DAD quantification method for piperazines–amphetamines mixtures in seized material

Egyptian Journal of Forensic Sciences (2014) xxx, xxx–xxx

Contents lists available at ScienceDirect

Egyptian Journal of Forensic Sciences journal homepage: www.ejfs.org

ORIGINAL ARTICLE

Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material Yacine Boumrah a,b,1, Martine Rosset c,*, Yannick Lecompte d, Sabrina Bouanani a, Kamel Khimeche e, Abdellah Dahmani b a

Drugs Laboratory, National Institute of Criminalistics and Criminology, Algiers, Algeria Laboratoire de thermodynamique et mode´lisation mole´culaire, Faculte´ de Chimie, USTHB, BP 32 El-Alia Bab-Ezzouar, 16111 Alger, Algeria c Etat-major de la marine, bureau maitrise des risques, 2 rue royale, 75008 Paris, France d Institut de recherche criminelle de la gendarmerie nationale (IRCGN), De´partement toxicologie, 1 boulevard The´ophile Sueur, 93111 Rosny-Sous-Bois, France e Ecole Militaire Polytechnique EMP, BP 17 Bordj–el-Bahri, Alger, Algeria b

Received 13 October 2013; revised 20 January 2014; accepted 23 March 2014

KEYWORDS Piperazines; Amphetamines; Designer drugs; b-Expectation tolerance interval; Benzylpiperazine; 1-(3-Trifluoromethylphenyl) piperazine

Abstract Piperazine related drugs are sold as party pills in the form of tablets, capsules, liquids or powders. These party pills can contain several piperazine derivatives, or even a mixture of piperazines and amphetamine derivatives. This paper therefore describes a screening method using a gas chromatography–mass spectrometry technique allowing the separation and the identification of active components within these mixtures by a combined silylation and acylation derivatization procedure. The studied substances namely: 1-benzylpiperazine (BZP), 1-(3,4-methylenedioxybenzyl)piperazine (MDBP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP), 1-(4-methoxyphenyl)piperazine (MeOPP), amphetamine, methamphetamine, ephedrine, pseudoephedrine, 3,4-methylenedioxy-N-methamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA) and N-methyl-1,3benzodioxolylbutanamine (MBDB), are separated.

* Corresponding author. Tel.: +33 1 42 92 19 42. E-mail addresses: [email protected] (Y. Boumrah), [email protected] (M. Rosset). 1 Tel.: +213 0663845657. Peer review under responsibility of Forensic Medicine Authority.

Production and hosting by Elsevier 2090-536X ª 2014 Production and hosting by Elsevier B.V. on behalf of Forensic Medicine Authority. http://dx.doi.org/10.1016/j.ejfs.2014.03.002 Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

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Y. Boumrah et al. A high performance liquid chromatography with a diode array detector quantitative method was validated following an approach using accuracy profiles based on b-expectation tolerance intervals for total error measurement. ª 2014 Production and hosting by Elsevier B.V. on behalf of Forensic Medicine Authority.

1. Introduction

2. Experimental

Synthetic drugs are among the most commonly abused drugs in the world. Some are derived from phenylethylamine, as is 3,4-methylenedioxymethamphetamine (MDMA). However, as these drugs became scheduled, new derivatives (like piperazines) have appeared on the market to bypass the law. Chemically, piperazines are synthesized from the same piperazine moiety and they can be classified into two classes: benzylpiperazines such as 1-benzylpiperazine (BZP) and its methylenedioxy analog 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) and phenylpiperazines such as 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-methoxyphenyl)piperazine (MeOPP). Piperazine-like compounds have spread around the world: according to Kovaleva et al. it was estimated that approximately 823,000 tablets of mCPP have been seized in 2008 in the European Union.1 In the Netherlands, the number of mCPP tablets seized alone or in combination with MDMA increased significantly.2 A recent survey in the United Kingdom, found that piperazines are among the most common active drugs in tablets purchased from internet supplier sites.3 These drugs have also been seized in New Zealand,4,5 Japan,6 Brazil7 and many other countries such as Bulgaria, Sweden and South Africa.8 Commonly, piperazines are sold as party pills in the form of tablets, capsules, liquids or powders on the black drug-market and in so-called headshops or over the internet under the names of ‘‘Rapture,’’ ‘‘Frenzy,’’ ‘‘Bliss,’’ ‘‘Charge,’’ ‘‘Herbal ecstasy,’’ ‘‘A2,’’ ‘‘Legal X’’ and ‘‘Legal E’’.9,10 They are also found in tablets sold as ecstasy or amphetamine.9–17 Generally, the party pills contain piperazine blends. Besides the most prevalent mixture of BZP with TFMPP,18,19 there are also combinations of up to four different piperazines which are sold to consumers as ‘‘party pills’’ or ‘‘P.E.P. pills’’ called X4.20–24 Furthermore, a large number of seized tablets containing mixtures of piperazine and amphetamine type stimulants (ATS) have been reported.2,11–13,25–35 Several methods related to the identification of piperazine like-compounds in both seized and biological samples have been reported12,36–42 but an identification method for amphetamine-piperazine mixtures, as encountered in a large number of seizures, has not been described yet. The aim of this work is therefore to develop a qualitative method using a gas chromatography-mass spectrometry technique which allows the separation and the identification of a mixture composed of piperazines and ATS. For the quantitative method, we have chosen to extend to piperazines an HPLC/DAD method, previously implemented for the quantification of amphetamine type stimulants.43 The method was validated following an approach using accuracy profiles based on b-expectation tolerance intervals for total error measurement.

