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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 4  |  Page : 181-190

A novel analytical method of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in fluids of drug addicts using liquid-liquid extraction-gas chromatographic/nitrogen-phosphorous detection


1 Institute of Forensic Science, Ministry of Public Security, Beijing, 100088, China
2 Collaborative Innovation Center of Judicial Civilization; Key Laboratory of Evidence Science, University of Political Science and Law, Ministry of Education, Beijing, 100088, China

Date of Web Publication11-Jan-2018

Correspondence Address:
Prof. Hongxia Hao
Collaborative Innovation Center of Judicial Civilization
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jfsm.jfsm_53_17

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  Abstract 

In accordance with the research specifications and guidelines in China, we developed a novel experimental method to detect new piperazine-type drugs, such as 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine. In this study, a new pretreatment method and gas chromatography (GC)/nitrogen-phosphorus detector detection technique were used to characterize these two kinds of drugs in urine and blood samples. For the purpose of isolation of these trace drugs from the samples, liquid-liquid extraction/solid-phase extraction was modified and validated for this specific study. The pretreatment method presented in this paper has many advantages, such as high recovery rate, high extraction efficiency, high detection sensitivity, low limit of detection, and simple operation. The GC/NPD instrument is popular in most laboratories because it can meet the routine requirements of forensic science. All these aspects make this combination of sample pretreatment and GC/NPD technique the most suitable choice for drug detection in biological samples.

Keywords: 1-(3-trifluoromethylphenyl) piperazine, 1-(3-chlorophenyl) piperazine, biological fluids, forensic drug, gas chromatography/nitrogen-phosphorus detector, piperazine derivatives, toxicology tests


How to cite this article:
Chang J, Hao B, Du J, Zhou H, Hao H. A novel analytical method of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in fluids of drug addicts using liquid-liquid extraction-gas chromatographic/nitrogen-phosphorous detection. J Forensic Sci Med 2017;3:181-90

How to cite this URL:
Chang J, Hao B, Du J, Zhou H, Hao H. A novel analytical method of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in fluids of drug addicts using liquid-liquid extraction-gas chromatographic/nitrogen-phosphorous detection. J Forensic Sci Med [serial online] 2017 [cited 2018 Jul 18];3:181-90. Available from: http://www.jfsmonline.com/text.asp?2017/3/4/181/222892


  Introduction Top


Piperazine is general name of a kind of psychotropic drugs, and it is also a kind of pharmaceutical intermediates. It was used for child antiparasitic in clinical trials in 1950.[1] In recent years went on in the market, the piperazine drugs mainly included two broad headings, one kind was benzylpiperazine drugs, such as 1-benzylpiperazine (BZP), one kind was phenylpiperazine drugs, such as 1-(3-trifluoromethylphenyl) piperazine (TFMPP) and 1-(3-chlorophenyl) piperazine (mCPP). The chemical structures and 3D single crystal structures are shown in [Figure 1] and [Figure 2]. The comments followed Hongkong (China) reported that the TFMPP which is derived from anthelmintics could make anyone hallucinate. Meanwhile, many countries such as New Zealand, America, and Japan have all reported the appearance of piperazine drug abuse because there is no one new framework of law to control the development of the new-type drugs.[2] They have been regulated in more and more countries, and BZP was in control list of first-generation psychotropics, but TFMPP and mCPP are still not controlled.
Figure 1: The chemical structure (a) and three-dimensional single crystal structure (b) for 1-(3-trifluoromethylphenyl) piperazine

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Figure 2: The chemical structure (a) and three-dimensional single crystal structure (b) for 1-(3-chlorophenyl) piperazine

