Stability of Synthetic Piperazines in Human Whole Blood

Stability of Synthetic Piperazines in Human Whole Blood Abstract While circumventing legislative controls, synthetic piperazines are encountered as “legal” alternatives to ecstasy. Unforeseeable challenges may delay quantitative analysis of these compounds in biological fluids. Enzymatic reactions, matrix interferences and limited knowledge of analyte stability further complicate interpretation of calculated concentrations. The objective of this study was to investigate the stability of synthetic piperazines in human blood under various storage conditions over time. All samples were prepared by spiking certified reference standards (Cayman Chemical, MI, U.S.A.) of eight synthetic piperazine into certified drug-free human whole blood (UTAK Laboratories, Inc., CA, U.S.A.) independently at 1000 ng/mL as well as mixtures containing all tested piperazines in this study. Samples were stored at room temperature (~20°C), 4°C and −20°C for 1, 3, 6, 9 and 12 months in dark sealed containers. Solid phase extraction (SPE) was performed using mixed-mode copolymeric cartridges (Clean Screen®, UCT Inc., PA, U.S.A.). Analytes were assessed on their degrees of degradation using a Shimadzu Ultra-Fast Liquid Chromatograph with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometer (UFLC-ESI-MS/MS) in positive ionization mode. Of the two categories, benzyl piperazines were more stable than phenyl piperazines under all storage conditions, in which 1-(4-methylbenzyl)-piperazine (MBZP) had more than 70% (769–1,047 ng/mL) remaining after 12 months. 1-(4-methoxyphenyl)-piperazine (MeOPP) was not detected under room and refrigerated temperatures after 6 months and was the least stable. Matrix interferences and drug–drug interaction were observed. Storing samples at room temperature should be avoided due to detrimental impacts on stability of piperazine compounds. For backlog situations, case samples suspected to contain synthetic piperazines should be kept frozen or refrigerated even for time periods as short as 30 days for optimal result. Phenyl piperazines stored for more than 6 months showed analyte degradation and loss of parent compounds after extended storage regardless of storage conditions. Introduction Novel psychoactive substances (NPS) cover a large number of recreational drugs that mimic the effects of existing controlled substances and are often sold as “legal highs” to circumvent legal authorities (1). One example of NPS is piperazines derivatives, which belong to a class of amphetamine-like compounds and have made a resurgence as “legal Ecstasy” (2). The first documented abuse of a piperazine-derived drug involved 1-benzylpiperazine (BZP) and was reported in the United States (U.S.A) in 1996 (3). In 2000, piperazine-related abuse had begun to spread in New Zealand and Australia (4). Reported by the U.S. National Forensic Laboratory Information System (NFLIS), about 38,230 piperazine-containing substances were seized from 2006 to 2010 and this class of drugs was one of the top 25 identified drugs in 2011 (5). In addition, there were at least twelve substituted synthetic piperazine drugs on the clandestine drug market as of 2013 in which few are currently regulated according to the United Nations Office on Drugs and Crime (UNODC) (6). Piperazine-based compounds are completely synthetic and were primarily used as anthelmintic agents in veterinary and clinical practices (3). They can be divided into two classes: benzyl piperazines, which include BZP, 1-(4-fluorobenzyl)-piperazine (FBZP) and 1-(4-methylbenzyl)-piperazine (MBZP); and phenyl piperazines, which include 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) (3). Several studies in the 1970s suggested that BZP could be a potential antidepressant medication but was subsequently rejected due to its amphetamine-like effects coupled with a high potential for abuse (2). Since the majority of the NPS are relatively new to the scientific community, TFMPP, BZP and mCPP are the only synthetic piperazines that have been studied in detail in terms of their pharmacodynamic and pharmacokinetic properties (7, 8). Synthetic piperazines can stimulate the release and also inhibit the reuptake of dopamine, serotonin (5-HT) and noradrenaline, in which serotonergic and dopaminergic effects predominate in most cases (4). Although post-consumption impact of BZP include stimulant effects such as elevated blood pressure, increased heart rate, increased euphoria, dysphoria, sociability and drug-liking, the variable hallucinogenic effect was found to be about 10-fold less potent than 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine or amphetamine. Piperazines are popular in youth populations because of their long-lasting psychoactive duration of effect, which is typically four to 6 h. In order to enhance the spectrum of effects, BZP may be mixed with TFMPP and drug-induced behavior may be observed and last up to 8 h (2, 4, 9). Laboratory backlogs often prevent analysts from obtaining reliable quantitative data due to variable time intervals between obtaining a sample and analysis. Moreover, storage conditions of specific drugs or compounds in biological samples may affect the analytical results due to ongoing enzymatic metabolism and postmortem redistribution (10, 11). Johnson and Botch-Jones’ study on the investigation of stability of TFMPP in blood demonstrated significant degradation after a 14-day storage period at room temperature (11). Thus, chemical degradation or even bacterial activity may potentially become an issue and may make the interpretation of analytical results difficult (10, 11). Since the stability of synthetic piperazines in whole blood samples over extended storage time periods remains unknown, a total of eight synthetic piperazines including BZP, FBZP, MBZP, MeOPP, pFPP, mCPP, DCPP and TFMPP (Figure 1) were assessed on their degrees of degradation. Figure 1. View largeDownload slide Chemical structures of synthetic piperazines and deuterated internal standards used in this study (13). Figure 1. View largeDownload slide Chemical structures of synthetic piperazines and deuterated internal standards used in this study (13). Material and Methods Instrumentation Analysis was performed on a Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC) System (Kyoto, Japan) and a SCIEX 4000 QTRAP tandem Mass Spectrometer (MS/MS) (Framingham, MA, U.S.A). The separation was achieved using a Kinetex® F5 2.6 μm, 100 Å, 150 mm × 3.0 mm ID column purchased from Phenomenex, Inc. (Torrance, CA, U.S.A). All data were collected using Analyst™ (version 1.6.2) software and quantitation was conducted with MultiQuant™ 3.0 (version 3.05373.0) software (SCIEX). Two mobile phases were used: 2 mM ammonium formate buffer with 0.2% formic acid (mobile phase A) and LC-grade methanol with 0.1% formic acid (mobile phase B). The flow rate was set to 0.4 mL/min in which the starting condition was 5% mobile phase B (Table I). The injection volume and run time were 5 μL and 6.5 min, respectively. The sensitivity of the multiple reactions monitoring (MRM) was optimized as shown in Table II. Ion source gas 1, ion source gas 2, curtain gas and collision gas flow were at instrument settings of 50 psi, 80 psi, 30 psi and at medium, respectively. The ionspray voltage was maintained at 2,500 V while the turbo gas temperature was at 600°C. Table I. LC time program Time (min)  Module  Event  Parameter (%)  0.01  Pumps  Pump B Concentration  5  0.30  Pumps  Pump B Concentration  5  3.50  Pumps  Pump B Concentration  80  6.50  Pumps  Pump B Concentration  80  6.51  Pumps  Pump B Concentration  5  10.00  Controller  Stop    Time (min)  Module  Event  Parameter (%)  0.01  Pumps  Pump B Concentration  5  0.30  Pumps  Pump B Concentration  5  3.50  Pumps  Pump B Concentration  80  6.50  Pumps  Pump B Concentration  80  6.51  Pumps  Pump B Concentration  5  10.00  Controller  Stop    Table II. Multiple reaction monitoring (MRM) table Name of Analyte  Q1 Mass (Da)  Q3 Mass (Da)  Dwell Time (msec)  DP (V)  EP (V)  CE (V)  CXP (V)  BZP-d7 IS 1  184.3  98.2  50  70.0  10  30.0  15.0  BZP 1  177.1  91.1  50  66.0  10  32.0  14.0  BZP 2  177.1  65.1  50  66.0  10  63.0  9.0  FBZP 1  195.2  109.1  50  72.0  10  29.0  18.0  FBZP 2  195.2  83.2  50  72.0  10  65.0  12.0  MBZP 1  191.2  91.1  50  75.0  10  31.0  15.0  MBZP 2  191.2  65.2  50  75.0  10  67.0  9.0  MeOPP 1  193.2  150.2  50  70.0  10  28.0  24.0  MeOPP 2  193.2  119.3  50  70.0  10  34.0  19.0  pFPP 1  181.2  138.2  50  75.0  10  29.0  23.0  pFPP 2  181.2  75.2  50  75.0  10  77.0  11.0  mCPP 1  197.1  154.2  50  75.0  10  28.0  26.0  mCPP 2  197.1  118.2  50  75.0  10  48.0  19.0  mCPP-d8 IS 1  205.4  158.2  50  86.0  10  31.0  26.0  DCPP 1  233.1  190.2  50  85.0  10  30.0  32.0  DCPP 2  233.1  117.2  50  85.0  10  67.0  19.0  TFMPP 1  231.1  188.1  50  80.0  10  32.0  33.0  TFMPP 2  231.1  118.3  50  80.0  10  54.0  19.0  TFMPP-d4 IS 1  235.4  190.2  50  84.0  10  32.0  32.0  Name of Analyte  Q1 Mass (Da)  Q3 Mass (Da)  Dwell Time (msec)  DP (V)  EP (V)  CE (V)  CXP (V)  BZP-d7 IS 1  184.3  98.2  50  70.0  10  30.0  15.0  BZP 1  177.1  91.1  50  66.0  10  32.0  14.0  BZP 2  177.1  65.1  50  66.0  10  63.0  9.0  FBZP 1  195.2  109.1  50  72.0  10  29.0  18.0  FBZP 2  195.2  83.2  50  72.0  10  65.0  12.0  MBZP 1  191.2  91.1  50  75.0  10  31.0  15.0  MBZP 2  191.2  65.2  50  75.0  10  67.0  9.0  MeOPP 1  193.2  150.2  50  70.0  10  28.0  24.0  MeOPP 2  193.2  119.3  50  70.0  10  34.0  19.0  pFPP 1  181.2  138.2  50  75.0  10  29.0  23.0  pFPP 2  181.2  75.2  50  75.0  10  77.0  11.0  mCPP 1  197.1  154.2  50  75.0  10  28.0  26.0  mCPP 2  197.1  118.2  50  75.0  10  48.0  19.0  mCPP-d8 IS 1  205.4  158.2  50  86.0  10  31.0  26.0  DCPP 1  233.1  190.2  50  85.0  10  30.0  32.0  DCPP 2  233.1  117.2  50  85.0  10  67.0  19.0  TFMPP 1  231.1  188.1  50  80.0  10  32.0  33.0  TFMPP 2  231.1  118.3  50  80.0  10  54.0  19.0  TFMPP-d4 IS 1  235.4  190.2  50  84.0  10  32.0  32.0  DP, declustering potential; CE, collision energy; CXP, cell exit potential; EP, entrance potential. Reference standards and reagents The following reagents were purchased from Fisher Scientific (Waltham, MA, U.S.A): LC-grade methanol, LC-grade 2-propanol, Optima grade acetonitrile, formic acid, methylene chloride, ammonium formate, concentrated ammonium hydroxide, concentrated hydrochloric acid, anhydrous disodium phosphate and monohydrate sodium dihydrogen phosphate. BZP, FBZP, MBZP, MeOPP, pFPP, mCPP, TFMPP and DCPP were purchased through Cayman Chemical Company (Ann Arbor, MI, U.S.A.). All piperazines were received in the form of 10 mg powder except for BZP, which was received as a 1 mg/mL standard in methanol. Three deuterated internal standards including BZP-d7, mCPP-d8 and TFMPP-d4 were