2.1. Reagents and standards Reference compounds 1-(4-methoxyphenyl)piperazine (MeOPP) dihydrochloride, 1-(4-methylphenyl)piperazine (pTP) dihydrochloride (internal standard), 1-(3-chlorophenyl)piperazine (mCPP) hydrochloride, 1-(3-trifluoromethylphenyl)piperazine (TFMPP) hydrochloride, 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) free base and 1-benzylpiperazine (BZP) were purchased from Lancaster (Frankfurt, Germany). Analytical standards of amphetamine, methamphetamine, ephedrine, pseudoephedrine, 3,4-methylenedioxy-N-methamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA) and N-methyl-1,3-benzodioxolylbutanamine (MBDB) were purchased from Promochem (Wesel, Germany) as 1 mg/mL methanolic solutions. Lactose was purchased from Fluka (Steinheim, Germany). Heptafluorobutyric anhydride (HFBA) and the silylation reagent SilPrep were purchased from Alltech (Templemars, France). Methanol and acetonitrile (HPLC grade), n-hexane, potassium dihydrogenphosphate (KH2PO4) and 85% ortho-phosphoric acid (analytical grade) were purchased from VWR Prolabo (Fontenay-sous-Bois, France). 2.2. Calibrators and specimen preparation for GC/MS qualitative analysis Stock solutions of BZP, MBDP, mCPP, TFMPP and MeOPP at a concentration of 2 mg/mL were prepared in methanol. Working solutions of amphetamine–piperazine mixtures were obtained by dilution in methanol. All these solutions were stored protected from light at +5 C. GC/MS analyses of the piperazine derivative mixture were performed first without derivatization at a concentration of 2 mg/mL, and then carried out after silylation and acylation at a concentration of 10 mg/L. The silylation was performed by adding 125 lL of SilPrep reagent to the evaporation residue of 125 lL of each 10 mg/L working solution. The acylation was performed by adding 25 lL of HFBA reagent to the evaporation residue of 300 lL of each 10 mg/L working solution, followed by 300 lL of hexane and 1 mL of 0.02 M potassium dihydrogen phosphate neutralizing solution. The mixture was centrifuged for 1 min at 302 g and the organic layer analyzed by GCMS. The combined silylation–acylation derivatization procedure was performed by adding 100 lL of the upper layers of each acylated solution to its silylated counterpart.

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

Development and validation of methods for piperazine–amphetamine mixture analysis 2.3. Calibrators and validation standards preparation for the validation of the HPLC/DAD quantification method Stock solutions of amphetamines, piperazines, internal standard and lactose at a concentration of 1 mg/mL were prepared in methanol/water 50:50 (v/v). Subsequent working solutions were prepared by proper dilutions of the stock solutions with methanol/water 50:50 (v/v). The calibration standard solutions were prepared from a different stock solution batch than that of the validation standards. Each operator prepared his own piperazine derivative mix calibration standards, by dilution of the stock solutions with methanol/water 50:50 (v/v) and 1 mg/mL internal standard (pTP) to obtain eight calibration standards of 2, 10, 20, 30, 40, 50, 80 and 100 mg/L (m = 8). This operation was repeated on three different days, by three different operators. The validation standards, were prepared once, and analyzed by each operator, with the calibration standards he had prepared. For routine work, tablets from the seized material were crushed in a mortar and a pestle and 50 mg of the resulting powder, were weighed on a precision scale (e = 10 4 g) and subsequently dissolved in 50 mL of the methanol/water 50:50 (v/v) mixture. The solution was mixed on vortex, sonicated for 15 min and centrifuged for 1 min at 302 g. One hundred (100) lL of the prepared solution was added to 100 lL of internal standard solution (1 mg/mL), and then filled up to 1 mL volume by adding methanol/water 50:50 (v/v) mixture solvent. Lactose is a substance known to be present in tablets (filling agent). Lactose has been added to the validation standards in order to mimic the matrix effect that can potentially be seen in the analysis of tablets. This is why the stock solutions were mixed with the lactose solution and 1 mg/mL internal standard (pTP) to obtain eleven validation standards of 2, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg/L (m = 11). Four replications of each validation standard were performed (n = 4). This operation was repeated on three different days (p = 3), by three different operators. 2.4. Validation software and principles of the quantification method In accordance to ISO/CEI 17025:2005 and the guidelines of the French standard NF V 03–110,44 the present method was fully validated using the total error approach.45–47