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Before analysis of the drug-abuse in biological fluids, one step is generally required: extraction and cleanup. If large impurities in biological samples (such as fat, hemocyte, protein, and so on) are not treated, they will cause some noisy data and the ion suppression phenomenon.[3] The most common methods of pretreatment are liquid-liquid extraction (LLE), solid-phase extraction (SPE),[4],[5],[6] protein precipitation,[7] solid-phase microextraction, and so on. On the other hand, the detection methods of TFMPP and mCPP are reported such as high-pressure liquid chromatography (HPLC)/V,[8],[9] HPLC/fluorescence detection,[7] LC/MS,[10] capillary zone electrophoresis (CZE)/ultraviolet (UV),[11] gas chromatography (GC)/MS,[12] LC/MS/MS,[13] and so on. However, capillary zone electrophoresis has complex operations, and the accuracy of results cannot meet the requirement for forensic science. The sensitivity and reproducibility of UV detector are not good enough to detect TFMPP and mCPP in biological fluids.

Because very few research studies have focused their investigation on the efficacy of various detection methods of piperazine drugs in China, it is essential that the forensic departments in China receive a complete overhaul. With an innovative strategy and better funding, the forensic departments in China can develop systematic, efficient, and convenient methods for the purpose of public security. For this purpose, the Chinese officials need to take into account the inspection methods, the actual situation of China, the laboratory department of public security that works in conjunction with judiciary and identifies various addicts and offenders. Thus, they would subsequently provide an insight into how a strong theoretical basis can be developed for conceptualizing and implementing a reliable method in forensic analysis; the novel method would have to be used extensively for the identification of domestic TFMPP, mCPP; the results of the aforementioned laboratory analyses would be used to comprehensively investigate and solve drug crime cases. In addition, the laboratory analyses obtained with the novel method would also serve as a reliable basis for domestic and foreign research studies focusing their investigating on new drugs, such as piperazine derivatives depending on the previous jobs.[14]


  Materials and Methods Top


Gas chromatographic method can be used for detection of most drug abuse.[15],[16] The selection of appropriate detector is very important after the drug abuse is separated through a GC. Nitrogen-phosphorus detector (NPD) is a kind of mass detectors for detection of nitrogen and phosphorus from pesticides and food. It can apply for microanalysis (medicine and pesticide, biochemistry, food inspection, etc.) with high selectivity and sensitivity and reduces interferences by substances. Benzylpiperazine has been detected from the hallucinogen “A2” by NPD.[1] With this method, nitrogen and phosphorus can be selectively detected with a sensitivity that is 104 times greater than that for carbon.

Instruments and material

Instruments

Agilent 7890A GC/nitrogen-phosphorous detector (Agilent technologies Co., Ltd); Thermo Scientific Biofuge Primo R High-speed centrifuge (Sorvall Inc.); Millipore Simplicity UPW SYSTEM (Millipore Corporation); EYELA HsOsc (Tokyo Rikakikai Co., Ltd); Cole-Parmer Ultrasonator (Cole-Parmer Corporation); Agilent Autosamper vials (Agilent technologies Co., Ltd); GILSON Pipette (10-5000 μL, Gilson Corporation); DSY-V Pressure Blowing Concentrator (Beijing Dongfang Jinghuayuan Analytical Instrument Co., Ltd); SUPELCO (Shanghai XiYou Analytical Instrument Co., Ltd); Disposable Sterile Syringe.

Reagents and standards

TFMPP (purity 99.0%, J and K scientific Co., Ltd); mCPP (purity 99.0%, J and K scientific Co., Ltd); high purity water (Millipore simplicity UPW System); methanol (TEDIA, Fisher Scientific Co., Ltd, No.: LOT122697); ethyl acetate (AR, Sinopharm Chemical Reagent Co., Ltd [SCRC], No.:10009492); trichloromethane (AR, SCRC, No.: 10006892); acetone (AR, SCRC, No.: 10000492); diethyl ether (AR, SCRC, No.: 10009392); isopropanol (AR, Beijing chemical reagent Co., Ltd); cyclohexane (AR, Beijing chemical reagent Co., Ltd); Benzene (AR, SCRC); Oasis® SCX, Oasis® mixed-mode cationic exchange (MCX), Oasis® polymeric cation exchange (PCX), Oasis® hydrophilic-lipophilic balance sorbent (HLB) (3 mL, Waters Corporation); Bond Elute certify (3 mL, Varian Corporation).