received as 100 μg/mL standards in methanol from Cerilliant Corporation (Round Rock, TX, U.S.A.). Fresh millipore water was obtained daily from the Synergy UV water filtration system from EMD Millipore/Merck (Darmstadt, Germany). Certified drug-free human whole blood was purchased from UTAK Laboratories, Inc. (Valencia, CA). Preparation of stock solution and calibrators A concentrated stock solution of each piperazine was prepared by dissolving 1 mg of each piperazine powder in 1 mL of methanol (except for BZP); they were further combined and serially diluted with 50:50 mixture of millipore water and methanol to give a 1 μg/mL stock solution (Stock 1). The second set of stock solutions (Stock 2) were prepared at a concentration of 100 ng/mL. An internal standard stock solution was prepared with 50:50 mixture of millipore water and methanol to a final concentration of 1 μg/mL by combining BZP-d7, mCPP-d8 and TFMPP-d4. Calibrators were prepared fresh daily by spiking 100 μL of whole blood with the appropriate volume of stock solutions to yield concentrations of 20, 50, 100, 200, 500, 1,000 and 2,000 ng/mL. Two quality control (QC) samples (30 and 1,500 ng/mL), a negative control and a double blank solution (without internal standards) were prepared for each run. A separate calibration curve was also required to accurately quantify the analyte of interest in order to avoid any possible underestimation of TFMPP. Preparation of storage samples on Day 0 Samples were stored in individual aliquots where eight different tubes of 10 mL whole blood each contained one of the synthetic piperazines were prepared independently at 1,000 ng/mL. A tube of 10 mL whole blood containing all eight synthetic piperazine standards (mixture sample) and a tube containing only 10 mL whole blood were prepared to serve as controls. Aliquots of each sample were then transferred into two separate amber glass vials (~3.3 mL each). They were then capped and placed at room temperature and 4°C for the designated period of time. The remaining ~3.4 mL of mixture solution in the tube was also stored and wrapped in opaque tape to prevent degradation by light, and placed in a freezer at −20°C. Temperatures for the freezer, refrigerator and the room where samples were stored were monitored and recorded daily. Internal standards were added on the day of analysis. Sample preparation All tested piperazines were extracted from whole blood by solid phase extraction (SPE). Since piperazines are basic in nature, Drugs of Abuse (DAU) mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.) were chosen for solid phase extraction. The mixed-mode sorbent exhibiting both hydrophobic and cation exchange characteristics serves to retain ionized basic compounds. First, 1 mL of phosphate buffer (100 mM, pH 6) was added to all samples prior to loading. Then, SPE cartridges were conditioned with 1 mL of methanol followed by 1 mL of phosphate buffer (100 mM, pH 6). Subsequently, samples (1 mL of the pre-added phosphate buffer and 100 μL of blood containing piperazines) were loaded into each designated pre-conditioned column and were allowed to drip with gravity flow. A series of wash steps were performed on each column in the order of 1 mL of millipore water, 1 mL of 0.1 N hydrochloric acid and 1 mL of LC-grade methanol. A flow of compressed nitrogen gas was applied for 5 min at 40 psi. Samples were then eluted in 2 mL of base elution solvent which was made fresh for each experiment. The base elution solvent was prepared in the order of adding 20% 2-propanol, 3% of concentrated ammonium hydroxide and 77% of methylene chloride. Eluents were evaporated to dryness at 65°C on a heating block. The residues were reconstituted in 250 μL of a 50:50 mixture of methanol and 2 mM ammonium formate buffer with 0.2% formic acid, and was directly injected into the chromatographic system. Stability analysis Stability of synthetic piperazines was examined by monitoring the amount loss of analytes over time at each storage condition. Quantification of synthetic piperazines in all samples was performed using LC-MS/MS and were compared to the calibration curves generated on both Day 0 and at the set analysis days for the duration of the study. Three replicates of each sample were analyzed and the average was obtained to serve as the initial concentration on Day 0 and at the end of the storage period. Results and Discussion Chromatography and specificity The UFLC method described above achieved excellent resolution and peak shape with full elution in 6.5 min. All benzyl piperazines eluted first at the following retention times: BZP at 3.82, FBZP at 4.48 and MBZP at 4.69 min (Figure 2). The phenyl piperazines then eluted at the followings: MeOPP at 5.09, pFPP at 5.33, mCPP at 5.80, DCPP at 6.11 and TFMPP at 6.14 min. Retention times were 3.71, 5.80 and 6.15 min for BZP-d7, mCPP-d8 and TFMPP-d4, respectively. Figure 2. View largeDownload slide Representative chromatogram of BZP, FBZP, MBZP, MeOPP, mCPP, TFMPP, pFPP, DCPP, BZP-d7, mCPP-d8 and TFMPP-d4 at 500 ng/mL extracted from whole blood on Day 0. Figure 2. View largeDownload slide Representative chromatogram of BZP, FBZP, MBZP, MeOPP, mCPP, TFMPP, pFPP, DCPP, BZP-d7, mCPP-d8 and TFMPP-d4 at 500 ng/mL extracted from whole blood on Day 0. Calibration and linearity The seven-point calibration curve was generated for each run by plotting the peak area ratio (y) of the target analyte to the internal standard versus analyte concentration (x). The coefficient of determination (R2 values) was calculated by MultiQuant™ software with a weighting factor of 1/x. Among the three deuterated internal standards, BZP-d7 was used to quantify BZP, FBZP and MBZP; mCPP-d8 was used to quantify MeOPP, pFPP, mCPP and DCPP; TFMPP-d4 was used to quantify TFMPP only. Accuracy and precision Accuracy of calibrators and QC samples were determined to be within the allowed ± 20% range by comparing the calculated concentration using calibration curves to the known concentration. All R2 values were above the minimum accepted value of 0.98 according to the Scientific Working Group for Forensic Toxicologists (SWGTOX) guidelines for quantitative methods (12). Precision of the assay was evaluated by calculating the standard deviation and the percent coefficient of variation (% CV) in three replicates. Stability All data, shown in Tables III through VII, was assessed based on storage conditions (~20°C, 4°C and −20°C) and time ranges (1, 3, 6, 9 and 12 months). The three benzyl piperazines that were kept frozen demonstrated minimal variation within the first 91 days (~3 months) and the overall loss was less than 15%. In contrast, phenyl piperazines that were kept frozen showed moderate degradations, especially for DCPP and TFMPP, for which only 42% and 52% of the parent compound remained on Day 91, respectively. As expected, the degree of loss for most synthetic piperazines stored at room temperature was larger than those that were kept at 4°C or −20°C. Most data showed a smaller degree of degradation if proper storage conditions were maintained and that is when samples were kept in either a refrigerator or a freezer. For crime laboratories that are facing backlog situations, it is recommended that specimens containing synthetic piperazines should be kept frozen or refrigerated for 30 days or less. Storing samples at room temperature should be avoided because of detrimental impacts on the stability of piperazine compounds. This information is also valuable when analyzing data for postmortem specimens that are collected from a decomposed body or a body that is found in an exceptionally warm environment. Table III. Stability data on Day 30 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  1105.43  62.60  5.66  1054.4–1175.28  BZP 4°C  949.15  53.16  5.60  887.76–980.12  BZP −20°C  1166.81  51.16  4.38  1107.74–1196.79  FBZP RT  787.14  10.27  1.30  776.82–797.36  FBZP 4°C  902.95  124.78  13.82  795.24–1039.68  FBZP −20°C  1295.06  38.63  2.98  1263.94–1338.29  MBZP RT  503.33  12.55  2.49  495.44–517.80  MBZP 4°C  606.33  26.30  4.34  582.41–634.49  MBZP −20°C  1144.13  60.45  5.28  1082.81–1203.68  MeOPP RT  1238.79  50.78  4.10  1189.35–1290.82  MeOPP 4°C  767.46  10.33  1.35  759.41–779.11  MeOPP −20°C  961.05  3.48  0.36  957.78–964.72  pFPP RT  1207.86  14.97  1.24  1192.02–1221.77  pFPP 4°C  917.57  37.17  4.05  878.09–951.89  pFPP −20°C  1073.07  34.24  3.19  1053.01–1112.60  mCPP RT  1010.19  37.80  3.74  966.73–1035.41  mCPP 4°C  973.45  13.62  1.40  964.31–989.11  mCPP −20°C  978.83  10.23  1.04  971.99–990.58  DCPP RT  1060.75  41.01  3.87  1018.16–1099.97  DCPP 4°C  705.49  59.09  8.38  641.63–758.22  DCPP −20°C  876.19  10.69  1.22  866.81–887.83  TFMPP RT  499.42  22.57  4.52  481.18–524.66  TFMPP 4°C  385.70  8.71  2.26  375.65–390.98  TFMPP −20°C  686.05  54.35  7.92  641.83–746.74  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  1105.43  62.60  5.66  1054.4–1175.28  BZP 4°C  949.15  53.16  5.60  887.76–980.12  BZP −20°C  1166.81  51.16  4.38  1107.74–1196.79  FBZP RT  787.14  10.27  1.30  776.82–797.36  FBZP 4°C  902.95  124.78  13.82  795.24–1039.68  FBZP −20°C  1295.06  38.63  2.98  1263.94–1338.29  MBZP RT  503.33  12.55  2.49  495.44–517.80  MBZP 4°C  606.33  26.30  4.34  582.41–634.49  MBZP −20°C  1144.13  60.45  5.28  1082.81–1203.68  MeOPP RT  1238.79  50.78  4.10  1189.35–1290.82  MeOPP 4°C  767.46  10.33  1.35  759.41–779.11  MeOPP −20°C  961.05  3.48  0.36  957.78–964.72  pFPP RT  1207.86  14.97  1.24  1192.02–1221.77  pFPP 4°C  917.57  37.17  4.05  878.09–951.89  pFPP −20°C  1073.07  34.24  3.19  1053.01–1112.60  mCPP RT  1010.19  37.80  3.74  966.73–1035.41  mCPP 4°C  973.45  13.62  1.40  964.31–989.11  mCPP −20°C  978.83  10.23  1.04  971.99–990.58  DCPP RT  1060.75  41.01  3.87  1018.16–1099.97  DCPP 4°C  705.49  59.09  8.38  641.63–758.22  DCPP −20°C  876.19  10.69  1.22  866.81–887.83  TFMPP RT  499.42  22.57  4.52  481.18–524.66  TFMPP 4°C  385.70  8.71  2.26  375.65–390.98  TFMPP −20°C  686.05  54.35  7.92  641.83–746.74  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL; TFMPP was at 502.83 ± 8.36 (mean ± standard deviation) on Day 0 as supposed to 1,000 ng/mL due to isotope interference. Table IV. Stability data on Day 91 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  934.90  110.27  11.79  807.60–1000.85  BZP 4°C  1059.16  35.21  3.32  1025.17–1095.48  BZP −20°C  1068.37  19.53  1.83  1046.83–1084.94  FBZP RT  851.75  21.09  2.48  827.62–866.66  FBZP 4°C  1017.92  13.31  1.31  1003.01–1028.62  FBZP −20°C  908.15  5.87  0.65  901.63–913.01  MBZP RT  1391.77  12.28  0.88  1380.41–1404.81  MBZP 4°C  1130.29  36.20  3.20  1105.81–1171.88  MBZP −20°C  1191.49  65.11  5.46  1118.61–1244.02  MeOPP RT  542.09  19.94  3.68  520.16–559.15  MeOPP 4°C  733.51  31.26  4.26  706.73–767.86  MeOPP −20°C  