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The b risk for the confidence interval has been set to 5%, and the ±k acceptance limits to 10%. Accuracy profiles and validation parameters have been computed with e.noval software V2.0 (Arlenda, Lie`ge, Belgium). The pre-validation step of the software, allows choosing the mathematical model that is closest to the response function. This protocol requires the preparation of two kinds of samples in two independent ways: calibration standards and validation standards, as described above. The experimental plan consisted of three series (reproducibility conditions) of four repetitions (repeatability conditions) of analyses. 2.5. GC/MS qualitative analysis GC/MS analyses were performed using an HP 5890 series II gas chromatograph (Hewlett–Packard, Agilent, Massy, France), equipped with a 5971A mass selective detector (Hewlett–Packard, Agilent, Massy, France). A 5% phenyl-methylsiloxane (HP-5MS) capillary column (30 m · 0.25 mm I.D., 0.25 lm film thickness), supplied by Hewlett–Packard (Agilent, Massy, France), was used. Chromatographic conditions were as follows: initial column temperature was 90 C for 0.5 min, increased by 20 C/ min to 200 C, then to 280 C at 15 C/min and finally to 320 C at 20 C/min for 3.67 min. The temperatures of the injection port and detector were set at 280 and 320 C, respectively. Injection of 1 lL of the sample solution was performed automatically in splitless mode, and helium (carrier gas) at a flow rate of 1.0 mL/min. The mass spectrometer was operated with a filament current of 300 lA and electron energy of 70 eV in the electron ionization (EI) mode. Positive ions were analyzed. Acquisition was carried out in the scan mode, and the entire mass range from 38 to 650 m/z was collected. 2.6. HPLC/DAD quantitative analysis Quantitative analysis was performed by high-performance liquid chromatography, using a liquid chromatograph equipped with a spectrophotometric diode-array detector (HPLC-DAD). The study was conducted on a Hewlett–Packard 1050 series HPLC. Chromatographic separation was carried out on a Thermo Hypersil C18 analytical column (3 lm · 125 mm · 3 mm i.d.), protected by a Thermo BDS

Figure 1 GCMS Total Ion Chromatograms of piperazine derivative mixture: (A) without derivatization (1: BZP, 2: TFMPP, 3: mCPP, 4: MeOPP and 5: MDBP); (B) after silylation (1: BZP-TMS, 2: TFMPP-TMS, 3: mCPP-TMS, 4: MeOPP-TMS and 5: MDBP-TMS); (C) after acylation (1: BZP-HFB, 2: TFMPP-HFB, 3: mCPP-HFB, 4: MeOPP-HFB and 5: MDBP-HFB).

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

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Y. Boumrah et al. Table 1

Retention times (RT), mass spectra (m/z) and the limits of detection (LODs) for five piperazine derivatives.

Without derivatization

After silylation

After acylation

Substance

RT (min)

m/z

LOD (lg/mL)

BZP TFMPP mCPP MeOPP MDBP BZP-TMS TFMPP-TMS mCPP-TMS MeOPP-TMS MDBP-TMS BZP-HFB TFMPP-HFB mCPP-HFB MeOPP-HFB MDBP-HFB

4.454 4.647 6.045 6.143 6.866 5.733 5.905 7.283 7.370 8.039 6.147 6.156 7.446 7.525 8.364

91;56 ;134 ;176 188;56;95;172;145;230 154;56;75;11 ;138;196 150;56;92;120;135;192 135;56;85;178;164;220 102;59;116;157;233;248 302;59;73;101;128;173 128;59;73;101;;226;268 264;59;73;101;135;162 135;59;73;102;;157;292 91;56;146;175;281;372 200;56;69;145;173;426 392;56;69;139;166;195 388;56;69;135;191 135;56;77;281;416

100.00 93.50 71.50 73.00 116.00 0.70 0.75 0.80 0.80 1.00 0.50 0.60 0.60 0.80 0.90

Figure 2 GCMS total ion chromatograms of amphetamine derivative mixture: (A) after silylation (1: Ephedrine-TMS, 2: Ephedrine2TMS, 3: Amphetamine-TMS, 4: Methamphetamine-TMS, 5: Ephedrine-2TMS and 6: MBDB-TMS); (B) after acylation (AmphetamineHFB, 2: Methamphetamine-HFB, 3: Ephedrine-HFB, 4: Pseudoephedrine-HFB, 5: MDA-HFB, 6: MDMA-HFB, 7: MDEA-HFB and 8: MBDB-HFB).

Figure 3 GCMS total ion chromatograms of a mixture containing piperazine and amphetamine derivatives: (A) after silylation (1: Ephedrine-TMS, 2: Ephedrine-2TMS, 3: Amphetamine-TMS, 4: BZP-TMS, 5: TFMPP-TMS, 6: Methamphetamine-TMS, 7: Ephedrine2TMS, 8: MBDB-TMS, 9: mCPP-TMS, 10: MeOPP-TMS and 11: MDBP-TMS); (B) after acylation (1: Amphetamine-HFB, 2: Methamphetamine-HFB, 3: Ephedrine-HFB, 4: Pseudoephedrine-HFB, 5: MDA-HFB, 6: BZP-HFB, 7: TFMPP-HFB, 8: MDMA-HFB, 9: MDEA-HFB, 10: MBDB-HFB, 11: mCPP-HFB, 12: MeOPP-HFB and 13: MDBP-HFB).