Biosamples

Blank urine samples (from the healthy volunteers who have no history of drug abuse); blank whole blood (from Fuxin Hospital).

Preparation of standard reserving solution and standard working solution

10.0 mg TFMPP and mCPP were taken into two 10 mL volumetric flasks, respectively, and then mixed with methanol. Prepared a standard reserving solution with 1.00 mg/mL concentration, and it was kept under a seal and frozen condition.

The TFMPP and mCPP standard reserving solutions were carefully diluted with methanol to form standard working solution at following concentrations: 100, 10, 1 μg/mL, 100, and 10 ng/mL. These standard working solutions were kept under seal and frozen condition.

The mixed standard working solution was made with a concentration of 10 μg/mL using TFMPP and mCPP standard reserving solution, and they were kept under seal and frozen condition.

Gas chromatography/nitrogen-phosphorus detector experimental instrument analysis

  • 7890 GC/NPD, Chemstation 3.1 Workstation (Agilent technologies Co., Ltd)
  • Chromatographic column: DB-5MS (30 m × 0.25 mm × 0.25 μm)
  • Autosampler: 7683B Series Injector
  • Column temperature (programmed heating up): Keeping 200°C for 3 min, raising to 230°C at speed of 3°C/min and keeping 1 min, raising to 260°C at speed of 10°C/min and keeping 1 min
  • Carrier gas: High purity nitrogen gas, at a speed of 15 mL/min
  • Injection temperature: 260°C
  • Injection mode: Split flow, split ratio is 15:1
  • Detector temperatures: 320°C.


The chromatogram of GC/NPD based on the above design methods are shown in [Figure 3].
Figure 3: Chromatogram of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine standard reserving solution using gas chromatography/nitrogen–phosphorus detector

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Sample pretreatment

0.1 mL TFMPP-mCPP mixed standard working solution (10 μg/mL) was added into 0.9 mL blank urine/blood and mixed together. The new working solution M (1 μg/mL TFMPP and 1 μg/mL mCPP) was obtained and let stand for 2 h.

Liquid-liquid extraction

Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine

The pH value of sample (1 ml from section sample pretreatment) was adjusted to 10.0, and it was extracted using 3 mL ethyl acetate. Centrifuge for 15 min at a speed of 8000r/min after 10 min oscillation. The collected supernatant should be dried under nitrogen at room temperature. It was diluted with methanol to 100 μL and moved it to sample vial (Sample N) for inspection pending. Repeat the above steps to obtain five Sample N. The gas chromatogram of TFMPP-mCPP in urine by LLE-GC/NPD is shown in [Figure 4].
Figure 4: The gas chromatogram of 1-(3-trifluoromethylphenyl)piperazine and 1-(3-chlorophenyl)piperazine in urine by liquid-liquid extraction–gas chromatography/nitrogen–phosphorus detector

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Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood

The pH value of Sample M (1 mL from section sample pretreatment) was adjusted to 11.0, and it was extracted using 3 mL chloroform/isopropanol (4/1, V/V). Centrifuge for 15 min at a speed of 8000r/min after 10 min oscillation. The collected supernatant should be dried under nitrogen at room temperature. It was diluted with methanol to 100 μL and moved it to sample vial (sample N) for inspection pending. Repeat the above steps to obtain five Sample N. The gas chromatogram of TFMPP-mCPP in blood by LLE-GC/NPD is shown in [Figure 5].
Figure 5: The gas chromatogram of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood by liquid-liquid extraction–gas chromatography/nitrogen–phosphorus detector

