Afterwards, a solution of 3.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). After hydrolysis, the fiber was submersed in the alkalinized urine (pH 10) containing 10% NaCl. Samples were then submitted to orbital shaking (2400 rpm) for 90 min. The acceptor phase was later withdrawn from the fiber, dried and the residue derivatized with trifluoroacetic anhydride (TFAA) for 10 min at 60 °C with further addition of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide containing 1% tert-butyldimethylchlorosilane (MTBSTFA) for 45 min at 90 °C followed by determination by gas chromatography-mass spectrometry (GC-MS). The calibration curves obtained showed linearity over the specified range, with a similar sensitivity to traditional techniques and a higher detection capability compared to most of the miniaturized methods described in the literature. The method has been developed and successfully validated and applied to urine samples from real cases of benzodiazepines intake.

Recent Advances in Chromatographic Methods to Detect Drugs of Abuse in Alternative Biological Matrices

In recent years, many studies have been developed with the aim of improving drug detection in both conventional specimens and alternative biological matrices. A large number of drug abuse studies, forensic toxicology analyses, drugs in the workplace and even in doping control in sports activities related to drug detection in biological samples have been reported in the literature. The interest in the development and optimization of analytical techniques to detect drugs of abuse in different specimens is explained by the several possi- bilities and information that they can provide. Conventional samples such as urine and blood and more recently, saliva and sweat, are of fundamental importance whenever recent exposure to drugs is under investigation. Human keratinized tissues such as hair and nails are especially important for obtaining data of chronic long-term exposure with the great advantage of being collected in a non-invasive way. Meconium can be a useful biological sample to evaluate fetal drug exposure following maternal drug use. This paper reviews chroma- tographic procedures for determination of amphetamines, cannabinoids, opiates, nicotine, cocaine and alcohol in alternative biological matrices. Gas chromatographic and liquid chromatographic procedures with different detectors (including mass spectrometry) and sample preparation techniques such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and solid phase microextraction (SPME) were considered.

Detecting Alcohol and Illicit Drugs in Oral Fluid Samples Collected from Truck Drivers in the State of Sao Paulo, Brazil

Objective: Alcohol and drug use by truck drivers is a current problem in Brazil. Though there is evidence that alcohol consumption is occurring in higher proportions, the use of stimulant drugs to avoid fatigue and to maintain the work schedule has also been reported. The purpose of this study was to estimate the incidence of alcohol and illicit drug use among truck drivers on São Paulo state roads. São Paulo is the most populous state in Brazil and has the largest industrial park and economic production in the country. Methods: Data were assessed not only using a questionnaire but also, and more reliably, through toxicological analysis of oral fluid samples. Between the years 2002 and 2008, 1250 oral fluid samples were collected from truck drivers on the roads during morning hours. The samples were tested for the presence of alcohol, cocaine, tetrahydrocannabinol (THC), and amphetamine/methamphetamine. A previously published, validated gas chromatographic (gas chromatography–flame ionization detection and gas chromatography–mass spectrometry) method was applied to the samples for alcohol and drug detection. Results: Of the total analyzed samples, 3.1 percent (n = 39) were positive: 1.44 percent (n = 18) were positive for alcohol, 0.64 percent (n = 8) for amphetamines, 0.56 percent (n = 7) for cocaine, and 0.40 percent (n = 5) for THC. In one case, cocaine and THC were detected. The results are indicative of the extent of alcohol and drug use by truck drivers in the state of São Paulo, Brazil. Conclusions: This research provides evidence that not only alcohol but also illicit drug use is a real problem among professional drivers. The use of these substances should be controlled to better promote safe driving conditions on Brazilian roads.