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

Development and validation of methods for piperazine–amphetamine mixture analysis C18 guard column, in reversed-phase mode, with a mobile phase gradient. The components of the mobile phase were: KH2PO4 buffer, 20 mM, adjusted to pH 3.2 with 85% orthophosphoric acid (A) and acetonitrile (B). The flow rate of the mobile phase was 0.4 mL/min. The total analysis time was 19 min.

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The gradient applied was increased from 20% to 40% B within 12 min, starting from 0 min after injection, then increased to 70% B in 2 min, and kept at 70% B for 4 min. Finally, the gradient was raised to 97% B from 18 to 19 min. The column temperature during the analysis run was maintained at 30 C. The volume of injection was 10 lL. The HP

Figure 4 GCMS total ion chromatogram of a mixture containing piperazines and amphetamines after a combined silylation and acylation: (1) Amphetamine-HFB, (2) Ephedrine-TMS, (3) Methamphetamine-HFB, (4) Ephedrine-HFB, (5) Pseudoephedrine-HFB, (6) Ephedrine-2TMS, (7) Ephedrine-2TMS, (8) Pseudoephedrine-HFB-TMS, (9) Amphetamine-TMS, (10) MDA-HFB, (11) BZP-TMS, (12) TFMPP-TMS, (13) Methamphetamine-TMS, (14) BZP-HFB, (15) MDMA-HFB, (16) MDEA-HFB, (17) MBDB-TMS, (18) MBDBHFB, (19) mCPP-TMS, (20) MeOPP-TMS, (21) mCPP-HFB, (22) MeOPP-HFB and (23) MDBP-HFB.

Figure 5 HPLC-UV/DAD chromatograms of piperazine derivative mixture: (A) Piperazine derivative mixture with internal standard PTP, detection at 210.4 nm [1: BZP; 2: MDBP; 3: MeOPP; 4: P-TP; 5: mCPP and 6: TFMPP]; (B) Piperazines mixed with amphetamines, detection at 230.4 nm [1: BZP; 2: MDBP; 3: MeOPP; 4: MDMA; 5: mCPP and 6: TFMPP].

Table 2

Integration criteria of the piperazine derivatives.

Dosing range (mg/L) Wave length (nm) Criteria of integration

BZP

BZP

MDBP

MeOPP

mCPP

TFMPP

2–30 210.4 Height

30–100 254.4 Area

2–100 286.4 Area

10–100 230.4 Area

10–100 210.4 Area

10–100 210.2 Area

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

– 394.10 68.88 1.000

– 157.10 – –

UV-DAD detector operates with a time response of 1 s, peak width taken >0.05 min and spectra of substances were recorded from 210 to 400 nm. 3. Results

– 137.80 175.60 1.000

3.1. GC/MS qualitative analysis

0.915 105.00 44.03 0.999

– 34.16 – –

– 47.70 – –

– 140.00 167.50 0.999

– 394.10 287.90 1.000

– 157.60 – –

0.878 94.54 17.39 0.999

– 36.50 – –

– 47.56 – –

In the applied GC/MS conditions, a good chromatographic separation for the silylated piperazines was obtained as shown in Fig. 1B. This was not the case for the acylated ones, where a total co-elution between BZP and TFMPP was observed (Fig. 1C). The derivatizing agents, acting on different sites (a mobile hydrogen for the silylation reaction, primary amine moiety for acylation) generate chemical structures that can easily be differentiated by mass spectrometry. Mass spectra are very specific, and allow unambiguous identification on the basis of major ions with m/z values given in Table 1. The limits of detection (LODs) for the GC/MS method were determined. A concentration causing a detector signal three times greater than the noise (S/N = 3) was accepted as the lower LOD. The LLODs for these piperazines were in the concentration range of 0.5–1.0 mg/L after acylation or silylation and 71.5–100 mg/L without derivatization. The acylated amphetamines were well separated and identified (Fig. 2B), whereas, this was not the case with the silylated ones (Fig. 2A). The mixture containing the studied piperazine and amphetamine derivatives is then well identified after HFBA-derivatization and the target components are well separated except those of BZP and TFMPP as shown in Fig. 3B. For this reason, a combined silylation–acylation derivatization protocol was carried out and the results show a better identification and a total separation of all the target components as shown in Fig. 4.

– 391.00 184.90 1.000

– 157.40 – – – 34.16 – – 0.973 109.60 29.83 1.000

– 139.50 51.51 0.999

3.2. HPLC/DAD validated method for quantitative analysis

Number of replicates (n). Number of amount levels (m). Number of series (Day) (k).

– 47.37 – – Quadratic term Slope Intercept r2

Day2

BZP MDBP MeOPP mCPP TFMPP BZP BZP

Response function (k = 3, m = 8, n = 1) Day1

Table 3

Validation results (response function) for the HPLC/DAD quantitative determination of piperazine derivatives.

