Investigation in stability of eight synthetic piperazines in human whole blood under various storage conditions over time

Over the past decade, synthetic piperazines have been associated with multiple fatalities and was one of the top 25 identified drugs in 2011. While circumventing legislative controls and preventing the detection in standard drug tests, synthetic piperazine derivatives are encountered in forensic casework as “legal” alternatives to ecstasy (3,4-methylenedioxymethamphetamine). These chemically-produced compounds share very similar pharmacological and psychological effects with ecstasy which in turn has led to their popularity as “party pills”. The long-lasting duration of synthetic piperazines, especially when 1-benzylpiperazine (BZP) is mixed with 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), has also made them desirable to drug users to receive enhanced hallucinogenic effects. Although most methods are optimized to accurately quantify the amount of drugs in biological specimens submitted for forensic toxicology testing, unforeseeable challenges may arise to complicate the analysis such as postmortem redistribution, enzymatic reactions, the presence of bacterial activities, chemical and matrix interferences as well as the lack of reference materials. Thus, the purpose of this research was to investigate the stability of synthetic piperazines in human whole blood under various storage conditions and time ranges. A total of eight synthetic piperazines were assessed on their degrees of degradation using a Shimadzu Ultra-Fast Liquid Chromatography (UFLC) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry in positive ionization mode. These analytes included: 1-benzylpiperazine (BZP), 1-(4-fluorobenzyl)-piperazine (FBZP), 1-(4-methylbenzyl)-piperazine (MBZP), 1-(4-methoxyphenyl)-piperazine (MeOPP), 1-(para-fluorophenyl)-piperazine (pFPP), 1-(3-chlorophenyl)-piperazine (mCPP), 2,3-dichlorophenylpiperazine (DCPP), and 1-(3-trifluoromethylphenyl)-piperazine (TFMPP). Individual unknown samples were prepared by spiking certified reference standards (Cayman Chemical, Ann Arbor, MI, U.S.A.) of each synthetic piperazine into certified drug-free human whole blood (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) independently at 1000 ng/mL. To closely monitor the stability of each compound and potential drug-drug interactions, mixed samples consisted of all eight piperazines were also stored at room temperature (~20°C), 4°C and -20°C for one, three, six, nine and twelve months in dark sealed containers. Solid phase extraction (SPE) was performed to remove unwanted components prior to the injection into the LC system. Drug of Abuse (DAU) mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.) were utilized with a positive pressure manifold rack followed by evaporating to dryness with low heat at 65°C. All samples were then reconstituted with 250 µL of 50:50 mixture of methanol and 2mM ammonium formate buffer with 0.2% formic acid (Fisher Scientific, Waltham, MA, U.S.A.). Analysis was performed in triplicate using a reversed-phase column (Kinetex® F5, Phenomenex®, Torrance, CA, U.S.A.) with a binary gradient of a 2mM ammonium formate buffer with 0.2% formic acid and methanol with 0.1% formic acid. The total run time was 11.5 minutes including equilibration and the flow rate was 0.4 mL/min. Three internal standards including BZP-d7, mCPP-d8 and TFMPP-d4 (Cerilliant, Round Rock, TX, U.S.A) were used to generate calibration curves that were ranged from 20 ng/mL to 2000 ng/mL. Results revealed that BZP, MBZP and FBZP were more stable than phenyl piperazines over time under all storage conditions, in which MBZP was consistently more stable and still had more than 70% remaining after 12 months. Data showed a smaller degree of degradation when samples were kept frozen or refrigerated; whereas storing at room temperature should be avoided to ensure minimal degradation and detrimental impacts on stability of piperazine compounds. For crime laboratories that are facing backlog situations, case samples with synthetic piperazines should be kept frozen or refrigerated even for time period as short as 30 days or less. However, storing them for too long will clearly affect the quantitation accuracy because phenyl piperazines are more susceptible to degrade completely after six months regardless of storage conditions. Additionally, matrix interference was present due to the outlier of MBZP quantified on Day 270. Drug-drug interaction was also observed in the analyte mixture but the exact stability pattern of phenyl piperazines when mixed together could not be determined from this data set alone due to discrepancies observed on Day 91 and 270. This research project had shown a solid method to examine how quickly or slowly synthetic piperazines degrade in blood at different storage conditions. To further this study, it would be also important to evaluate the number of freeze-thaw cycles on each specimen in order to minimize the effect of non-metabolic degradation

