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.

"Benzo Fury" compounds and derivatives are enactogens similar to 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in various aspects. These compounds are similar in structure to MDMA and MDA, as well as, elicit similar effects such as elevated mood, euphoria and hallucinations. This similarity in effect increases the potential for abuse as MDMA has become less prevalent in some regions as the use of these new psychoactive substances (NPSs) has increased. The benzofury compounds are used as legal alternatives to MDMA because of their marketing as “not for human consumption”. With the relative ease in obtaining NPSs via the Internet, it is possible that these drugs may soon be prevalent in the United States. The project’s goal was to separate, detect, and quantitate the benzofury compounds and derivatives as well as MDA and MDMA in one method of analysis using ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). The project also examined which method of sample preparation is more effective for these compounds. Six benzofury compounds were researched: 5-(2-aminopropyl)benzofuran) (5-APB), 6-(2-aminopropyl)benzofuran) (6-APB), 5-(2-aminopropyl)-2,3-dihydrobenzofuran (5-APDB), 6-(2-aminopropyl)-2,3-dihydrobenzofuran (6-APDB), 1-(benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) and 1-(benzofuran-6-yl)-N-methylpropan-2-amine (6-MAPB) as well as MDMA and MDA. These drugs were analyzed in blood. A liquid-liquid extraction (LLE) method and solid phase extraction (SPE) method were examined to determine which would be better for the separation, detection and quantitation of the benzofury compounds. For the development of the overall method, accuracy, precision, calibration curve, carryover, limit of detection, limit of quantitation, analyte stability, and recovery were examined. The accuracy of the methods examined was greater than +/- 20%. For most analytes, the precision within-run and between-run did not exceed 20%, regardless of the sample preparation method used. A weighting of 1/x was applied to the calibration curve regardless of sample preparation method utilized. The carryover was less than 2% with the SPE method having less carryover (0.02% to 0.50%) than the LLE method (0.05% to 1.56%). The limit of quantitation was determined to be greater than 10 ng/mL. While this was unexpected, the limit of detection calculations determined that this was correct. Using the LLE method in combination with the UFLC-MS/MS method developed, the limit of detection was determined to be at least 9.98 ng/mL. Compared to the LLE method, the SPE limit of detection was lower and calculated to be 3.75 ng/mL. The percent recovery was examined for each of the analytes. It was determined that the SPE was capable of recovering 80% or more of the benzofury compounds and derivatives regardless of the concentration level. The LLE was not as successful in the recovering the benzofury compounds, the best recovery occurred at the 200 ng/mL level with only 65% or less recovered. Analyte stability exhibited a general decrease with variation prior to day 7 and then remains relatively stable until day 14. It was anticipated that the quantitation of the drugs might be complicated due to the similarity in structure between the isomers as well as the similarity of structure between all of the compounds. While this may still be the case, the difficult separation resulted in a re-evaluation and alterations to the UFLC-MS/MS method to correct for these issues. With the change in the UFLC-MS/MS method, further method optimization is required to achieve the appropriate accuracy and limit of quantitation. It was found that the best combination of sample preparation and detection of the benzofury compounds and derivatives is to use SPE followed by an UFLC-MS/MS method.