Day3

Y. Boumrah et al. BZP MDBP MeOPP mCPP TFMPP BZP BZP MDBP MeOPP mCPP TFMPP

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Typical HPLC/DAD chromatograms of the piperazine derivatives and of the piperazines mixed with amphetamine derivatives are shown in Fig. 5. The achieved separation was satisfactory in both analyses with a small co-elution between BZP and MDBP. The five piperazine derivatives are well separated from the amphetamines, especially from MDMA which is the most encountered substance in an admixture with the piperazine derivatives. Because of the BZP-MDBP co-elution, the quantification of BZP has to be carried out for lower concentrations by considering the peak height at the absorption maximum (210.4 nm), then by considering the area for upper concentrations, at 254.4 nm (Table 2). However, these two substances are rarely simultaneously present in the same tablet. 3.3. Method validation A weighted (1/X2) quadratic regression model was selected for the quantitative determination of BZP from 2 to 30 mg/L and a linear regression through 0 fitted with the level 100 model, from 30 to 100 mg/L. A linear regression through 0 fitted with the level 100 was selected for both MBDP and TFMPP from 2 to 100 mg/L. For mCPP and MeOPP, a linear regression

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

Development and validation of methods for piperazine–amphetamine mixture analysis model was selected. The validation results for the response functions in the present study are presented in Table 3. According to our acceptance criteria (acceptance limits ±10%, expectation tolerance limits of 90%), the selected method was shown to be sufficiently accurate since the bias did not exceed the value of 15% (Table 3). In addition, no systematic bias was observed, and this confirms the absence of a matrix effect. The relative standard deviation for both repeatability and intermediate precision was between 0.37% and 7.18%. 3.3.1. Accuracy The upper and the lower b-expectation tolerance limit expressed in mg/L are presented in Table 4 as a function of

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the introduced amounts. As can be seen from these results, the tolerance limits did not exceed the acceptation limits for all concentration levels tested: the proposed method is accurate in the range from 8.5 to 100 mg/L for BZP, 17.5 to 100 mg/L for MDBP, 10 to 100 mg/L for both MeOPP and mCPP piperazines and 6 to 100 mg/L for TFMPP. Furthermore, the risk of having future assay results exceeding the 10% limits of the targeted amount is strictly controlled.48–52 The linearity of the analytical method was tested. The regression equations are presented in Table 5. Limits of quantification are obtained by calculating the smallest and highest concentration beyond which the accuracy limits or b-expectation limits exceed the acceptance limits. The results are presented in Table 5.

Table 4 Validation results (trueness, precision, and accuracy) for the HPLC/DAD quantitative determination of piperazine derivatives. BZP

BZP

MDBP

MeOPP

TFMPP

BZP

MeOPP

mCPP

TFMPP

– 1.2

6.6 2.3

Recovery (%) 99.4 – 93.7 99.0 – 97.0

– 101.5

– 101.2

106.6 102.3

1.4 2.9 2.9 1.2 1.7 2.1 1.2 1.1 2.4

1.3 2.0 2.2 0.9 1.8 1.4 1.1 0.8 0.5

1.7 3.0 2.9 13 2.1 2.0 1.6 1.6 1.6

100.2 99.9 – – – – – – –

101.4 102.9 102.9 101.2 101.7 102.1 101.2 101.1 102.4

101.3 102.0 102.2 100.9 101.8 101.4 101.1 100.8 100.5

101.7 103.0 102.9 101.3 102.1 102.0 101.6 101.6 101.6

(RSD,%) 4.7 – 2.0 2.1 1.9 1.9 2.1 1.5 0.6 0.6 0.7 1.0 0.9 1.0 0.7 1.6 0.4 0.5 0.4 0.6 0.6 0.6

– 1.5 1.3 1.6 0.4 0.7 0.7 0.6 0.6 0.6 0.7

4.3 1.6 1.6 17 0.4 0.5 0.7 0.7 0.7 0.6 0.8

Intermediate precision (RSD,%) 7.2 – 5.9 – – 3.0 – 3.8 2.2 2.2 3.7 – 3.5 1.9 1.3 3.4 1.5 2.0 1.9 1.9 – 1.3 2.0 1.0 1.2 – 0.9 2.0 1.6 1.3 – 1.0 1.3 1.0 1.0 – 1.0 1.4 1.6 0.8 – 0.7 1.5 1.2 1.3 – 1.2 1.8 1.4 1.4 – 0.8 1.4 1.2 1.0

Trueness (k = 3, m = 11, n = 4) 2 10

Relative bias (%) 0.6 – 6.3 1.0 – 3.0

– 1.5

20 30 40 50 60 70 80 90 100

0.2 0.1 – – – – – – –

Precision (k = 3, m = 11, n = 4) 2 10 20 30 40 50 60 70 80 90 100

Repeatability 5.2 – 1.6 – 3.7 – 1.7 1.3 – 0.5 – 0.9 – 0.8 – 0.7 – 0.7 – 0.8 – 0.8

Accuracy (k = 3, m = 11, n = 4)