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Solid-phase extraction

Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine

The pH value of sample M (1 mL from section sample pretreatment) was adjusted using phosphate buffer to 6.0, and it was extracted using 3 mL chloroform/isopropanol (4/1, V/V). Centrifuge for 15 min at a speed of 8000r/min after 10 min oscillation. 1 mL methanol, 1 mL deionized water, and 1 mL phosphate buffer (pH = 6.0) were used in turn to active Oasis® MCX SPE cartridge. Extracts of treated sample M were obtained, and 3 mL water and 3 mL methanol were used to leach it, then SPE cartridge was centrifuged to remove the leachate. Finally, the eluent was collected after treating it using 5 mL 5% ammonium methanol. The eluent should be dried under nitrogen at room temperature. It was diluted with methanol to 100 μL, and it was moved to sample vial (Sample N) for inspection pending. Repeat the above steps to obtain five Sample N. The gas chromatogram of TFMPP-mCPP in blood by LLE-GC/NPD is shown in [Figure 6].
Figure 6: The gas chromatogram of urine samples by mixed mode cationic exchange solid-phase extraction–gas chromatography/nitrogen–phosphorus detector

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Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood

Four milliliter ionized water was added into 1 mL sample M (from section sample pretreatment) and shook well. Centrifuge for 15 min at a speed of 8000 r/min after 10 min oscillation; then, the supernatant was removed. 1 mL methanol and 1 mL deionized water were used in turn to active Oasis® HLB cartridge. Extracts of treated Sample M were obtained, and 1 mL water and 1 mL methanol were used to leach it, then HLB cartridge was centrifuged to remove the leachate.

Finally, the eluent was collected after treating it using 5 mL methanol. The eluent should be dried under nitrogen at room temperature. It was diluted with methanol to 100 μL, and it was moved to sample vial (Sample N) for inspection pending. Repeat the above steps to obtain five Sample N. The gas chromatogram of TFMPP-mCPP in blood by LLE-GC/NPD is shown in [Figure 7].
Figure 7: The gas chromatogram of blood samples by hydrophilic-lipophilic balance sorbent–gas chromatography/nitrogen–phosphorus detector

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Specificity tests

One milliliter blank urine and blood, respectively, were treated depending on section LLE, and TFMPP and mCPP were analyzed by section Gas chromatography/nitrogen-phosphorus detector experimental instrument analysis, then the chromatograms of TFMPP and mCPP were obtained from blank urine sample [Figure 8], blank blood sample [Figure 9], and TFMPP-mCPP standard mixture [Figure 3]. The results show that the impurities in urine cannot interfere the detection of TFMPP and mCPP by GC/NPD, so this method has exclusive property in identifying experiments.
Figure 8: The chromatograms of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine from blank urine sample by gas chromatography/nitrogen–phosphorus detector

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Figure 9: The chromatograms of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine from blank blood sample by gas chromatography/nitrogen–phosphorus detector

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  Results Top


The method and selection optimization of the analytical conditions

Standard curve

A mixture of TFMPP and mCPP was dissolved and diluted to fixed volume (0.1, 0.5, 1, 5, 10, 25, 50, 100 μg/mL) with methanol for GC/NPD analysis depending on section gas chromatography/nitrogen-phosphorus detector experimental instrument analysis. A homogenous solution shall be sampled and injected for 3 times, and standard curve equation is obtained in [Table 1]; working curves are in [Figure 10] and [Figure 11].
Table 1: The linear equation for detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine standard using gas chromatography/nitrogen-phosphorus detector

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Figure 10: Standard curve of 1-(3-trifluoromethylphenyl) piperazine

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Figure 11: Standard curve of 1-(3-chlorophenyl) piperazine

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Quantitation limit and detection limit

Quantitation limit and detection limit for TFMPP and mCPP by GC/NPD are shown in [Table 2].
Table 2: Quantitative line and detection limit of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine using gas chromatography/nitrogen-phosphorus detector

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Relative standard deviation

Took high concentration (10 μg/mL) and low concentration (1 μg/mL) of TFMPP and mCPP standard solutions to calculate the intraday precision by injecting them five times a day. Took the same solutions to calculate the interday precision by injecting them once a day, lasting for 5 days [Table 3]. The results of investigation show that the method has a low standard deviation and high precision.
Table 3: Relative standard deviation (n=5)