645.49  18.74  2.90  633.43–667.08  pFPP RT  513.79  10.09  1.96  502.27–521.03  pFPP 4°C  744.71  22.15  2.97  719.13–758.15  pFPP −20°C  910.21  41.96  4.61  864.18–946.32  mCPP RT  788.11  2.90  0.37  785.13–790.93  mCPP 4°C  755.23  21.67  2.87  730.42–770.42  mCPP −20°C  738.95  14.50  1.96  724.18–753.18  DCPP RT  497.08  13.49  2.71  485.29–511.79  DCPP 4°C  648.46  16.45  2.54  629.75–660.64  DCPP −20°C  483.55  8.96  1.85  477.78–493.87  TFMPP RT  686.02  12.80  1.87  675.00–700.06  TFMPP 4°C  668.36  13.56  2.03  657.23–683.47  TFMPP −20°C  580.24  13.60  2.34  565.76–592.75  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  934.90  110.27  11.79  807.60–1000.85  BZP 4°C  1059.16  35.21  3.32  1025.17–1095.48  BZP −20°C  1068.37  19.53  1.83  1046.83–1084.94  FBZP RT  851.75  21.09  2.48  827.62–866.66  FBZP 4°C  1017.92  13.31  1.31  1003.01–1028.62  FBZP −20°C  908.15  5.87  0.65  901.63–913.01  MBZP RT  1391.77  12.28  0.88  1380.41–1404.81  MBZP 4°C  1130.29  36.20  3.20  1105.81–1171.88  MBZP −20°C  1191.49  65.11  5.46  1118.61–1244.02  MeOPP RT  542.09  19.94  3.68  520.16–559.15  MeOPP 4°C  733.51  31.26  4.26  706.73–767.86  MeOPP −20°C  645.49  18.74  2.90  633.43–667.08  pFPP RT  513.79  10.09  1.96  502.27–521.03  pFPP 4°C  744.71  22.15  2.97  719.13–758.15  pFPP −20°C  910.21  41.96  4.61  864.18–946.32  mCPP RT  788.11  2.90  0.37  785.13–790.93  mCPP 4°C  755.23  21.67  2.87  730.42–770.42  mCPP −20°C  738.95  14.50  1.96  724.18–753.18  DCPP RT  497.08  13.49  2.71  485.29–511.79  DCPP 4°C  648.46  16.45  2.54  629.75–660.64  DCPP −20°C  483.55  8.96  1.85  477.78–493.87  TFMPP RT  686.02  12.80  1.87  675.00–700.06  TFMPP 4°C  668.36  13.56  2.03  657.23–683.47  TFMPP −20°C  580.24  13.60  2.34  565.76–592.75  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Table V. Stability data on Day 182 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  253.42  0.94  0.37  252.53–254.40  BZP 4°C  240.88  3.01  1.25  237.45–243.07  BZP −20°C  443.01  4.11  0.93  439.13–447.33  FBZP RT  67.76  1.20  1.77  66.63–69.02  FBZP 4°C  81.39  1.49  1.83  80.26–83.08  FBZP −20°C  317.75  7.57  2.38  311.63–326.21  MBZP RT  1144.30  13.53  1.18  1128.98–1154.64  MBZP 4°C  959.84  32.84  3.42  922.76–985.26  MBZP −20°C  1189.62  77.19  6.49  1100.91–1241.49  MeOPP RT  0.00  0.00  0.00  0.00  MeOPP 4°C  0.00  0.00  0.00  0.00  MeOPP −20°C  3.42  5.93  173.21  0.00–10.27  pFPP RT  76.25  7.13  9.36  70.12–84.09  pFPP 4°C  17.33  0.97  5.62  16.61–18.44  pFPP −20°C  105.61  10.33  9.78  98.69–117.49  mCPP RT  9.84  0.12  1.25  9.75–9.98  mCPP 4°C  15.59  3.15  20.20  13.24–19.17  mCPP −20°C  56.39  1.16  2.06  55.15–57.44  DCPP RT  13.19  2.92  22.13  9.84–15.15  DCPP 4°C  16.64  1.54  9.27  15.45–18.38  DCPP −20°C  78.14  17.14  21.94  65.71–97.70  TFMPP RT  13.33  0.91  6.82  12.56–14.33  TFMPP 4°C  15.72  1.40  8.88  14.77–17.32  TFMPP −20°C  63.57  4.22  6.63  58.72–66.39  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  253.42  0.94  0.37  252.53–254.40  BZP 4°C  240.88  3.01  1.25  237.45–243.07  BZP −20°C  443.01  4.11  0.93  439.13–447.33  FBZP RT  67.76  1.20  1.77  66.63–69.02  FBZP 4°C  81.39  1.49  1.83  80.26–83.08  FBZP −20°C  317.75  7.57  2.38  311.63–326.21  MBZP RT  1144.30  13.53  1.18  1128.98–1154.64  MBZP 4°C  959.84  32.84  3.42  922.76–985.26  MBZP −20°C  1189.62  77.19  6.49  1100.91–1241.49  MeOPP RT  0.00  0.00  0.00  0.00  MeOPP 4°C  0.00  0.00  0.00  0.00  MeOPP −20°C  3.42  5.93  173.21  0.00–10.27  pFPP RT  76.25  7.13  9.36  70.12–84.09  pFPP 4°C  17.33  0.97  5.62  16.61–18.44  pFPP −20°C  105.61  10.33  9.78  98.69–117.49  mCPP RT  9.84  0.12  1.25  9.75–9.98  mCPP 4°C  15.59  3.15  20.20  13.24–19.17  mCPP −20°C  56.39  1.16  2.06  55.15–57.44  DCPP RT  13.19  2.92  22.13  9.84–15.15  DCPP 4°C  16.64  1.54  9.27  15.45–18.38  DCPP −20°C  78.14  17.14  21.94  65.71–97.70  TFMPP RT  13.33  0.91  6.82  12.56–14.33  TFMPP 4°C  15.72  1.40  8.88  14.77–17.32  TFMPP −20°C  63.57  4.22  6.63  58.72–66.39  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL; TFMPP was at 548.59 ± 4.22 (mean ± standard deviation) on Day 0. Table VI. Stability data on Day 270 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  600.41  10.58  1.76  590.36–611.45  BZP 4°C  671.34  40.60  6.05  630.82–712.01  BZP −20°C  576.30  31.97  5.55  545.85–609.60  FBZP RT  63.75  1.03  1.61  63.08–64.93  FBZP 4°C  81.48  5.87  7.20  75.38–87.09  FBZP −20°C  331.61  9.39  2.83  320.98–338.80  MBZP RT  1223.00  11.17  0.91  1210.14–1230.32  MBZP 4°C  1530.80  50.88  3.32  1486.61–1586.42  MBZP −20°C  1262.52  16.33  1.29  1244.10–1275.24  MeOPP RT  63.06  17.85  28.31  51.15–83.58  MeOPP 4°C  143.96  40.22  27.94  117.15–190.21  MeOPP −20°C  179.06  40.41  22.57  132.70–206.77  pFPP RT  167.07  46.91  28.08  139.24–221.23  pFPP 4°C  236.47  62.92  26.61  199.48–309.12  pFPP −20°C  172.96  42.75  24.72  124.09–203.45  mCPP RT  26.12  0.45  1.73  25.60–26.43  mCPP 4°C  69.02  3.07  4.45  66.47–72.43  mCPP −20°C  151.02  5.46  3.61  147.58–157.31  DCPP RT  9.10  1.50  16.49  8.23–10.83  DCPP 4°C  26.52  9.26  34.92  19.71–37.07  DCPP −20°C  106.07  22.58  21.28  80.10–120.98  TFMPP RT  0.71  1.22  173.21  0.00–2.12  TFMPP 4°C  22.46  30.63  136.38  4.63–57.83  TFMPP −20°C  88.80  34.47  38.81  60.68–127.25  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  600.41  10.58  1.76  590.36–611.45  BZP 4°C  671.34  40.60  6.05  630.82–712.01  BZP −20°C  576.30  31.97  5.55  545.85–609.60  FBZP RT  63.75  1.03  1.61  63.08–64.93  FBZP 4°C  81.48  5.87  7.20  75.38–87.09  FBZP −20°C  331.61  9.39  2.83  320.98–338.80  MBZP RT  1223.00  11.17  0.91  1210.14–1230.32  MBZP 4°C  1530.80  50.88  3.32  1486.61–1586.42  MBZP −20°C  1262.52  16.33  1.29  1244.10–1275.24  MeOPP RT  63.06  17.85  28.31  51.15–83.58  MeOPP 4°C  143.96  40.22  27.94  117.15–190.21  MeOPP −20°C  179.06  40.41  22.57  132.70–206.77  pFPP RT  167.07  46.91  28.08  139.24–221.23  pFPP 4°C  236.47  62.92  26.61  199.48–309.12  pFPP −20°C  172.96  42.75  24.72  124.09–203.45  mCPP RT  26.12  0.45  1.73  25.60–26.43  mCPP 4°C  69.02  3.07  4.45  66.47–72.43  mCPP −20°C  151.02  5.46  3.61  147.58–157.31  DCPP RT  9.10  1.50  16.49  8.23–10.83  DCPP 4°C  26.52  9.26  34.92  19.71–37.07  DCPP −20°C  106.07  22.58  21.28  80.10–120.98  TFMPP RT  0.71  1.22  173.21  0.00–2.12  TFMPP 4°C  22.46  30.63  136.38  4.63–57.83  TFMPP −20°C  88.80  34.47  38.81  60.68–127.25  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Table VII. Stability data on Day 365 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  284.83  13.00  4.56  269.94–293.85  BZP 4°C  585.98  6.80  1.16  581.91–593.83  BZP −20°C  662.86  22.46  3.39  640.48–685.41  FBZP RT  63.72  3.62  5.68  60.91–67.80  FBZP 4°C  166.41  9.44  5.67  155.52–172.03  FBZP −20°C  442.59  5.57  1.26  436.68–447.73  MBZP RT  874.87  31.06  3.55  843.32–905.41  MBZP 4°C  713.65  49.57  6.95  673.20–768.96  MBZP −20°C  966.52  69.82  7.22  917.80–1046.51  MeOPP RT  398.36  4.84  1.21  394.67–403.83  MeOPP 4°C  474.92  5.07  1.07  469.10–478.31  MeOPP −20°C  366.65  5.58  1.52  360.49–371.34  pFPP RT  314.91  3.86  1.22  311.80–319.22  pFPP 4°C  711.98  1.97  0.28  710.04–713.99  pFPP −20°C  590.29  21.05  3.57  567.86–609.61  mCPP RT  75.59  2.19  2.90  73.14–77.36  mCPP 4°C  621.57  4.15  0.67  616.79–624.31  mCPP −20°C  408.93  17.93  4.38  398.15–429.63  DCPP RT  20.07  1.74  8.67  18.70–22.03  DCPP 4°C  79.98  2.95  3.69  77.79–83.34  DCPP −20°C  283.65  7.92  2.79  275.08–290.70  TFMPP RT  73.32  4.34  5.92  68.89–77.57  TFMPP 4°C  420.73  4.87  1.16  416.98–426.23  TFMPP −20°C  343.50  13.00  3.78  333.01–358.04  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  284.83  13.00  4.56  269.94–293.85  BZP 4°C  585.98  6.80  1.16  581.91–593.83  BZP −20°C  662.86  22.46  3.39  640.48–685.41  FBZP RT  63.72  3.62  5.68  60.91–67.80  FBZP 4°C  166.41  9.44  5.67  155.52–172.03  FBZP −20°C  442.59  5.57  1.26  436.68–447.73  MBZP RT  874.87  31.06  3.55  843.32–905.41  MBZP 4°C  713.65  49.57  6.95  673.20–768.96  MBZP −20°C  966.52  69.82  7.22  917.80–1046.51  MeOPP RT  398.36  4.84  1.21  394.67–403.83  MeOPP 4°C  474.92  5.07  1.07  469.10–478.31  MeOPP −20°C  366.65  5.58  1.52  360.49–371.34  pFPP RT  314.91  3.86  1.22  311.80–319.22  pFPP 4°C  711.98  1.97  0.28  710.04–713.99  pFPP −20°C  590.29  21.05  3.57  567.86–609.61  mCPP RT  75.59  2.19  2.90  73.14–77.36  mCPP 4°C  621.57  4.15  0.67  616.79–624.31  mCPP −20°C  408.93  17.93  4.38  398.15–429.63  DCPP RT  20.07  1.74  8.67  18.70–22.03  DCPP 4°C  79.98  2.95  3.69  77.79–83.34  DCPP −20°C  283.65  7.92  2.79  275.08–290.70  TFMPP RT  73.32  4.34  5.92  68.89–77.57  TFMPP 4°C  420.73  4.87  1.16  416.98–426.23  TFMPP −20°C  343.50  13.00  3.78  333.01–358.04  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Our study showed that most piperazines in blood experienced the highest rate of degradation between Day 91 (~3 months) and 182 (~6 months); where the amount loss was significantly larger than those observed on Day 30 (~1 month) and Day 91. At −20°C, benzyl piperazines overall had a smaller degree of loss as opposed to phenyl piperazines (Figure 3). Under both room temperature and at 4°C on Day 182 (Figures 4 and 5), the degree of degradation for BZP and FBZP revealed a 75% and 90% loss respectively. However, MBZP was found to be extremely stable regardless of storage conditions in which more than 70% of its parent analyte was still detected after 12 months under all conditions. In general, all phenyl piperazines were slightly more stable at −20°C in comparison to room temperature and 4°C. Phenyl piperazines stored for more than 6 months showed analyte degradation of parent compounds after extended storage regardless of the storage conditions. On Day 182, MeOPP experienced the largest degradation in which only approximately 3 ng/mL remained in the blood sample when stored at −20°C; while no MeOPP was detected under room or refrigerated temperature after 6 months. Figure 3. View largeDownload slide Concentration versus time with samples stored at −20°C. Figure 3. View largeDownload slide Concentration versus time with samples stored at −20°C. Figure 4. View largeDownload slide Concentration versus time with samples stored at 4°C. Figure 4. View largeDownload slide Concentration versus time with samples stored at 4°C. Figure 5. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C). Figure 5. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C). Analyte interference in mixture Mixtures of synthetic piperazines were also evaluated and the following results refer to the mixture samples. Benzyl piperazines in mixture were relatively stable over time, but most phenyl piperazines showed moderate to severe degradation after only 1 month at −20°C. Within the first 6 months, phenyl piperazines when in the presence of other piperazine derivatives appeared to be more stable at room and refrigerated temperatures as opposed to −20°C. Data from Days 91 and 182 indicated that DCPP and TFMPP were consistently the most stable at room temperature (Figures 6 and 7). Although this contradicted the hypothesis that synthetic piperazines in biological specimens are more likely to degrade at a slower rate when stored in a freezer or refrigerator, this piece of information is very useful for laboratories that have limited freezer storage; as detectable levels of analytes in whole blood may exist after three to 6 months without freezing. Nonetheless, the findings obtained from Days 270 and 365 reflected that all phenyl piperazine derivatives were the most stable in freezer especially for mCPP which had more than 80% remaining on Day 270 (~9 months). Therefore, for cases involving a mixture of BZP with other piperazine drugs such as TFMPP, data suggested that both of these piperazines might still be detectable in 3 months or even a longer period of time regardless if a proper storage condition is provided and support Johnson and Botch-Jones’ previously published work (11). Figure 6. View largeDownload slide Concentration versus time with samples stored at −20°C in mixture. Figure 6. View largeDownload slide Concentration versus time with samples stored at −20°C in mixture. Figure 7. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C) in mixture. Figure 7. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C) in mixture. There was also a dramatic loss of MeOPP in mixture samples under all storage conditions on Day 91. Less than 20% of MeOPP was recovered in blood at room temperature, while the sample also lost 60–80% of its parent analyte at the other two conditions (Figure 8). Only 74 ng/mL of MeOPP remained in blood when kept at −20°C after 6 months. This particular analyte demonstrated a degree of degradation of more than 95% loss over a 182-day trial under freezing conditions. In addition, data revealed that all phenyl piperazines had dramatic declines after 12 months at room temperature under mix-mode in which MeOPP, pFPP, mCPP and TFMPP were not detectable. Figure 8. View largeDownload slide Concentration versus time with samples stored at 4°C in mixture. Figure 8. View largeDownload slide Concentration versus time with samples stored at 4°C in mixture. Isotopic influence of DCPP on TFMPP In the presence of two principle isotopes of chlorine, chlorine-35 and 37, DCPP has a stable isotope that shares the same mass-to-charge ratio with TFMPP (Figures 9 and 10). In this study, the ratio between the internal standard and the calibrators as well as the ratio between the internal standard and unknown were kept constant. Since the ratio of the DCPP isotope influence was maintained, the result of quantification (when TFMPP and DCPP were both present in the calibrators and in the mixture samples) was accurate. To avoid underestimating TFMPP’s concentration due to its absence of the “231” portion from DCPP, a separate calibration curve (with TFMPP only) was required to obtain a more accurate concentration in non-mixture samples. However due to the nature of this work and the lack of a separate calibration curve with TFMPP, final concentration levels of TFMPP under all storage conditions during the 30-day trial (Table III) were based off of an initial quantitated value of 502.83 ± 8.36 (mean ± standard deviation). Figure 9. View largeDownload slide Virtual Q1 MS scan of TFMPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level of the peak should be at approximately e^5 or higher. Figure 9. View largeDownload slide Virtual Q1 MS scan of TFMPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level of the peak should be at approximately e^5 or higher. Figure 10. View largeDownload slide Virtual Q1 MS scan of DCPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level peak “231” should be at approximately e^5 or higher; peak “232” should be at about e^3; peak “233” should be at approximately e^4 or higher. Figure 10. View largeDownload slide Virtual Q1 MS scan of DCPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level peak “231” should be at approximately e^5 or higher; peak “232” should be at about e^3; peak “233” should be at approximately e^4 or higher. Matrix interference MBZP in mixture samples, which were originally spiked at 1,000 ng/mL, had reached 1,420 ng/mL after storage at 4°C for 270 days. Since slight coagulation was observed in some blood samples after 9 months, matrix interference could not be excluded as one of the causes of this exceptionally high level of MBZP. Although limited information is currently available on synthetic piperazines’ metabolic pathways, the excess amount of MBZP quantified could also be a result of the presence of metabolites that share the same m/z and having the same structure with MBZP. Furthermore, the possibility of having MBZP as the metabolite derived from other piperazines in mixture could confound this result as well. Similarly, having metabolites sharing the same m/z ratio with parent compounds might also be able to explain the significant increase of BZP concentration from Day 182 to Day 270 under room and refrigerated temperature (Figures 4 and 5). Conclusions Data from this stability investigation revealed that BZP, MBZP and FBZP were generally more stable than phenyl piperazines over time under all storage conditions, in which MBZP was extremely stable and still had at least 70% (range 769–1,047 ng/mL) remaining after 12 months. It was determined that the stability of benzyl piperazines were less likely to be affected by analyte degradation in blood over time. Analyte interactions were observed but the exact stability pattern of phenyl piperazines in mixture samples could not be determined from this data set alone due to analyte concentration variations found on Days 91 and 270. Matrix interferences could not be ruled out due to the outlier of MBZP quantified on Day 270. Results emphasized that further work is needed to explore the most stable storage condition and necessary precautions for specimens containing more than one synthetic piperazines in order to ensure minimal degradation. As the number, categories and analogs of designer drugs continues to grow, a greater understanding of the stability of synthetic piperazines is crucial in terms of data analyses and interpretation for forensic casework. This study exhibited a solid method to examine synthetic piperazines degradation patterns in blood. Although samples stored in the freezer (−20°C) were subdivided so that only one freeze-thaw cycle occurred, this study could be expanded to evaluate the potential impact of additional freeze-thaw cycles may have on analyte concentration in order to minimize the effect of non-metabolic degradation. Moreover, sample preservation with sodium fluoride (NaF) should also be examined as a future experiment. At practical levels, the stability of piperazines when stored at −60°C to −80°C should also be investigated to support the hypothesis that lower temperature freezing conditions may be the most stable environment for piperazines in blood. As a result, it is highly recommended to store matrices containing synthetic piperazines at an appropriate storage condition rather than at room temperature. This might help to retain more of the compound(s) when samples are retained for extended periods of time. Acknowledgments Sincere thanks to the support from the Boston University School of Medicine’s Biomedical Forensic Sciences graduate program. The authors would like to acknowledge the constructive comments from Dr. Yun-Kwok Wing and Dr. Joav Prives. The authors would also like to thank SCIEX and UCT for their technical support and applications. References 1 Rosenbaum, C.D., Carreiro, S.P., Babu, K.M. ( 2012) Here today, gone tomorrow…and back again? A review of herbal marijuana alternatives (K2, spice), synthetic cathinones (bath salts), kratom, salvia divinorum, methoxetamine, and piperazines. Journal of Medical Toxicology , 8, 15– 32. Google Scholar CrossRef Search ADS PubMed  2 Nikolova, I., Danchev, N. ( 2008) Piperazine based substances of abuse: a new party pills on bulgarian drug market. Biotechnology & Biotechnological Equipment , 22, 652– 655. Google Scholar CrossRef Search ADS   3 Arbo, M.D., Bastos, M.L., Carmo, H.F. ( 2012) Piperazine compounds as drugs of abuse. Drug Alcohol Dependence , 122, 174– 185. Google Scholar CrossRef Search ADS PubMed  4 Elliott, S. ( 2011) Current awareness of piperazines: pharmacology and toxicology. Drug Testing and Analysis , 3, 430– 438. Google Scholar CrossRef Search ADS PubMed  5 National Forensic Laboratory Information System. ( 2012) Special report: Emerging 2C-phenethylamines, piperazines, and tryptamines in NFLIS, 2006−2011. https://www.deadiversion.usdoj.gov/nflis/spec_rpt_emerging_2012.pdf (accessed May 18, 2016). 6 Smith, J, Sutcliffe, O., Banks, C. ( 2015) An overview of recent developments in the analytical detection of new psychoactive substances (NPSs). Royal Society of Chemistry , 140, 4932– 4948. 7 Kerrigan, S., Savage, M., Cavazos, C., Bella, P. ( 2016) Thermal degradation of synthetic cathinones: implications for forensic toxicology. Journal of Analytical Toxicology , 40, 1– 11. Google Scholar CrossRef Search ADS PubMed  8 Monteiro, M., Bastos, M., Guedes de Pinho, P., Carvalho, M. ( 2013) Update on 1- benzylpiperazine (BZP) party pills. Archives of Toxicology , 87, 929– 947. Google Scholar CrossRef Search ADS PubMed  9 King, L.A., Kicman, A.T. ( 2011) A brief history of ‘new psychoactive substances’. Drug Testing and Analysis , 3, 401– 403. Google Scholar CrossRef Search ADS PubMed  10 Clauwaert, K.M., Van Bocxlaer, J.F., De Leenheer, A.P. ( 2001) Stability study of the designer drugs “MDA, MDMA and MDEA” in water, serum, whole blood, and urine under various storage temperatures. Forensic Science International , 124, 36– 42. Google Scholar CrossRef Search ADS PubMed  11 Johnson, R.D., Botch-Jones, S. ( 2013) The stability of four designer drugs: MDPV, mephedrone, BZP and TFMPP in three biological matrices under various storage conditions. Journal of Analytical Toxicology , 37, 51– 55. Google Scholar CrossRef Search ADS PubMed  12 Scientific Working Group for Forensic Toxicology (SWGTOX). ( 2013) Standard Practices for Method Validation in Forensic Toxicology. http://swgtox.org/documents/Validation3.pdf (accessed April 20, 2016). 13 LeBlanc, R. (ed). Method Development and Validation for the Quantification of Eight Synthetic Piperazines in Blood and Urine using Liquid Chromatography-tandem Mass Spectrometry (UFLC-ESI-MS/MS) . Boston University Libraries: Boston, MA, 2016; pp. 14– 15. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Analytical Toxicology Oxford University Press