– 1.1 0.2 1.5 0.9 1.0 1.3 1.6 2.3

0.4 4.4 2.7 1.0 2.0 1.5 1.4 1.5 1.6

BZP

– 98.9 99.8 98.5 99.0 99.0 98.8 98.4 97.7

MDBP

99.6 104.4 102.7 101.0 102.0 101.5 101.4 101.5 101.6

4.3 1.6 1.6 1.7 1.1 1.2 1.1 1.2 1.3 1.8 1.5

b-expectation tolerance limits (ng) BZP

2 10 20 30 40 50 60 70 80 90 100

mCPP

[01.7, [09.1, [18.6, [27.2, – – – – – – –

02.3] 10.6] 21.4] 32.7]

BZP

MDBP

MeOPP

mCPP

TFMPP

– – – [28.8, [38.4, [48.4, [58.1, [67.5, [77.9, [85.3, [96.1,

[01.6, [08.7, [18.2, [29.4, [38.4, [47.4, [59.1, [68.3, [77.2, [85.9, [97.5,

– [09.8, [19.7, [30.0, [40.5, [49.1, [60.5, [70.1, [79.3, [88.4, [99.4,

– [09.6, [19.9, [29.6, [39.7, [49.1, [60.2, [70.4, [78.5, [87.8, [99.0,

[01.9, [09.8, [19.6, [29.7, [39.5, [48.6, [59.2, [68.7, [77.9, [86.1, [96.8,

30.5] 41.4] 50.1] 60.8] 71.1] 80.1] 91.7] 99.3]

02.1] 10.6] 21.7] 31.7] 43.6] 53.3] 62.8] 73.4] 84.7] 96.3] 105.2]

10.7] 21.2] 32.3] 42.5] 53.1] 62.7] 74.2] 84.4] 95.6] 105.4]

10.6] 20.8] 32.0] 42.7] 52.5] 62.9] 72.8] 84.3] 95.0] 103.7]

02.3] 10.4] 20.8] 31.6] 42.2] 52.1] 62.5] 73.0] 83.5] 95.4] 104.9]

Number of replicates (n). Number of amount levels (m). Number of series (Day) (k). Relative standard deviation (RSD).

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

8

Y. Boumrah et al. Table 5

Validation results (linearity, limits of quantification) for the quantitative determination of piperazine derivatives.

Linearity (k = 3,m = 11, n = 4) Range (ng) Slope Intercept r2

BZP

BZP

MDBP

MeOPP

mCPP

TFMPP

8.545–30 1.0010 0.04276 0.9970

30–100 0.9746 0.74460 0.9991

17.54–100 1.0170 0.04606 0.9992

10–100 1.0160 0.09157 0.9992

10–100 1.0060 0.3459 0.9994

5.95–100 1.0140 0.2102 0.9994

30 100

17.54 100

10.10 100

10 100

5.95 100

Limits of quantification (k = 3,m = 11, n = 4) Lower limit of quantification (lg/mL) 8.545 Upper limit of quantification (lg/mL) 30 Number of replicates (n). Number of amount levels (m). Number of series (Day) (k).

4. Discussion The use of accuracy profiles for the validation based on total error concept is compliant with the fundamental demands of the ISO/CEI 17025 standard, for example validation and determination of measurement uncertainty. This method is based on the determination of confidence interval of measurements, with a risk b. This interval, also called b expectation interval, is computed from accuracy and intermediate precision. For each concentration level included in the experiment, the standard deviation of intermediate precision is calculated by adding the repeatability variance to the inter-series variance (S2IF = S2R + S2IS). The graphical representation of these b

expectation tolerance limits builds up the accuracy profile of the quantification method. On a practical point of view, the validation decision is based on the graphical comparison of the accuracy profile obtained previously with the acceptance limits ±k that are set prior to the beginning of the experiment. The acceptance limits correspond to the maximal uncertainty of measurement that is accepted for the results obtained by the analytical method. These limits have to be set according to the characteristic constraints of the domain or activity in which the dosing will be implemented. For example, in forensic toxicology the limit is ±k = 40%. For the analysis of illicit substances in seized material described in this case, we have chosen ±k of 10%. The overall performance of the method described in this

Figure 6 Accuracy profiles of piperazine derivatives (amount in mg/L): (1) Accuracy profile of BZP from 02 to 30 mg/L using a weighted (1/X2) quadratic regression model; (2) Accuracy profile of BZP from 30 to 100 mg/L using a linear regression through 0 fitted with the level 100 only model; (3) Accuracy profile of MDBP from 02 to 100 mg/L using a linear regression through 0 fitted with the level 100 only model; (4) Accuracy profile of MeOPP from 10 to 100 mg/L using a linear regression model; (5) Accuracy profile of mCPP from 10 to 100 mg/L using a linear regression model; (6) Accuracy profile of TFMPP from 02 to 100 mg/L using a linear regression through 0 fitted with the level 100 only model. The plain line is the relative bias, the dashed lines are the b-expectation tolerance limits and the dotted lines represent the acceptance limits (10%). The dots represent the relative back-calculated concentrations and are plotted with respect to their targeted concentration.