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Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine

Standard curve, quantitation limit, and detection limit

In this experimental analysis, 0.9 ml aliquot of five samples was taken from the blank urine. Then, mixed standard solutions of 0.1 mL were prepared using different concentrations of TFMPP-mCPP mixed solution; the various concentrations of TFMPP and mCPP were as follows: 1μg/mL, 3μg/mL, 5μg/mL, 8μg/mL, and 20μg/mL in the five samples, respectively. Analyzed the quantitation limit and detection limit according to section gas chromatography/nitrogen-phosphorus detector experimental instrument analysis, and the solutions with same concentration were injected three times. Experiments show that this method has good linear relationship for TFMPP and mCPP in a certain concentration range. The linear equation is in [Table 4] and quantitation limit and detection limit are shown in [Figure 12] and [Figure 13].
Table 4: The linear equation, limit of quantification, and limit of detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine

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Figure 12: Standard curve of 1-(3-trifluoromethylphenyl) piperazine in urine

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Figure 13: Standard curve of 1-(3-chlorophenyl) piperazine in urine

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Recovery rate and relative standard deviation

Took two blank urine samples (0.9 mL), and different levels of TFMPP-mCPP working standard solutions (0.1 mL) are added in, respectively, to prepare the solutions with different concentration of TFMPP and mCPP (1, 10 μg/mL), preprocessed them depending on section LLE and analyzed the quantitation limit and detection limit according to section gas chromatography/nitrogen-phosphorus detector experimental instrument analysis. These samples should be injected five times a day and last 5 days. [Table 5] means the recovery and relative standard deviation (RSD) of TFMPP and mCPP using GC/NPD. The results of investigation show that the method has a low standard deviation and high precision.
Table 5: The recovery and relative standard deviation of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine using gas chromatography/nitrogen-phosphorus detector

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Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood

Standard curve, quantitation limit, and detection limit

Took five blank blood samples (0.9 mL), and different levels of TFMPP-mCPP working standard solutions (0.1 mL) are added in, respectively, to prepare the solutions with different concentration of TFMPP and mCPP (3, 5, 10, 15, 20 μg/mL) and then made a standard curve. The quantitation limit and detection limit were analyzed according to section gas chromatography/nitrogen-phosphorus detector experimental instrument analysis, and the solutions with same concentration were injected three times. Experiments show that this method has good linear relationship for TFMPP and mCPP in a certain concentration range. The linear equation is in [Table 6] and quantitation limit and detection limit are shown in [Figure 14] and [Figure 15].
Table 6: The linear equation, limit of quantification, and limit of detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood

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Figure 14: Standard curve of 1-(3-trifluoromethylphenyl) piperazine in blood

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Figure 15: Standard curve of 1-(3-chlorophenyl) piperazine in blood

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Recovery rate and relative standard deviation

Took two blank blood samples (0.9 mL), and different levels of TFMPP-mCPP working standard solutions (0.1 mL) are added in, respectively, to prepare the solutions with different concentration of TFMPP and mCPP (1, 10 μg/mL), preprocessed them depending on section LLE, and the quantitation limit and detection Limit were analyzed according to section gas chromatography/nitrogen-phosphorus detector experimental instrument analysis. These samples should be injected five times a day and last 5 days. [Table 7] means the recovery and RSD of TFMPP and mCPP using GC/NPD. The results of investigation show that the method has a low standard deviation and high precision.
Table 7: The recovery and relative standard deviation of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine using gas chromatography/nitrogen-phosphorus detector

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  Discussion Top


The selection of gas chromatography

The choice of stationary phases is a very important condition for GC. Choose the GC which have stationary phase with similar polarity depending on the polarity of the sample that can help the sample to show peaks in appropriate retention time and reduce interference by substances. Another condition is column length of GC. The longer the column is, the longer the sample flow through the column, and the retention time is longer. The column efficiency will be reduced and it will consume long time if the column length is too long. Besides that, the diameter of chromatographic column and film thickness also affects the column efficiency.