Stability of Synthetic Piperazines in Human Whole Blood

Loading next page...
 
/lp/ou_press/stability-of-synthetic-piperazines-in-human-whole-blood-TwkjlvaKyu
Publisher
Oxford University Press
Copyright
© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
ISSN
0146-4760
eISSN
1945-2403
D.O.I.
10.1093/jat/bkx090
Publisher site
See Article on Publisher Site

Abstract

Abstract While circumventing legislative controls, synthetic piperazines are encountered as “legal” alternatives to ecstasy. Unforeseeable challenges may delay quantitative analysis of these compounds in biological fluids. Enzymatic reactions, matrix interferences and limited knowledge of analyte stability further complicate interpretation of calculated concentrations. The objective of this study was to investigate the stability of synthetic piperazines in human blood under various storage conditions over time. All samples were prepared by spiking certified reference standards (Cayman Chemical, MI, U.S.A.) of eight synthetic piperazine into certified drug-free human whole blood (UTAK Laboratories, Inc., CA, U.S.A.) independently at 1000 ng/mL as well as mixtures containing all tested piperazines in this study. Samples were stored at room temperature (~20°C), 4°C and −20°C for 1, 3, 6, 9 and 12 months in dark sealed containers. Solid phase extraction (SPE) was performed using mixed-mode copolymeric cartridges (Clean Screen®, UCT Inc., PA, U.S.A.). Analytes were assessed on their degrees of degradation using a Shimadzu Ultra-Fast Liquid Chromatograph with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometer (UFLC-ESI-MS/MS) in positive ionization mode. Of the two categories, benzyl piperazines were more stable than phenyl piperazines under all storage conditions, in which 1-(4-methylbenzyl)-piperazine (MBZP) had more than 70% (769–1,047 ng/mL) remaining after 12 months. 1-(4-methoxyphenyl)-piperazine (MeOPP) was not detected under room and refrigerated temperatures after 6 months and was the least stable. Matrix interferences and drug–drug interaction were observed. Storing samples at room temperature should be avoided due to detrimental impacts on stability of piperazine compounds. For backlog situations, case samples suspected to contain synthetic piperazines should be kept frozen or refrigerated even for time periods as short as 30 days for optimal result. Phenyl piperazines stored for more than 6 months showed analyte degradation and loss of parent compounds after extended storage regardless of storage conditions. Introduction Novel psychoactive substances (NPS) cover a large number of recreational drugs that mimic the effects of existing controlled substances and are often sold as “legal highs” to circumvent legal authorities (1). One example of NPS is piperazines derivatives, which belong to a class of amphetamine-like compounds and have made a resurgence as “legal Ecstasy” (2). The first documented abuse of a piperazine-derived drug involved 1-benzylpiperazine (BZP) and was reported in the United States (U.S.A) in 1996 (3). In 2000, piperazine-related abuse had begun to spread in New Zealand and Australia (4). Reported by the U.S. National Forensic Laboratory Information System (NFLIS), about 38,230 piperazine-containing substances were seized from 2006 to 2010 and this class of drugs was one of the top 25 identified drugs in 2011 (5). In addition, there were at least twelve substituted synthetic piperazine drugs on the clandestine drug market as of 2013 in which few are currently regulated according to the United Nations Office on Drugs and Crime (UNODC) (6). Piperazine-based compounds are completely synthetic and were primarily used as anthelmintic agents in veterinary and clinical practices (3). They can be divided into two classes: benzyl piperazines, which include BZP, 1-(4-fluorobenzyl)-piperazine (FBZP) and 1-(4-methylbenzyl)-piperazine (MBZP); and phenyl piperazines, which include 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) (3). Several studies in the 1970s suggested that BZP could be a potential antidepressant medication but was subsequently rejected due to its amphetamine-like effects coupled with a high potential for abuse (2). Since the majority of the NPS are relatively new to the scientific community, TFMPP, BZP and mCPP are the only synthetic piperazines that have been studied in detail in terms of their pharmacodynamic and pharmacokinetic properties (7, 8). Synthetic piperazines can stimulate the release and also inhibit the reuptake of dopamine, serotonin (5-HT) and noradrenaline, in which serotonergic and dopaminergic effects predominate in most cases (4). Although post-consumption impact of BZP include stimulant effects such as elevated blood pressure, increased heart rate, increased euphoria, dysphoria, sociability and drug-liking, the variable hallucinogenic effect was found to be about 10-fold less potent than 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine or amphetamine. Piperazines are popular in youth populations because of their long-lasting psychoactive duration of effect, which is typically four to 6 h. In order to enhance the spectrum of effects, BZP may be mixed with TFMPP and drug-induced behavior may be observed and last up to 8 h (2, 4, 9). Laboratory backlogs often prevent analysts from obtaining reliable quantitative data due to variable time intervals between obtaining a sample and analysis. Moreover, storage conditions of specific drugs or compounds in biological samples may affect the analytical results due to ongoing enzymatic metabolism and postmortem redistribution (10, 11). Johnson and Botch-Jones’ study on the investigation of stability of TFMPP in blood demonstrated significant degradation after a 14-day storage period at room temperature (11). Thus, chemical degradation or even bacterial activity may potentially become an issue and may make the interpretation of analytical results difficult (10, 11). Since the stability of synthetic piperazines in whole blood samples over extended storage time periods remains unknown, a total of eight synthetic piperazines including BZP, FBZP, MBZP, MeOPP, pFPP, mCPP, DCPP and TFMPP (Figure 1) were assessed on their degrees of degradation. Figure 1. View largeDownload slide Chemical structures of synthetic piperazines and deuterated internal standards used in this study (13). Figure 1. View largeDownload slide Chemical structures of synthetic piperazines and deuterated internal standards used in this study (13). Material and Methods Instrumentation Analysis was performed on a Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC) System (Kyoto, Japan) and a SCIEX 4000 QTRAP tandem Mass Spectrometer (MS/MS) (Framingham, MA, U.S.A). The separation was achieved using a Kinetex® F5 2.6 μm, 100 Å, 150 mm × 3.0 mm ID column purchased from Phenomenex, Inc. (Torrance, CA, U.S.A). All data were collected using Analyst™ (version 1.6.2) software and quantitation was conducted with MultiQuant™ 3.0 (version 3.05373.0) software (SCIEX). Two mobile phases were used: 2 mM ammonium formate buffer with 0.2% formic acid (mobile phase A) and LC-grade methanol with 0.1% formic acid (mobile phase B). The flow rate was set to 0.4 mL/min in which the starting condition was 5% mobile phase B (Table I). The injection volume and run time were 5 μL and 6.5 min, respectively. The sensitivity of the multiple reactions monitoring (MRM) was optimized as shown in Table II. Ion source gas 1, ion source gas 2, curtain gas and collision gas flow were at instrument settings of 50 psi, 80 psi, 30 psi and at medium, respectively. The ionspray voltage was maintained at 2,500 V while the turbo gas temperature was at 600°C. Table I. LC time program Time (min)  Module  Event  Parameter (%)  0.01  Pumps  Pump B Concentration  5  0.30  Pumps  Pump B Concentration  5  3.50  Pumps  Pump B Concentration  80  6.50  Pumps  Pump B Concentration  80  6.51  Pumps  Pump B Concentration  5  10.00  Controller  Stop    Time (min)  Module  Event  Parameter (%)  0.01  Pumps  Pump B Concentration  5  0.30  Pumps  Pump B Concentration  5  3.50  Pumps  Pump B Concentration  80  6.50  Pumps  Pump B Concentration  80  6.51  Pumps  Pump B Concentration  5  10.00  Controller  Stop    Table II. Multiple reaction monitoring (MRM) table Name of Analyte  Q1 Mass (Da)  Q3 Mass (Da)  Dwell Time (msec)  DP (V)  EP (V)  CE (V)  CXP (V)  BZP-d7 IS 1  184.3  98.2  50  70.0  10  30.0  15.0  BZP 1  177.1  91.1  50  66.0  10  32.0  14.0  BZP 2  177.1  65.1  50  66.0  10  63.0  9.0  FBZP 1  195.2  109.1  50  72.0  10  29.0  18.0  FBZP 2  195.2  83.2  50  72.0  10  65.0  12.0  MBZP 1  191.2  91.1  50  75.0  10  31.0  15.0  MBZP 2  191.2  65.2  50  75.0  10  67.0  9.0  MeOPP 1  193.2  150.2  50  70.0  10  28.0  24.0  MeOPP 2  193.2  119.3  50  70.0  10  34.0  19.0  pFPP 1  181.2  138.2  50  75.0  10  29.0  23.0  pFPP 2  181.2  75.2  50  75.0  10  77.0  11.0  mCPP 1  197.1  154.2  50  75.0  10  28.0  26.0  mCPP 2  197.1  118.2  50  75.0  10  48.0  19.0  mCPP-d8 IS 1  205.4  158.2  50  86.0  10  31.0  26.0  DCPP 1  233.1  190.2  50  85.0  10  30.0  32.0  DCPP 2  233.1  117.2  50  85.0  10  67.0  19.0  TFMPP 1  231.1  188.1  50  80.0  10  32.0  33.0  TFMPP 2  231.1  118.3  50  80.0  10  54.0  19.0  TFMPP-d4 IS 1  235.4  190.2  50  84.0  10  32.0  32.0  Name of Analyte  Q1 Mass (Da)  Q3 Mass (Da)  Dwell Time (msec)  DP (V)  EP (V)  CE (V)  CXP (V)  BZP-d7 IS 1  184.3  98.2  50  70.0  10  30.0  15.0  BZP 1  177.1  91.1  50  66.0  10  32.0  14.0  BZP 2  177.1  65.1  50  66.0  10  63.0  9.0  FBZP 1  195.2  109.1  50  72.0  10  29.0  18.0  FBZP 2  195.2  83.2  50  72.0  10  65.0  12.0  MBZP 1  191.2  91.1  50  75.0  10  31.0  15.0  MBZP 2  191.2  65.2  50  75.0  10  67.0  9.0  MeOPP 1  193.2  150.2  50  70.0  10  28.0  24.0  MeOPP 2  193.2  119.3  50  70.0  10  34.0  19.0  pFPP 1  181.2  138.2  50  75.0  10  29.0  23.0  pFPP 2  181.2  75.2  50  75.0  10  77.0  11.0  mCPP 1  197.1  154.2  50  75.0  10  28.0  26.0  mCPP 2  197.1  118.2  50  75.0  10  48.0  19.0  mCPP-d8 IS 1  205.4  158.2  50  86.0  10  31.0  26.0  DCPP 1  233.1  190.2  50  85.0  10  30.0  32.0  DCPP 2  233.1  117.2  50  85.0  10  67.0  19.0  TFMPP 1  231.1  188.1  50  80.0  10  32.0  33.0  TFMPP 2  231.1  118.3  50  80.0  10  54.0  19.0  TFMPP-d4 IS 1  235.4  190.2  50  84.0  10  32.0  32.0  DP, declustering potential; CE, collision energy; CXP, cell exit potential; EP, entrance potential. Reference standards and reagents The following reagents were purchased from Fisher Scientific (Waltham, MA, U.S.A): LC-grade methanol, LC-grade 2-propanol, Optima grade acetonitrile, formic acid, methylene chloride, ammonium formate, concentrated ammonium hydroxide, concentrated hydrochloric acid, anhydrous disodium phosphate and monohydrate sodium dihydrogen phosphate. BZP, FBZP, MBZP, MeOPP, pFPP, mCPP, TFMPP and DCPP were purchased through Cayman Chemical Company (Ann Arbor, MI, U.S.A.). All piperazines were received in the form of 10 mg powder except for BZP, which was received as a 1 mg/mL standard in methanol. Three deuterated internal standards including BZP-d7, mCPP-d8 and TFMPP-d4 were received as 100 