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

Development and validation of methods for piperazine–amphetamine mixture analysis paper is much better than expected, as can be seen in Fig. 6 for MeOPP and mCPP. Moreover, during the pre-validation step, the best mathematical model of response function which is chosen is the one which gives the best accuracy index, or the best analytical performance in terms of LOQ and uncertainty measurement. 5. Conclusions The GC/MS method developed in this work has allowed the separation and the identification of a large number of piperazine and amphetamine type substances. The HPLC/DAD quantitative method is fully validated following the concept of total error, by means of accuracy profiles. This graphical approach allows to study in a global way accuracy and intermediate reproducibility, and eventually to easily set LOQ and dosing range. The quantitative method has shown to be linear within the adopted ranges for all the studied piperazines. Furthermore, both qualitative and quantitative methods can be useful to laboratories for performing routine analysis of seized material containing piperazine and amphetamine type substances.

7.

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13.

Funding None.

14.

Conflict of interest

15.

None declared.

16. 17.

Ethical approval 18.

Necessary ethical approval was obtained from the institute ethics committee.

19.

Acknowledgements

20.

The author would like to thank Aziz Khiari and Salah Haroun for their helpful comments on an early draft of this manuscript.

21.

22.

References 23. 1. Kovaleva J, Devuyst E, De Paepe P, Verstraete A. Acute chlorophenylpiperazine overdose: a case report and review of the literature. Ther Drug Monit 2008;30:394. 2. Bossong MG, Brunt TM, Van Dijk JP, Rigter SM, Hoek J, Goldschmidt HMJ, et al. MCPP: an undesired addition to the ecstasy market. J Psychopharmacol 2010;24:1395. 3. Davies S, Wood DM, Smith G, Button J, Ramsey J, Archer R, et al. Purchasing ‘legal highs’ on the Internet––is there consistency in what you get? Q J Med 2010;103:489. 4. Gee P, Richardson S, Woltersdorf W, Moore G. Toxic effects of BZP-based herbal party pills in humans: a prospective study in Christchurch. NZ Med J 2005;118:1784. 5. Sheridan J, Butler R, Wilkins C, Russel B. Legal piperazinecontaining party pills–a new trend in substance misuse. Drug Alcohol Rev 2007;26:335. 6. Takahashi M, Nagashima M, Suzuki J, Seto T, Yasuda I, Yoshida T. Creation and application of psychoactive designer drugs data library using liquid chromatography with photodiode array

24. 25.

26.

27. 28. 29.

9

spectrophotometry detector and gas chromatography-mass spectrometry. Talanta 2009;77(4):1245–72. Lanaro R, Costa JL, Zanolli-Filho LA, Cazenave SOS. Identificacß a˜oquı´ mica da clorofenilpiperazina (CPP) emcomprimidosapreendidos. Quim Nova 2010;33:725–9. Cohen BMZ, Butler R. BZP-party pills: a review of research on benzylpiperazine as recreational drug. Int J Drug Policy 2011;22:95–101. Gee P, Richardson S, Woltersdorf W, Moore G. Toxic effects of BZP-based herbal party pills in humans: a prospective study in Christchurch, New Zealand. NZ Med J 2005;118:1784–94. Drug Enforcement Administration Office of Diversion Control Drug & Chemical Evaluation Section. 1-3-(Trifluoro-methyl)phenyl piperazine (Street Names: ‘‘TFMPP’’ or ‘‘Molly’’. Often found in combination with BZP: ‘‘A2’’, ‘‘Legal E’’ or ‘‘Legal X’’). Available at: ; 2013 [accessed 01.09.13]. Yarosh HL, Katz EB, Coop A, Fantegrossi WE. MDMA-like behavioral effects of N-substituted piperazines in the mouse. Pharmacol Biochem Behav 2007;88:18–27. De Boer D, Bosman IJ, Hidve´gi E, Manzoni C, Benko AA, dos Reys LAL, et al. Piperazine-like compounds: a new group of designer drugs-of abuse on the European market. Forensic Sci Int 2001;121:47–56. European monitoring centre for drugs and drug addiction. Annual report on the state of drugs problem in Europe. Available at: ; 2012 accessed 10.01.13. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2007;40(9):91. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(4):37. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2008;41(5):48. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2008;41(2):18. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2008;41(1):1–4. United States Department of Justice/Drug enforcement administration, system to retrieve information from drug evidence II (STRIDE), 2003. Cuddy L. Common drugs of abuse–Part II. J Pract Nurse 2004;54(1):25–31. Maurer HH, Kraemer T, Springer D, Staack RF. Chemistry, pharmacology, toxicology and hepatic metabolism of designer drugs of the amphetamine (ecstasy), piperazine, and pyrrolidinophenone types. Drug Monit 2004;26:127–31. Wikstrom M, Holmgren P, Ahlner J. A2 (N-benzylpiperazine) a new drug of abuse in Sweden. J Anal Toxicol 2004;28:67–70. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2003;36(6):118–9. Arbo MD, Bastos ML, Carmo HF. Piperazine compounds as drugs of abuse. Drug Alcohol Depend 2012;122:174–85. United Nations of Drugs and Crime. World Drug Report. Available at: ; 2013 accessed 09.08.13. EMCDDA–Europol New drugs in Europe, 2012. EMCDDA– Europol 2012 annual report on the implementation of council decision 2005/387/JHA. Available at: ; 2012 accessed 11.12.12. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(6):53. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(7):64. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(8):68–70.

