Forensic botany and forensic chemistry working together: application of plant DNA barcoding as a complement to forensic chemistry-a case study in Brazil.

Recently, Brazilian Federal Police used forensic chemistry and forensic botany techniques on a case. Two packets containing fragmented plant matter were seized and sent for forensic analysis. Forensic chemistry, the gold standard for evaluating plant material suspected to contain illicit substances, did not find illicit materials. Gas chromatography coupled mass spectrometry (GC-MS) identified thujone in the botanical material. Thujone is a chemical compound naturally found in many plant species, notably Artemisia absinthium. Because doubt remained, we next used plant DNA barcoding methods. Total DNA from plant tissue fragments was extracted and five different DNA regions were amplified, sequenced, and analyzed using plant DNA barcoding methods. Genetic analysis yielded 30 good quality sequences representing five taxa. Most specimens were identified as A. absinthium. Few studies focus on practical forensic applications of plant DNA barcoding methods using a case solved in a forensic laboratory with its difficulties and limitations. To the best of our knowledge, this is the first study to report an effective joint effort of forensic chemistry and botany techniques to assess plant material in Brazil. The availability of a new technical approach for the genetic sequencing of plant species will enhance many forensic investigations and inspire similar initiatives.

Screening of seized cocaine samples using electrophoresis microchips with integrated contactless conductivity detection.

This study describes the development of a new analytical method for the separation and detection of cocaine (COC) and its adulterants, or cutting agents, using microchip electrophoresis (ME) devices coupled with capacitively coupled contactless conductivity detection (C4 D). All the experiments were carried out using a glass commercial ME device containing two pairs of integrated sensing electrodes. The running buffer composed of 20 mmol/L amino-2-(hydroxymethyl) propane-1,3-diol and 10 mmol/L 3,4-dimethoxycinnamic acid provided the best separation conditions for COC and its adulterants with baseline resolution (R > 1.6), separation efficiencies ranging from (2.9 ± 0.1) to (3.2 ± 0.2) × 105 plates/m, and estimated LOD values between 40 and 150 µmol/L. The quantification of COC was successfully performed in four samples seized by the Brazilian Federal Police Department and all predicted values agree with values estimated by the reference method. Some other interfering species were detected in the seized samples during the screening procedure on ME-C4 D devices. While lidocaine was detected in sample 3, the presence of levamisole was observed in samples 2 and 4. However, their concentrations were estimated to be below the LOQ. ME-C4 D devices have proved to be quite efficient for the identification and quantification of COC with errors lower than 10% when compared to the data obtained by a reference method. The approach herein reported offers great potential to be used for on-site COC screening in seized samples.

Discrimination and quantification of cocaine and adulterants in seized drug samples by infrared spectroscopy and PLSR.

Middle infrared spectroscopy and multivariate analysis have been applied for the development of methods to perform both quantitative and qualitative analysis of real drug samples seized by the Brazilian Police Federal (BPF). Currently, quantification of cocaine and determination of adulterants in seizures is performed using gas chromatography with flame ionization detection. However, this technique requires a relatively complex sample preparation, higher time of analysis, the destruction of sample and a high cost. In this context, this paper presents a simpler method to quantify cocaine and its major adulterants in seized materials. Out of 375 seizures, taken within a time frame of 2009-2013. A total of 1085 samples were analyzed of which 500 were selected for the calibration set and 585 for the validation set. Cocaine concentration in seized samples was determined by using middle infrared spectroscopy and partial least squares regression (PLSR), obtaining an average prediction error of 3.0% (w/w), precision of 2.0 and 11.8% (w/w) of minimum detectable cocaine concentration in a range varying from 24.2 to 99.9% (w/w). Results indicate that the developed method is able to discriminate between cocaine hydrochloride and free base samples, to quantify cocaine content as well as to estimate the concentration of main adulterants phenacetin, benzocaine, caffeine, lidocaine and aminopyrine.

Demystifying "oxi" cocaine: Chemical profiling analysis of a "new Brazilian drug" from Acre State.

Recent information from various sources suggests that a new illicit drug, called "oxi", is being spread across Brazil. It would be used in the smoked form and it would look like to crack cocaine: usually small yellowish or light brown stones. As fully released in the media, "oxi" would differ from crack cocaine in the sense that crack would contain carbonate or bicarbonate salts whereas "oxi" would include the addition of calcium oxide and kerosene (or gasoline). In this context, this work presents a chemical profiling comparative study between "oxi" street samples seized by the Civil Police of the State of Acre (CP/AC) and samples associated with both international and interstate drug trafficking seized by the Brazilian Federal Police in Acre (FP/AC). The outcome of this work assisted Brazilian authorities to stop inaccurate and alarmist releases on this issue. It may be of good use by the forensic community in order to better understand matters in their efforts to guide local law enforcement agencies in case such claims reach the international illicit market.

Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom.

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.