μg/mL standards in methanol from Cerilliant Corporation (Round Rock, TX, U.S.A.). Fresh millipore water was obtained daily from the Synergy UV water filtration system from EMD Millipore/Merck (Darmstadt, Germany). Certified drug-free human whole blood was purchased from UTAK Laboratories, Inc. (Valencia, CA). Preparation of stock solution and calibrators A concentrated stock solution of each piperazine was prepared by dissolving 1 mg of each piperazine powder in 1 mL of methanol (except for BZP); they were further combined and serially diluted with 50:50 mixture of millipore water and methanol to give a 1 μg/mL stock solution (Stock 1). The second set of stock solutions (Stock 2) were prepared at a concentration of 100 ng/mL. An internal standard stock solution was prepared with 50:50 mixture of millipore water and methanol to a final concentration of 1 μg/mL by combining BZP-d7, mCPP-d8 and TFMPP-d4. Calibrators were prepared fresh daily by spiking 100 μL of whole blood with the appropriate volume of stock solutions to yield concentrations of 20, 50, 100, 200, 500, 1,000 and 2,000 ng/mL. Two quality control (QC) samples (30 and 1,500 ng/mL), a negative control and a double blank solution (without internal standards) were prepared for each run. A separate calibration curve was also required to accurately quantify the analyte of interest in order to avoid any possible underestimation of TFMPP. Preparation of storage samples on Day 0 Samples were stored in individual aliquots where eight different tubes of 10 mL whole blood each contained one of the synthetic piperazines were prepared independently at 1,000 ng/mL. A tube of 10 mL whole blood containing all eight synthetic piperazine standards (mixture sample) and a tube containing only 10 mL whole blood were prepared to serve as controls. Aliquots of each sample were then transferred into two separate amber glass vials (~3.3 mL each). They were then capped and placed at room temperature and 4°C for the designated period of time. The remaining ~3.4 mL of mixture solution in the tube was also stored and wrapped in opaque tape to prevent degradation by light, and placed in a freezer at −20°C. Temperatures for the freezer, refrigerator and the room where samples were stored were monitored and recorded daily. Internal standards were added on the day of analysis. Sample preparation All tested piperazines were extracted from whole blood by solid phase extraction (SPE). Since piperazines are basic in nature, Drugs of Abuse (DAU) mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.) were chosen for solid phase extraction. The mixed-mode sorbent exhibiting both hydrophobic and cation exchange characteristics serves to retain ionized basic compounds. First, 1 mL of phosphate buffer (100 mM, pH 6) was added to all samples prior to loading. Then, SPE cartridges were conditioned with 1 mL of methanol followed by 1 mL of phosphate buffer (100 mM, pH 6). Subsequently, samples (1 mL of the pre-added phosphate buffer and 100 μL of blood containing piperazines) were loaded into each designated pre-conditioned column and were allowed to drip with gravity flow. A series of wash steps were performed on each column in the order of 1 mL of millipore water, 1 mL of 0.1 N hydrochloric acid and 1 mL of LC-grade methanol. A flow of compressed nitrogen gas was applied for 5 min at 40 psi. Samples were then eluted in 2 mL of base elution solvent which was made fresh for each experiment. The base elution solvent was prepared in the order of adding 20% 2-propanol, 3% of concentrated ammonium hydroxide and 77% of methylene chloride. Eluents were evaporated to dryness at 65°C on a heating block. The residues were reconstituted in 250 μL of a 50:50 mixture of methanol and 2 mM ammonium formate buffer with 0.2% formic acid, and was directly injected into the chromatographic system. Stability analysis Stability of synthetic piperazines was examined by monitoring the amount loss of analytes over time at each storage condition. Quantification of synthetic piperazines in all samples was performed using LC-MS/MS and were compared to the calibration curves generated on both Day 0 and at the set analysis days for the duration of the study. Three replicates of each sample were analyzed and the average was obtained to serve as the initial concentration on Day 0 and at the end of the storage period. Results and Discussion Chromatography and specificity The UFLC method described above achieved excellent resolution and peak shape with full elution in 6.5 min. All benzyl piperazines eluted first at the following retention times: BZP at 3.82, FBZP at 4.48 and MBZP at 4.69 min (Figure 2). The phenyl piperazines then eluted at the followings: MeOPP at 5.09, pFPP at 5.33, mCPP at 5.80, DCPP at 6.11 and TFMPP at 6.14 min. Retention times were 3.71, 5.80 and 6.15 min for BZP-d7, mCPP-d8 and TFMPP-d4, respectively. Figure 2. View largeDownload slide Representative chromatogram of BZP, FBZP, MBZP, MeOPP, mCPP, TFMPP, pFPP, DCPP, BZP-d7, mCPP-d8 and TFMPP-d4 at 500 ng/mL extracted from whole blood on Day 0. Figure 2. View largeDownload slide Representative chromatogram of BZP, FBZP, MBZP, MeOPP, mCPP, TFMPP, pFPP, DCPP, BZP-d7, mCPP-d8 and TFMPP-d4 at 500 ng/mL extracted from whole blood on Day 0. Calibration and linearity The seven-point calibration curve was generated for each run by plotting the peak area ratio (y) of the target analyte to the internal standard versus analyte concentration (x). The coefficient of determination (R2 values) was calculated by MultiQuant™ software with a weighting factor of 1/x. Among the three deuterated internal standards, BZP-d7 was used to quantify BZP, FBZP and MBZP; mCPP-d8 was used to quantify MeOPP, pFPP, mCPP and DCPP; TFMPP-d4 was used to quantify TFMPP only. Accuracy and precision Accuracy of calibrators and QC samples were determined to be within the allowed ± 20% range by comparing the calculated concentration using calibration curves to the known concentration. All R2 values were above the minimum accepted value of 0.98 according to the Scientific Working Group for Forensic Toxicologists (SWGTOX) guidelines for quantitative methods (12). Precision of the assay was evaluated by calculating the standard deviation and the percent coefficient of variation (% CV) in three replicates. Stability All data, shown in Tables III through VII, was assessed based on storage conditions (~20°C, 4°C and −20°C) and time ranges (1, 3, 6, 9 and 12 months). The three benzyl piperazines that were kept frozen demonstrated minimal variation within the first 91 days (~3 months) and the overall loss was less than 15%. In contrast, phenyl piperazines that were kept frozen showed moderate degradations, especially for DCPP and TFMPP, for which only 42% and 52% of the parent compound remained on Day 91, respectively. As expected, the degree of loss for most synthetic piperazines stored at room temperature was larger than those that were kept at 4°C or −20°C. Most data showed a smaller degree of degradation if proper storage conditions were maintained and that is when samples were kept in either a refrigerator or a freezer. For crime laboratories that are facing backlog situations, it is recommended that specimens containing synthetic piperazines should be kept frozen or refrigerated for 30 days or less. Storing samples at room temperature should be avoided because of detrimental impacts on the stability of piperazine compounds. This information is also valuable when analyzing data for postmortem specimens that are collected from a decomposed body or a body that is found in an exceptionally warm environment. Table III. Stability data on Day 30 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  1105.43  62.60  5.66  1054.4–1175.28  BZP 4°C  949.15  53.16  5.60  887.76–980.12  BZP −20°C  1166.81  51.16  4.38  1107.74–1196.79  FBZP RT  787.14  10.27  1.30  776.82–797.36  FBZP 4°C  902.95  124.78  13.82  795.24–1039.68  FBZP −20°C  1295.06  38.63  2.98  1263.94–1338.29  MBZP RT  503.33  12.55  2.49  495.44–517.80  MBZP 4°C  606.33  26.30  4.34  582.41–634.49  MBZP −20°C  1144.13  60.45  5.28  1082.81–1203.68  MeOPP RT  1238.79  50.78  4.10  1189.35–1290.82  MeOPP 4°C  767.46  10.33  1.35  759.41–779.11  MeOPP −20°C  961.05  3.48  0.36  957.78–964.72  pFPP RT  1207.86  14.97  1.24  1192.02–1221.77  pFPP 4°C  917.57  37.17  4.05  878.09–951.89  pFPP −20°C  1073.07  34.24  3.19  1053.01–1112.60  mCPP RT  1010.19  37.80  3.74  966.73–1035.41  mCPP 4°C  973.45  13.62  1.40  964.31–989.11  mCPP −20°C  978.83  10.23  1.04  971.99–990.58  DCPP RT  1060.75  41.01  3.87  1018.16–1099.97  DCPP 4°C  705.49  59.09  8.38  641.63–758.22  DCPP −20°C  876.19  10.69  1.22  866.81–887.83  TFMPP RT  499.42  22.57  4.52  481.18–524.66  TFMPP 4°C  385.70  8.71  2.26  375.65–390.98  TFMPP −20°C  686.05  54.35  7.92  641.83–746.74  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  1105.43  62.60  5.66  1054.4–1175.28  BZP 4°C  949.15  53.16  5.60  887.76–980.12  BZP −20°C  1166.81  51.16  4.38  1107.74–1196.79  FBZP RT  787.14  10.27  1.30  776.82–797.36  FBZP 4°C  902.95  124.78  13.82  795.24–1039.68  FBZP −20°C  1295.06  38.63  2.98  1263.94–1338.29  MBZP RT  503.33  12.55  2.49  495.44–517.80  MBZP 4°C  606.33  26.30  4.34  582.41–634.49  MBZP −20°C  1144.13  60.45  5.28  1082.81–1203.68  MeOPP RT  1238.79  50.78  4.10  1189.35–1290.82  MeOPP 4°C  767.46  10.33  1.35  759.41–779.11  MeOPP −20°C  961.05  3.48  0.36  957.78–964.72  pFPP RT  1207.86  14.97  1.24  1192.02–1221.77  pFPP 4°C  917.57  37.17  4.05  878.09–951.89  pFPP −20°C  1073.07  34.24  3.19  1053.01–1112.60  mCPP RT  1010.19  37.80  3.74  966.73–1035.41  mCPP 4°C  973.45  13.62  1.40  964.31–989.11  mCPP −20°C  978.83  10.23  1.04  971.99–990.58  DCPP RT  1060.75  41.01  3.87  1018.16–1099.97  DCPP 4°C  705.49  59.09  8.38  641.63–758.22  DCPP −20°C  876.19  10.69  1.22  866.81–887.83  TFMPP RT  499.42  22.57  4.52  481.18–524.66  TFMPP 4°C  385.70  8.71  2.26  375.65–390.98  TFMPP −20°C  686.05  54.35  7.92  641.83–746.74  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL; TFMPP was at 502.83 ± 8.36 (mean ± standard deviation) on Day 0 as supposed to 1,000 ng/mL due to isotope interference. Table IV. Stability data on Day 91 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  934.90  110.27  11.79  807.60–1000.85  BZP 4°C  1059.16  35.21  3.32  1025.17–1095.48  BZP −20°C  1068.37  19.53  1.83  1046.83–1084.94  FBZP RT  851.75  21.09  2.48  827.62–866.66  FBZP 4°C  1017.92  13.31  1.31  1003.01–1028.62  FBZP −20°C  908.15  5.87  0.65  901.63–913.01  MBZP RT  1391.77  12.28  0.88  1380.41–1404.81  MBZP 4°C  1130.29  36.20  3.20  1105.81–1171.88  MBZP −20°C  1191.49  65.11  5.46  1118.61–1244.02  MeOPP RT  542.09  19.94  3.68  520.16–559.15  MeOPP 4°C  733.51  31.26  4.26  706.73–767.86  MeOPP −20°C  645.49  18.74  2.90  633.43–667.08  pFPP RT  513.79  10.09  1.96  502.27–521.03  pFPP 4°C  744.71  22.15  2.97  719.13–758.15  pFPP −20°C  910.21  41.96  4.61  864.18–946.32  mCPP RT  788.11  2.90  0.37  785.13–790.93  mCPP 4°C  755.23  21.67  2.87  730.42–770.42  mCPP −20°C  738.95  14.50  