Please cite this article in press as: Boumrah Y et al. Development of a targeted GC/MS screening method and validation of an HPLC/DAD quantification method for piperazines–amphetamines mixtures in seized material, Egypt J Forensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2014.03.002

10 30. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(5):45–9. 31. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2009;42(2):16–7. 32. U.S. Department of Justice-Drug Enforcement Administration. Microgram Bull 2007;40(4):39. 33. Perrin M, Lecompte Y, Roussel O, Le Boisselier R, Bourgine J, Coquerel A. Nouvelles drogues in Traite´ de toxicologie me´dicojudiciaire. Paris, 2012, (ISBN 978-2-994-71561-7) p. 517–585. 34. Sheridan J, Butler R, Wilkins C, Russell B. Legal piperazinecontaining party pills – a new trend in substance misuse. Drug Alcohol Rev 2007;26:335–43. 35. Bishop SC, McCord BR, Gratz SR, Loeliger JR, Witkowski MR. Simultaneous separation of different types of amphetamine and piperazine designer drugs by capillary electrophoresis with a chiral selector. J Forensic Sci 2005;50:326–35. 36. Staack RF, Fritschi G, Maurer HH. Studies on the metabolism and toxicological detection of the new designer drug N-benzylpiperazine in urine using gas chromatography-mass spectrometry. J. Chromatogr B 2002;773(1):35–46. 37. Peters FT, Schaefer S, Staack RF, Kraemer T, Maurer HH. Screening for and validated quantification of amphetamines and of amphetamine and piperazine-derived designer drugs in human blood plasma by gas chromatography/mass spectrometry. J Mass Spectrom 2003;38:659–76. 38. Barroso M, Costa S, Dias M, Vieira DN, Queiroz JA, LopezRivadulla M. Analysis of phenylpiperazine-like stimulants in human hair as trimethylsilyl derivatives by gas chromatography– mass spectrometry. J. Chromatogr A 2010;1217:6274–80. 39. Navaneeswari R, Raveendra RP. Analytical method for piperazine in an active pharmaceutical ingredient using chemical derivatization and HPLC-UV. J Chem Pharm Res 2012;4:2854–9. 40. Tsutsumi H, Katagi M, Miki A, Shima N, Kamata T, Nishikaw, et al. Development of simultaneous gas chromatography mass spectrometric and liquid chromatography–electrospray ionization mass spectrometric determination method for the new designer drugs Nbenzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine. J Chromatogr B 2005;819:315–22. 41. Vorce SP, Holler JM, Levine B, Past MR. Detection of 1benzylpiperazine and 1-(3-trifluoromethylphenyl)-piperazine in urine analysis specimens using GC–MS and LC–ESI-MS. J Anal Toxicol 2008;32:444–50.

Y. Boumrah et al. 42. Moreno IED, da Fonseca BM, Magalha˜es AR, Geraldes VS, Queiroz JA, Barroso M, et al. Rapid determination of piperazinetype stimulants in human urine by microextraction in packed sorbent after method optimization using a multivariate approach. J Chromatogr A 2012;1222:116–20. 43. Sadeghipour F, Giroud C, Rivier L, Veuthey JL. Rapid determination of amphetamines by high-performance liquid chromatography with UV detection. J Chromatogr A 1997;761:71–8. 44. Norme Franc¸aise: NF V 03–110. Analysis of agri-foodstuffs –– Protocol of characterization for the validation of a quantitative method of analysis by construction of an accuracy profile, 2010. 45. Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ. Workshop/conference report –– quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays. AAPS J. 2007;9:30–42. 46. Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Cohen N, Compagnon PA, et al. Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal–part III. J Pharm Biomed Anal 2007;45(1):82–96. 47. Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Chiap P, Cohen N, et al. Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal–part II. J Pharm Biomed Anal 2007;45(1):70–81. 48. Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Chiap P, Cohen N, et al. Validation des proce´dures analytiques quantitatives. Harmonisation des de´marches. STP Pharma Pratiques 2003;13:101–38. 49. Food and Drug Administration: US Department of Health and Human Services. Guidance for Industry, Bioanalytical Method Validation, FDA. Available at: ; 2001 accessed 19.03.13. 50. Shah VP, Midha KK, Dighe S, McGilneray IJ, Skelly JP, Yakobi A, et al. Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetic studies. J Pharm Sci 1992;81:309–12. 51. Hubert Ph, Chiap P, Crommen J, Boulanger B, Chapuzet E, Mercier N, et al. The SFSTP guide on the validation of chromatographic methods for drug bioanalysis: from the Washington Conference to the laboratory. Anal Chim Acta 1999;391:135–48. 52. ISO, 5725–1. Application of statistics. Accuracy (trueness and precision) of measurement methods and results––part 1: general principles and definitions. International Organization for Standardization, Geneva 1994.

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