Synthetic piperazines are chemically-produced compounds that contain a six-member ring with two opposing nitrogen atoms. Several piperazine derivatives, namely 1- benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), and 1-(3- chlorophenyl)-piperazine (mCPP), have fallen into the “designer drugs” category due to their increasing recreational use as a “legal” alternative to ecstasy (3,4-methylenedioxymethamphetamine). These compounds share similar stimulant and physiological effects with amphetamines which make them desirable to young adults in party-type atmospheres. BZP, a Schedule I drug for its high abuse potential and no accepted medical use, is the only recreationally-abused synthetic piperazine currently federally controlled in the United States. The purpose of this research was to develop and validate a reliable method to identify and quantify eight forensically significant synthetic piperazines in blood and urine using ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry (UFLC-ESI-MS/MS). The method was validated according to the Scientific Working Group for Forensic Toxicologists (SWGTOX) guidelines for quantitative analysis for both matrices and includes the following analytes: 1-benzylpiperazine (BZP), 1-(4-fluorobenzyl)-piperazine (FBZP), 4-methyl-1-benzylpiperazine (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). All samples were prepared by fortifying 100 µL of certified drug-free whole blood and urine (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) with certified reference standards (Cayman Chemical, Ann Arbor, MI, U.S.A.) of each analyte at desired concentrations and standard additions of 1-benzylpiperazine-d7, 1-(3-chlorophenyl)-piperazine-d8, and 1(-3-trifluoromethylphenyl)-piperazine-d4 internal standards (Cerilliant, Round Rock, TX, U.S.A). After pretreatment with 1 mL phosphate buffer, samples underwent solid phase extraction (SPE) on mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.). Eluents were evaporated to dryness with low heat (65°C) and nitrogen gas. Samples were reconstituted with a 50:50 mixture of methanol and 2mM ammonium formate buffer with 0.2% formic acid before being analyzed by a UFLC (Shimadzu Corporation, Kyoto, Japan) and 4000 QTRAP ESIMS/MS (SCIEX, Framingham, MA, U.S.A.) system. Analyses were performed with multiple reaction monitoring scans in positive ionization mode using ions and voltages obtained from a manual compound optimization. Analytes were separated on a reversed phase column (Kinetex® F5, Phenomenex®, Torrance, CA, U.S.A.) with a binary gradient consisting of a 2mM ammonium formate buffer with 0.2% formic acid and methanol with 0.1% formic acid. The flow rate was 0.400 mL/min. Analyst™ (SCIEX) software was used for data collection and MultiQuant™ (SCIEX) software was used for quantitation. The total run time was 11.5 minutes with equilibrations. All calibration curves in both matrices exhibited R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 20-2000 ng/mL was used for all analytes in both matrices, except for BZP in urine which ranged from 50-2000 ng/mL. In blood, the limit of quantitation was 10 ng/mL for mCPP and TFMPP and 20 ng/mL for BZP, FBZP, MBZP, MeOPP, pFPP and DCPP. In urine, the limit of quantitation was 10 ng/mL for MeOPP, mCPP, TFMPP and DCPP, 20 ng/mL for FBZP, MBZP and pFPP and 50 ng/mL for BZP. When a 200 ng/mL concentration was evaluated, the SPE procedure showed percent recoveries ranging from 80-95% for blood; except for BZP, FBZP, and MeOPP which had recoveries of 60%, 60%, and 105%, respectively. Percent recoveries ranged from 82-94% for urine; except for BZP and FBZP which had recoveries of 66% and 68%, respectively. Bias and precision were assessed at concentrations of 50, 200, and 700 ng/mL. All samples were calculated within ±20% bias and within ±20% coefficient of variation. The highest concentration evaluated that did not produce carryover in subsequent matrix blanks was 5000 ng/mL. Ionization was suppressed for all analytes in both matrices by 45-95%. Matrix effects were present but were determined to be insignificant. Of the drugs evaluated, caffeine, dibenzylpiperazine, and 1-(4-chlorophenyl)-piperazine (pCPP) produced chromatographic peaks in the method; however, pCPP was the only substance that affected quantitation of an analyte. It increased the peak area of mCPP by almost 50% when present at the same concentration which suggests this method is unable to differentiate between isomeric pairs. This is a sensitive, reliable, and robust method with a wide linear dynamic range to account for the presence of these analytes in both blood and urine. This research will provide for the identification and quantitation of these substances in forensic casework.

In the last few years, the prevalence of legal party pills in New Zealand has risen dramatically. These pills contain new piperazine designer drugs, two of the more common being 1-benzylpiperazine (BZP) and m-trifluoromethylphenylpiperazine (TFMPP). This thesis describes an optimised LC-MS/MS method for the detection of BZP and TFMPP in whole blood, using an automated solid phase extraction (SPE) for sample clean-up. The method was validated on three different days using five replicate samples each day. The standard curve was linear from 7 - 7000 ng/mL for BZP and 10 - 10,000 ng/mL for TFMPP, with coefficients of variation (CV) below 10%, and accuracy greater than 90% for both drugs. The method was used to quantitate samples provided by the Medical Research Institute of New Zealand. Blood levels were used to show concentrations in the blood over time, and relate these to performance of subjects on a driving simulator. The study was stopped after 41% of the participants who received BZP and TFMPP had adverse reactions to the pills, including vomiting and migraines. The LC-MS/MS method was also used to detect and quantitate methamphetamine, amphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, morphine, codeine and 6-monoacetylmorphine in hair. The drugs were extracted from 20 mg of hair using hydrochloric acid in a water bath overnight, then purified using SPE. Validation on three days with five replicate samples gave coefficients of variation (CV) below 12% and acceptable accuracy for all drugs. The method was tested on three samples, previously reported by Environmental Science and Research (ESR) using gas chromatography mass spectrometry (GC-MS) giving results in good agreement. This thesis describes a sensitive, accurate, reproducible LC-MS/MS method easily adapted to analyse drugs of abuse in different biological matrices. It demonstrates the versatility of LC-MS/MS and its applications in forensic work.

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