1.96  724.18–753.18  DCPP RT  497.08  13.49  2.71  485.29–511.79  DCPP 4°C  648.46  16.45  2.54  629.75–660.64  DCPP −20°C  483.55  8.96  1.85  477.78–493.87  TFMPP RT  686.02  12.80  1.87  675.00–700.06  TFMPP 4°C  668.36  13.56  2.03  657.23–683.47  TFMPP −20°C  580.24  13.60  2.34  565.76–592.75  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  934.90  110.27  11.79  807.60–1000.85  BZP 4°C  1059.16  35.21  3.32  1025.17–1095.48  BZP −20°C  1068.37  19.53  1.83  1046.83–1084.94  FBZP RT  851.75  21.09  2.48  827.62–866.66  FBZP 4°C  1017.92  13.31  1.31  1003.01–1028.62  FBZP −20°C  908.15  5.87  0.65  901.63–913.01  MBZP RT  1391.77  12.28  0.88  1380.41–1404.81  MBZP 4°C  1130.29  36.20  3.20  1105.81–1171.88  MBZP −20°C  1191.49  65.11  5.46  1118.61–1244.02  MeOPP RT  542.09  19.94  3.68  520.16–559.15  MeOPP 4°C  733.51  31.26  4.26  706.73–767.86  MeOPP −20°C  645.49  18.74  2.90  633.43–667.08  pFPP RT  513.79  10.09  1.96  502.27–521.03  pFPP 4°C  744.71  22.15  2.97  719.13–758.15  pFPP −20°C  910.21  41.96  4.61  864.18–946.32  mCPP RT  788.11  2.90  0.37  785.13–790.93  mCPP 4°C  755.23  21.67  2.87  730.42–770.42  mCPP −20°C  738.95  14.50  1.96  724.18–753.18  DCPP RT  497.08  13.49  2.71  485.29–511.79  DCPP 4°C  648.46  16.45  2.54  629.75–660.64  DCPP −20°C  483.55  8.96  1.85  477.78–493.87  TFMPP RT  686.02  12.80  1.87  675.00–700.06  TFMPP 4°C  668.36  13.56  2.03  657.23–683.47  TFMPP −20°C  580.24  13.60  2.34  565.76–592.75  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Table V. Stability data on Day 182 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  253.42  0.94  0.37  252.53–254.40  BZP 4°C  240.88  3.01  1.25  237.45–243.07  BZP −20°C  443.01  4.11  0.93  439.13–447.33  FBZP RT  67.76  1.20  1.77  66.63–69.02  FBZP 4°C  81.39  1.49  1.83  80.26–83.08  FBZP −20°C  317.75  7.57  2.38  311.63–326.21  MBZP RT  1144.30  13.53  1.18  1128.98–1154.64  MBZP 4°C  959.84  32.84  3.42  922.76–985.26  MBZP −20°C  1189.62  77.19  6.49  1100.91–1241.49  MeOPP RT  0.00  0.00  0.00  0.00  MeOPP 4°C  0.00  0.00  0.00  0.00  MeOPP −20°C  3.42  5.93  173.21  0.00–10.27  pFPP RT  76.25  7.13  9.36  70.12–84.09  pFPP 4°C  17.33  0.97  5.62  16.61–18.44  pFPP −20°C  105.61  10.33  9.78  98.69–117.49  mCPP RT  9.84  0.12  1.25  9.75–9.98  mCPP 4°C  15.59  3.15  20.20  13.24–19.17  mCPP −20°C  56.39  1.16  2.06  55.15–57.44  DCPP RT  13.19  2.92  22.13  9.84–15.15  DCPP 4°C  16.64  1.54  9.27  15.45–18.38  DCPP −20°C  78.14  17.14  21.94  65.71–97.70  TFMPP RT  13.33  0.91  6.82  12.56–14.33  TFMPP 4°C  15.72  1.40  8.88  14.77–17.32  TFMPP −20°C  63.57  4.22  6.63  58.72–66.39  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  253.42  0.94  0.37  252.53–254.40  BZP 4°C  240.88  3.01  1.25  237.45–243.07  BZP −20°C  443.01  4.11  0.93  439.13–447.33  FBZP RT  67.76  1.20  1.77  66.63–69.02  FBZP 4°C  81.39  1.49  1.83  80.26–83.08  FBZP −20°C  317.75  7.57  2.38  311.63–326.21  MBZP RT  1144.30  13.53  1.18  1128.98–1154.64  MBZP 4°C  959.84  32.84  3.42  922.76–985.26  MBZP −20°C  1189.62  77.19  6.49  1100.91–1241.49  MeOPP RT  0.00  0.00  0.00  0.00  MeOPP 4°C  0.00  0.00  0.00  0.00  MeOPP −20°C  3.42  5.93  173.21  0.00–10.27  pFPP RT  76.25  7.13  9.36  70.12–84.09  pFPP 4°C  17.33  0.97  5.62  16.61–18.44  pFPP −20°C  105.61  10.33  9.78  98.69–117.49  mCPP RT  9.84  0.12  1.25  9.75–9.98  mCPP 4°C  15.59  3.15  20.20  13.24–19.17  mCPP −20°C  56.39  1.16  2.06  55.15–57.44  DCPP RT  13.19  2.92  22.13  9.84–15.15  DCPP 4°C  16.64  1.54  9.27  15.45–18.38  DCPP −20°C  78.14  17.14  21.94  65.71–97.70  TFMPP RT  13.33  0.91  6.82  12.56–14.33  TFMPP 4°C  15.72  1.40  8.88  14.77–17.32  TFMPP −20°C  63.57  4.22  6.63  58.72–66.39  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL; TFMPP was at 548.59 ± 4.22 (mean ± standard deviation) on Day 0. Table VI. Stability data on Day 270 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  600.41  10.58  1.76  590.36–611.45  BZP 4°C  671.34  40.60  6.05  630.82–712.01  BZP −20°C  576.30  31.97  5.55  545.85–609.60  FBZP RT  63.75  1.03  1.61  63.08–64.93  FBZP 4°C  81.48  5.87  7.20  75.38–87.09  FBZP −20°C  331.61  9.39  2.83  320.98–338.80  MBZP RT  1223.00  11.17  0.91  1210.14–1230.32  MBZP 4°C  1530.80  50.88  3.32  1486.61–1586.42  MBZP −20°C  1262.52  16.33  1.29  1244.10–1275.24  MeOPP RT  63.06  17.85  28.31  51.15–83.58  MeOPP 4°C  143.96  40.22  27.94  117.15–190.21  MeOPP −20°C  179.06  40.41  22.57  132.70–206.77  pFPP RT  167.07  46.91  28.08  139.24–221.23  pFPP 4°C  236.47  62.92  26.61  199.48–309.12  pFPP −20°C  172.96  42.75  24.72  124.09–203.45  mCPP RT  26.12  0.45  1.73  25.60–26.43  mCPP 4°C  69.02  3.07  4.45  66.47–72.43  mCPP −20°C  151.02  5.46  3.61  147.58–157.31  DCPP RT  9.10  1.50  16.49  8.23–10.83  DCPP 4°C  26.52  9.26  34.92  19.71–37.07  DCPP −20°C  106.07  22.58  21.28  80.10–120.98  TFMPP RT  0.71  1.22  173.21  0.00–2.12  TFMPP 4°C  22.46  30.63  136.38  4.63–57.83  TFMPP −20°C  88.80  34.47  38.81  60.68–127.25  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  600.41  10.58  1.76  590.36–611.45  BZP 4°C  671.34  40.60  6.05  630.82–712.01  BZP −20°C  576.30  31.97  5.55  545.85–609.60  FBZP RT  63.75  1.03  1.61  63.08–64.93  FBZP 4°C  81.48  5.87  7.20  75.38–87.09  FBZP −20°C  331.61  9.39  2.83  320.98–338.80  MBZP RT  1223.00  11.17  0.91  1210.14–1230.32  MBZP 4°C  1530.80  50.88  3.32  1486.61–1586.42  MBZP −20°C  1262.52  16.33  1.29  1244.10–1275.24  MeOPP RT  63.06  17.85  28.31  51.15–83.58  MeOPP 4°C  143.96  40.22  27.94  117.15–190.21  MeOPP −20°C  179.06  40.41  22.57  132.70–206.77  pFPP RT  167.07  46.91  28.08  139.24–221.23  pFPP 4°C  236.47  62.92  26.61  199.48–309.12  pFPP −20°C  172.96  42.75  24.72  124.09–203.45  mCPP RT  26.12  0.45  1.73  25.60–26.43  mCPP 4°C  69.02  3.07  4.45  66.47–72.43  mCPP −20°C  151.02  5.46  3.61  147.58–157.31  DCPP RT  9.10  1.50  16.49  8.23–10.83  DCPP 4°C  26.52  9.26  34.92  19.71–37.07  DCPP −20°C  106.07  22.58  21.28  80.10–120.98  TFMPP RT  0.71  1.22  173.21  0.00–2.12  TFMPP 4°C  22.46  30.63  136.38  4.63–57.83  TFMPP −20°C  88.80  34.47  38.81  60.68–127.25  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Table VII. Stability data on Day 365 at different storage conditions Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  284.83  13.00  4.56  269.94–293.85  BZP 4°C  585.98  6.80  1.16  581.91–593.83  BZP −20°C  662.86  22.46  3.39  640.48–685.41  FBZP RT  63.72  3.62  5.68  60.91–67.80  FBZP 4°C  166.41  9.44  5.67  155.52–172.03  FBZP −20°C  442.59  5.57  1.26  436.68–447.73  MBZP RT  874.87  31.06  3.55  843.32–905.41  MBZP 4°C  713.65  49.57  6.95  673.20–768.96  MBZP −20°C  966.52  69.82  7.22  917.80–1046.51  MeOPP RT  398.36  4.84  1.21  394.67–403.83  MeOPP 4°C  474.92  5.07  1.07  469.10–478.31  MeOPP −20°C  366.65  5.58  1.52  360.49–371.34  pFPP RT  314.91  3.86  1.22  311.80–319.22  pFPP 4°C  711.98  1.97  0.28  710.04–713.99  pFPP −20°C  590.29  21.05  3.57  567.86–609.61  mCPP RT  75.59  2.19  2.90  73.14–77.36  mCPP 4°C  621.57  4.15  0.67  616.79–624.31  mCPP −20°C  408.93  17.93  4.38  398.15–429.63  DCPP RT  20.07  1.74  8.67  18.70–22.03  DCPP 4°C  79.98  2.95  3.69  77.79–83.34  DCPP −20°C  283.65  7.92  2.79  275.08–290.70  TFMPP RT  73.32  4.34  5.92  68.89–77.57  TFMPP 4°C  420.73  4.87  1.16  416.98–426.23  TFMPP −20°C  343.50  13.00  3.78  333.01–358.04  Analyte/storage condition  Average  Standard deviation  % CV  Range (ng/mL)  BZP RT  284.83  13.00  4.56  269.94–293.85  BZP 4°C  585.98  6.80  1.16  581.91–593.83  BZP −20°C  662.86  22.46  3.39  640.48–685.41  FBZP RT  63.72  3.62  5.68  60.91–67.80  FBZP 4°C  166.41  9.44  5.67  155.52–172.03  FBZP −20°C  442.59  5.57  1.26  436.68–447.73  MBZP RT  874.87  31.06  3.55  843.32–905.41  MBZP 4°C  713.65  49.57  6.95  673.20–768.96  MBZP −20°C  966.52  69.82  7.22  917.80–1046.51  MeOPP RT  398.36  4.84  1.21  394.67–403.83  MeOPP 4°C  474.92  5.07  1.07  469.10–478.31  MeOPP −20°C  366.65  5.58  1.52  360.49–371.34  pFPP RT  314.91  3.86  1.22  311.80–319.22  pFPP 4°C  711.98  1.97  0.28  710.04–713.99  pFPP −20°C  590.29  21.05  3.57  567.86–609.61  mCPP RT  75.59  2.19  2.90  73.14–77.36  mCPP 4°C  621.57  4.15  0.67  616.79–624.31  mCPP −20°C  408.93  17.93  4.38  398.15–429.63  DCPP RT  20.07  1.74  8.67  18.70–22.03  DCPP 4°C  79.98  2.95  3.69  77.79–83.34  DCPP −20°C  283.65  7.92  2.79  275.08–290.70  TFMPP RT  73.32  4.34  5.92  68.89–77.57  TFMPP 4°C  420.73  4.87  1.16  416.98–426.23  TFMPP −20°C  343.50  13.00  3.78  333.01–358.04  RT = room temperature (~20°C); n = 3; average concentrations are shown in ng/mL. Our study showed that most piperazines in blood experienced the highest rate of degradation between Day 91 (~3 months) and 182 (~6 months); where the amount loss was significantly larger than those observed on Day 30 (~1 month) and Day 91. At −20°C, benzyl piperazines overall had a smaller degree of loss as opposed to phenyl piperazines (Figure 3). Under both room temperature and at 4°C on Day 182 (Figures 4 and 5), the degree of degradation for BZP and FBZP revealed a 75% and 90% loss respectively. However, MBZP was found to be extremely stable regardless of storage conditions in which more than 70% of its parent analyte was still detected after 12 months under all conditions. In general, all phenyl piperazines were slightly more stable at −20°C in comparison to room temperature and 4°C. Phenyl piperazines stored for more than 6 months showed analyte degradation of parent compounds after extended storage regardless of the storage conditions. On Day 182, MeOPP experienced the largest degradation in which only approximately 3 ng/mL remained in the blood sample when stored at −20°C; while no MeOPP was detected under room or refrigerated temperature after 6 months. Figure 3. View largeDownload slide Concentration versus time with samples stored at −20°C. Figure 3. View largeDownload slide Concentration versus time with samples stored at −20°C. Figure 4. View largeDownload slide Concentration versus time with samples stored at 4°C. Figure 4. View largeDownload slide Concentration versus time with samples stored at 4°C. Figure 5. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C). Figure 5. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C). Analyte interference in mixture Mixtures of synthetic piperazines were also evaluated and the following results refer to the mixture samples. Benzyl piperazines in mixture were relatively stable over time, but most phenyl piperazines showed moderate to severe degradation after only 1 month at −20°C. Within the first 6 months, phenyl piperazines when in the presence of other piperazine derivatives appeared to be more stable at room and refrigerated temperatures as opposed to −20°C. Data from Days 91 and 182 indicated that DCPP and TFMPP were consistently the most stable at room temperature (Figures 6 and 7). Although this contradicted the hypothesis that synthetic piperazines in biological specimens are more likely to degrade at a slower rate when stored in a freezer or refrigerator, this piece of information is very useful for laboratories that have limited freezer storage; as detectable levels of analytes in whole blood may exist after three to 6 months without freezing. Nonetheless, the findings obtained from Days 270 and 365 reflected that all phenyl piperazine derivatives were the most stable in freezer especially for mCPP which had more than 80% remaining on Day 270 (~9 months). Therefore, for cases involving a mixture of BZP with other piperazine drugs such as TFMPP, data suggested that both of these piperazines might still be detectable in 3 months or even a longer period of time regardless if a proper storage condition is provided and support Johnson and Botch-Jones’ previously published work (11). Figure 6. View largeDownload slide Concentration versus time with samples stored at −20°C in mixture. Figure 6. View largeDownload slide Concentration versus time with samples stored at −20°C in mixture. Figure 7. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C) in mixture. Figure 7. View largeDownload slide Concentration versus time with samples stored at room temperature (~20°C) in mixture. There was also a dramatic loss of MeOPP in mixture samples under all storage conditions on Day 91. Less than 20% of MeOPP was recovered in blood at room temperature, while the sample also lost 60–80% of its parent analyte at the other two conditions (Figure 8). Only 74 ng/mL of MeOPP remained in blood when kept at −20°C after 6 months. This particular analyte demonstrated a degree of degradation of more than 95% loss over a 182-day trial under freezing conditions. In addition, data revealed that all phenyl piperazines had dramatic declines after 12 months at room temperature under mix-mode in which MeOPP, pFPP, mCPP and TFMPP were not detectable. Figure 8. View largeDownload slide Concentration versus time with samples stored at 4°C in mixture. Figure 8. View largeDownload slide Concentration versus time with samples stored at 4°C in mixture. Isotopic influence of DCPP on TFMPP In the presence of two principle isotopes of chlorine, chlorine-35 and 37, DCPP has a stable isotope that shares the same mass-to-charge ratio with TFMPP (Figures 9 and 10). In this study, the ratio between the internal standard and the calibrators as well as the ratio between the internal standard and unknown were kept constant. Since the ratio of the DCPP isotope influence was maintained, the result of quantification (when TFMPP and DCPP were both present in the calibrators and in the mixture samples) was accurate. To avoid underestimating TFMPP’s concentration due to its absence of the “231” portion from DCPP, a separate calibration curve (with TFMPP only) was required to obtain a more accurate concentration in non-mixture samples. However due to the nature of this work and the lack of a separate calibration curve with TFMPP, final concentration levels of TFMPP under all storage conditions during the 30-day trial (Table III) were based off of an initial quantitated value of 502.83 ± 8.36 (mean ± standard deviation). Figure 9. View largeDownload slide Virtual Q1 MS scan of TFMPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level of the peak should be at approximately e^5 or higher. Figure 9. View largeDownload slide Virtual Q1 MS scan of TFMPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level of the peak should be at approximately e^5 or higher. Figure 10. View largeDownload slide Virtual Q1 MS scan of DCPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level peak “231” should be at approximately e^5 or higher; peak “232” should be at about e^3; peak “233” should be at approximately e^4 or higher. Figure 10. View largeDownload slide Virtual Q1 MS scan of DCPP. This scan shows mass-to-charge ratios in Da on the x-axis and intensity in counts per second on the y-axis. Intensity level peak “231” should be at approximately e^5 or higher; peak “232” should be at about e^3; peak “233” should be at approximately e^4 or higher. Matrix interference MBZP in mixture samples, which were originally spiked at 1,000 ng/mL, had reached 1,420 ng/mL after storage at 4°C for 270 days. Since slight coagulation was observed in some blood samples after 9 months, matrix interference could not be excluded as one of the causes of this exceptionally high level of MBZP. Although limited information is currently available on synthetic piperazines’ metabolic pathways, the excess amount of MBZP quantified could also be a result of the presence of metabolites that share the same m/z and having the same structure with MBZP. Furthermore, the possibility of having MBZP as the metabolite derived from other piperazines in mixture could confound this result as well. Similarly, having metabolites sharing the same m/z ratio with parent compounds might also be able to explain the significant increase of BZP concentration from Day 182 to Day 270 under room and refrigerated temperature (Figures 4 and 5). Conclusions Data from this stability investigation revealed that BZP, MBZP and FBZP were generally more stable than phenyl piperazines over time under all storage conditions, in which MBZP was extremely stable and still had at least 70% (range 769–1,047 ng/mL) remaining after 12 months. It was determined that the stability of benzyl piperazines were less likely to be affected by analyte degradation in blood over time. Analyte interactions were observed but the exact stability pattern of phenyl piperazines in mixture samples could not be determined from this data set alone due to analyte concentration variations found on Days 91 and 270. Matrix interferences could not be ruled out due to the outlier of MBZP quantified on Day 270. Results emphasized that further work is needed to explore the most stable storage condition and necessary precautions for specimens containing more than one synthetic piperazines in order to ensure minimal degradation. As the number, categories and analogs of designer drugs continues to grow, a greater understanding of the stability of synthetic piperazines is crucial in terms of data analyses and interpretation for forensic casework. This study exhibited a solid method to examine synthetic piperazines degradation patterns in blood. Although samples stored in the freezer (−20°C) were subdivided so that only one freeze-thaw cycle occurred, this study could be expanded to evaluate the potential impact of additional freeze-thaw cycles may have on analyte concentration in order to minimize the effect of non-metabolic degradation. Moreover, sample preservation with sodium fluoride (NaF) should also be examined as a future experiment. At practical levels, the stability of piperazines when stored at −60°C to −80°C should also be investigated to support the hypothesis that lower temperature freezing conditions may be the most stable environment for piperazines in blood. As a result, it is highly recommended to store matrices containing synthetic piperazines at an appropriate storage condition rather than at room temperature. This might help to retain more of the compound(s) when samples are retained for extended periods of time. Acknowledgments Sincere thanks to the support from the Boston University School of Medicine’s Biomedical Forensic Sciences graduate program. The authors would like to acknowledge the constructive comments from Dr. Yun-Kwok Wing and Dr. Joav Prives. The authors would also like to thank SCIEX and UCT for their technical support and applications. References 1 Rosenbaum, C.D., Carreiro, S.P., Babu, K.M. ( 2012) Here today, gone tomorrow…and back again? A review of herbal marijuana alternatives (K2, spice), synthetic cathinones (bath salts), kratom, salvia divinorum, methoxetamine, and piperazines. Journal of Medical Toxicology , 8, 15– 32. Google Scholar CrossRef Search ADS PubMed  2 Nikolova, I., Danchev, N. ( 2008) Piperazine based substances of abuse: a new party pills on bulgarian drug market. Biotechnology & Biotechnological Equipment , 22, 652– 655. Google Scholar CrossRef Search ADS   3 Arbo, M.D., Bastos, M.L., Carmo, H.F. ( 2012) Piperazine compounds as drugs of abuse. Drug Alcohol Dependence , 122, 174– 185. Google Scholar CrossRef Search ADS PubMed  4 Elliott, S. ( 2011) Current awareness of piperazines: pharmacology and toxicology. Drug Testing and Analysis , 3, 430– 438. Google Scholar CrossRef Search ADS PubMed  5 National Forensic Laboratory Information System. ( 2012) Special report: Emerging 2C-phenethylamines, piperazines, and tryptamines in NFLIS, 2006−2011. https://www.deadiversion.usdoj.gov/nflis/spec_rpt_emerging_2012.pdf (accessed May 18, 2016). 6 Smith, J, Sutcliffe, O., Banks, C. ( 2015) An overview of recent developments in the analytical detection of new psychoactive substances (NPSs). Royal Society of Chemistry , 140, 4932– 4948. 7 Kerrigan, S., Savage, M., Cavazos, C., Bella, P. ( 2016) Thermal degradation of synthetic cathinones: implications for forensic toxicology. Journal of Analytical Toxicology , 40, 1– 11. Google Scholar CrossRef Search ADS PubMed  8 Monteiro, M., Bastos, M., Guedes de Pinho, P., Carvalho, M. ( 2013) Update on 1- benzylpiperazine (BZP) party pills. Archives of Toxicology , 87, 929– 947. Google Scholar CrossRef Search ADS PubMed  9 King, L.A., Kicman, A.T. ( 2011) A brief history of ‘new psychoactive substances’. Drug Testing and Analysis , 3, 401– 403. Google Scholar CrossRef Search ADS PubMed  10 Clauwaert, K.M., Van Bocxlaer, J.F., De Leenheer, A.P. ( 2001) Stability study of the designer drugs “MDA, MDMA and MDEA” in water, serum, whole blood, and urine under various storage temperatures. Forensic Science International , 124, 36– 42. Google Scholar CrossRef Search ADS PubMed  11 Johnson, R.D., Botch-Jones, S. ( 2013) The stability of four designer drugs: MDPV, mephedrone, BZP and TFMPP in three biological matrices under various storage conditions. Journal of Analytical Toxicology , 37, 51– 55. Google Scholar CrossRef Search ADS PubMed  12 Scientific Working Group for Forensic Toxicology (SWGTOX). ( 2013) Standard Practices for Method Validation in Forensic Toxicology. http://swgtox.org/documents/Validation3.pdf (accessed April 20, 2016). 13 LeBlanc, R. (ed). Method Development and Validation for the Quantification of Eight Synthetic Piperazines in Blood and Urine using Liquid Chromatography-tandem Mass Spectrometry (UFLC-ESI-MS/MS) . Boston University Libraries: Boston, MA, 2016; pp. 14– 15. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Journal

Journal of Analytical ToxicologyOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

Monthly Plan

  • Read unlimited articles
  • Personalized recommendations
  • No expiration
  • Print 20 pages per month
  • 20% off on PDF purchases
  • Organize your research
  • Get updates on your journals and topic searches

$49/month

Start Free Trial

14-day Free Trial

Best Deal — 39% off

Annual Plan

  • All the features of the Professional Plan, but for 39% off!
  • Billed annually
  • No expiration
  • For the normal price of 10 articles elsewhere, you get one full year of unlimited access to articles.

$588

$360/year

billed annually
Start Free Trial

14-day Free Trial