Applying rotorcraft modelling technology to renewable energy research

The perceived need to reduce mankind's impact on the global climate motivates towards a future society in which a significant proportion of its energy needs will be extracted from the winds and the tides of the planet. This paper shows several examples of the application of Brown's Vorticity Transport Model, originally developed to perform simulations of helicopter aeromechanics and wake dynamics, to the analysis of the performance of renewable energy devices and their possible impact on the environment. Prediction of the loading on wind turbines introduces significant additional challenges to such a model, including the need to account fully for the effects of radial flow on blade stall. The wake-mediated aerodynamic interactions that occur within a wind farm can reduce its power output significantly, but this problem is very similar to that where the aerodynamic unsteadiness of the coupled wake of the main and tail rotors of a helicopter can result in significantly increased pilot workload. The helicopter-related problem of brownout, encountered during operations in desert conditions, has its analogue in the entrainment of sediment into the wakes of tidal turbines. In both cases it may be possible to ameliorate the influence of the rotor on its environment by careful and well-informed design. Finally, calculations of the distortion and dispersal of the exhaust plumes of a helicopter by the wake of its rotor allow insight into how wind turbines might interfere with the dispersal of pollutants from nearby industrial sites. These examples show how cross-disciplinary information transfer between the rotorcraft field and the renewable energy community is helping to develop the technologies that will be required by our future society, as well as helping to understand the environmental issues that might need to be faced as these technologies become more prevalent

Seasonal and spatial dynamics of the primary vector of plasmodium knowlesi within a major transmission focus in Sabah, Malaysia

Background The simian malaria parasite Plasmodium knowlesi is emerging as a public health problem in Southeast Asia, particularly in Malaysian Borneo where it now accounts for the greatest burden of malaria cases and deaths. Control is hindered by limited understanding of the ecology of potential vector species. Methodology/Principal Findings We conducted a one year longitudinal study of P. knowlesi vectors in three sites within an endemic area of Sabah, Malaysia. All mosquitoes were captured using human landing catch. Anopheles mosquitoes were dissected to determine, oocyst, sporozoites and parous rate. Anopheles balabacensis is confirmed as the primary vector of. P. knowlesi (using nested PCR) in Sabah for the first time. Vector densities were significantly higher and more seasonally variable in the village than forest or small scale farming site. However An. balabacensis survival and P. knowlesi infection rates were highest in forest and small scale farm sites. Anopheles balabacensis mostly bites humans outdoors in the early evening between 1800 to 2000hrs. Conclusions/Significance This study indicates transmission is unlikely to be prevented by bednets. This combined with its high vectorial capacity poses a threat to malaria elimination programmes within the region. Author Summary The first natural infection of Plasmodium knowlesi was reported 40 years ago. At that time it was perceived that the infection would not affect humans. However, now P. knowlesi is the predominant malaria species (38% of the cases) infecting people in Malaysia and is a notable obstacle to malaria elimination in the country. Plasmodium knowlesi has also been reported from all countries in Southeast Asia with the exception of Lao PDR and Timor Leste. In Sabah, Malaysian Borneo cases of human P. knowlesi are increasing. Thus, a comprehensive understanding of the bionomics of the vectors is required so as to enable proper control strategies. Here, we conducted a longitudinal study in Kudat district, Sabah, to determine and characterize the vectors of P. knowlesi within this transmission foci. Anopheles balabacensis was the predominant mosquito in all study sites and is confirmed as vector for P. knowlesi and other simian malaria parasites. The peak biting time was in the early part of the evening between1800 to 2000. Thus, breaking the chain of transmission is an extremely challenging task for the malaria elimination programme