Use of Illicit Drugs by Truck Drivers Arriving at Paranaguá Port Terminal, Brazil

Objective: The purpose of this study was to estimate the prevalence of recent use of illicit drugs among truck drivers who had parked their vehicles at the terminal port in Paranaguá City at Paraná State, southern Brazil. Methods: This cross-sectional study was part of a larger research project conducted among drivers at a regional Brazilian port. Data on professional characteristics, involvement in road traffic injuries, sleep, and use of alcohol and illicit drugs were collected using a questionnaire. Urine samples were collected and analyzed for amphetamines, cocaine, and cannabis using gas chromatography with mass spectrometric detection. Results: Sixty-two drivers were included in the study. Toxicological analyses showed that 8.1 percent (95% confidence interval [CI], 2.7–17.8%) of the urine samples were positive for drugs (4.8% for cocaine, 1.6% for amphetamine, and 1.6% for both); 8.1 percent reported drug use during the preceding 30 days in the questionnaire and only one tested positive for the drug in the urine sample. No sample was positive for cannabinoids. In total, at least 14.5 percent (95% CI, 6.9–25.8%) had used illicit drugs during the preceding 30 days based on self-reports and urine testing. Drivers who reported involvement in traffic injuries the year before more often tested positive for drugs in biological samples (P <.05). Conclusions: This research provides preliminary evidence that the use of illicit stimulants was common among professional truck drivers transporting grain loads. Thus, actions are needed to reduce drug use among truck drivers in order to prevent drug-related road traffic injuries.

Enzymatic-spectrophotometric determination of paraquat in urine samples: A method based on its toxic mechanism

Paraquat is a broad-spectrum contact herbicide that has been encountered worldwide in several cases of accidental, homicidal, and suicidal poisonings. The pulmonary toxicity of this compound is related to the depletion of NADPH in the pneumocytes, which is continuously consumed by the reduction/oxidation of paraquat and reductase enzyme systems in the presence of O2 (redox cycling). Based on this mechanism, an enzymatic-spectrophotometric method was developed for the determination of paraquat in urine samples. The velocity of NADPH consumption was monitored at 340 nm, every 10 s during 15 min. The velocity of NADPH oxidation correlated with the paraquat levels found in samples. The enzymatic-spectrophotometric method showed to be sensitive, making possible the detection of paraquat in urine samples at concentrations as low as 0.05 mg/L.

Simultaneous accelerated solvent extraction and hydrolysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide in meconium samples for gas chromatography–mass spectrometry analysis

Cannabis misuse during pregnancy is associated with severe impacts on the mother and baby health, such as newborn low birth weight, growth restriction, pre-term birth, neurobehavioral and developmental deficits. In most of the cases, drug abuse is omitted or denied by the mothers. Thus, toxicological analyzes using maternal-fetal matrices takes place as a suitable tool to assess drug use. Herein, meconium was the chosen matrix to evaluate cannabis exposure through identification and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic (THCCOOH). Accelerated solvent extraction (ASE) was applied for sample preparation technique to simultaneously extract and hydrolyze conjugated THCCOOH from meconium, followed by a solid-phase extraction (SPE) procedure. The method was developed and validated for gas chromatography-mass spectrometry (GC-MS), reaching hydrolysis efficiency of 98%. Limits of detection (LOD) and quantification (LOQ) were, respectively, 5 and 10 ng/g. The range of linearity was LOQ to 500 ng/g. Inter and intra-batch coefficients of variation were <8.4% for all concentration levels. Accuracy was in 101.7-108.9% range. Recovery was on average 60.3%. Carryover effect was not observed. The procedure was applied in six meconium samples from babies whose mothers were drug users and showed satisfactory performance to confirm fetal cannabis exposure.

Determination of Amphetamine, Amfepramone and Fenproporex in Urine Samples by HPLC-DAD: Application to a Population of Brazilian Truck Drivers

Commercially available immunoassay tests are designed to detect the presence of amphetamine/methamphetamine or methylenodioxyamphetamines. However, it is known that Brazilian truck drivers also report the use of other illicit amphetamines, such as amfepramone and fenproporex. Thus, a method was developed and validated in order to quantify amphetamine-type stimulants (amphetamine, fenproporex and amfepramone) in urine by high performance liquid chromatography with diode array detection (HPLC-DAD). Prior to this, a liquid-liquid extraction (LLE) with diethyl ether was performed in order to extract the analytes. The limit of detection was 150 ng mL-1. The method showed to be precise (relative standard deviation, RSD 0.99). Urine samples randomly collected from 385 truck drivers in Brazilian roads were submitted to the developed method. Nine samples were tested positive for amphetamine and one was tested positive for fenproporex and amphetamine.