Nozzle Sensor for In-System Chemical Concentration Monitoring

Chemical concentration is a vital parameter for determining appropriate chemical application. This study describes the design and testing of a sensor that attempted to monitor concentration of chemicals upstream from each nozzle body. The sensor is based on an LED and photodiode pair. Its ability to detect chemical concentration within the main carrier was tested with a 2,4-D formulation, a glyphosate formulation, and a powdered Acid Blue 9 dye. The liquid herbicide formulations of glyphosate and 2,4-D were tested across common application concentrations of 0% to 12.5% by volume. The powdered dye produced a much stronger effect on the sensor and was only tested at the much lower concentrations of 0 to 50 mg L-1. Further tests were conducted in which the dye was mixed with the herbicide formulation before the combined solution was added to the carrier. While this enabled establishment of pre-determined sensor outputs based on given concentrations of the pre-mixed solution, the sensor may have been responding to the predominance of a dye mixed with a herbicide formulation and not directly to the concentration of the herbicide. While the sensor did not appreciably respond to the concentration of the glyphosate formulation, it did respond in a consistent manner to the 2,4-D formulation and the dye. The sensor‘s response to the concentration of these chemicals was a rational (1/x type) relationship, and the R2 values for the rational models describing these relationships were greater than 0.99. With the mixed dye and herbicide formulation, the effects of the dye and the 2,4-D formulation combined independently, and the total sensor output was a multiplication of the percent effect of each alone. The test with the pre-mixed dye and 2,4-D formulation produced the expected 1 V output at a 12.5% by volume concentration of the 2,4-D formulation, proving that dye can be added to a herbicide to produce a desired response from the sensor. Overall, the sensor‘s response was remarkably stable, with a maximum standard deviation of 42.2 mg L-1 of 2,4-D active ingredient for samples taken at a constant chemical concentration. These tests confirmed that the sensor could respond to chemical formulations and dye in a consistent and predictable manner. However, use of the sensor for herbicide monitoring will require sensor calibration for each combination of herbicide and dye mixture, as the light transmittance properties of the tested mixtures were not quantified and the light transmittance properties of formulations and dyes can be arbitrarily changed by manufacturers

Role of Pseudogenes in Cancer Stem Creation Via High Nitric Oxide (HNO) Adaptation

Gene chip analysis of ten HNO adapted cell lines (Squamous cells: SCC-016, SCC-040, SCC-056, SCC-114, SCC-116; Adenocarcinomas: A549, BT20, Hs578, MCF7, and T47D) was carried out. Known pseudogenes were identified in each line, as well as their coding counterparts. The adenocarcinoma cell lines had no up regulated pseudogenes, while they had the following down regulated pseudogenes: RP6-159A1.2, RP11-255N24.3, AC004490.1, LDHBP, RP11-572H4.2. The squamous cell carcinomas (SCCs) had the following up regulated pseudogenes: RPL37AP1, AC138972.1, RP11-641D5.1, AC005534.6, AC022431.1, RPL26P12, and they had these down regulated pseudogenes: RP6-159A1.2, RP11-255N24.3, RBMXP1, RP11-20O23.1, RP11-551G24.2. All cell lines adhered to the hypothesis that an increase in a pseudogene expression also had an increase in the corresponding gene. The high level of pseudogenes could be due to low levels of microRNA; low expression of microRNA could then be due to high levels of ceRNA. In cases when the pseudogenes increase in expression (possibly due to HNO interference) they, like BRAF, take the functionality of ceRNA which in turn decreases microRNA expression. Although a pseudogene may not have any direct translational significance, it can act as ceRNA to facilitate the over expression of the coding gene in a feedback loop