Agilent DB-5MS is a nonpolar column utilizing 5% phenyl-arylene and 95% dimethylpolysiloxane as the stationary phase, and it had a good retention and separating effect. Due to TFMPP and mCPP are weak polar compounds containing phenyl and halogen, DB-5MS column was selected to establish GC method. It can be shown from the research the best condition was as follows: column length 30 m, inner diameter 0.25 mm, and liquid film thickness 0.25 μm. This method is of high sensitivity and separating effect.

Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine

The optimization of liquid-liquid extraction method

The comparison of extraction rate in different extractants

TFMPP and mCPP are soluble in organic solvents such as acetone, ethanol, diethyl ether, benzene, hexamethylene, trichloromethane, and so on. This paper examines the extraction effects of ethyl acetate, hexamethylene, ethyl acetate/hexamethylene (1/1, V/V), ethyl acetate/hexamethylene (2/1, V/V), ethyl acetate/hexamethylene (4/1, V/V), trichloromethane, etc., The experiment was conducted in accordance with “section LLE,” and the results are shown in [Figure 16]. It indicates that the extraction rate and the recovery are low by hexamethylene; however, it is high by trichloromethane, but there are too many impure peaks. Ethyl acetate is the best extraction agent.
Figure 16: The comparative recoveries of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in urine using various extraction agents

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From the chromatogram of blank urine, it can be seen that the impurity content is low in urine, and ethyl acetate was the most effective in the extraction. Therefore, benzene, ethyl ether, and other solvents are not considered because of their significant toxicity and uncontrollable parallelism. Ethyl acetate was selected in this method.

The impacts of solution pH value on extraction rate

Compounds TFMPP and mCPP present weak alkalinity, so in this paper, the extraction efficiency under different pH value is studied, such as pH = 3.0, pH = 5.0, pH = 8.0, pH = 9.0, pH = 10.0, pH = 11.0, and pH = 12.0. The experiment was conducted in accordance with section LLE, and the results are shown in [Figure 17]. The results indicate that the recovery is the best in pH = 10.0; however, the compounds cannot be detected in pH = 3.0 and 5.0. It seems that pH = 10.0 is the best condition.
Figure 17: The recovery rates of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in urine under different pH values

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The comparison of extraction and purification effect of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in urine using solid-phase extraction

This paper inspects the purification effect of SPE cartridge such as Oasis® SCX, Oasis® MCX, Oasis® PCX, Oasis® HLB, Bond Elute certify. Oasis® SCX and Oasis® PCX have a better selectivity of weak anion compounds due to their quaternary ammonium salts. Oasis® MCX containing sulfonic acid ion exchange groups has a better extraction efficiency of alkali compounds. Oasis® HLB contains a kind of reverse adsorbent which is often used to be general adsorbents for acidic, neutral, and alkaline compounds due to its good infiltration performance. The adsorbent of Bond Elute Certify is a mixed mode with the properties of ion exchange and nonpolar action, and it can extract alkaline (kation) drugs from urine, adtevak, serum, blood, and other biological matrix.

Extraction experimental processes of Oasis® SCX, Oasis® MCX, Oasis® PCX, and Bond Elute certify were based on section solid-phase extraction. The main process of Oasis® HLB is as follows: added 4 mL deionized water to 1 mL treated sample (from 2.3 Sample pretreatment) and centrifuged for 15 min at a speed of 8000 r/min after 10 min oscillation. Used 1 mL methanol and 1 mL deionized water in turn to active HLB SPE cartridge. Took extracts of treated sample, and used 1 mL water and 1 mL methanol to leach it, then SPE cartridge was centrifuged to remove the leachate. Finally, collected the eluent after treated it using 5 mL methyl alcohol. The eluent should be dried under nitrogen at room temperature. Diluted with methanol to 100 μL and moved it to sample vial for detection. From [Figure 18], it can be seen that all the eluents from these solid-phase extraction cartridges were colorless, and for spikes, recovery rate, and parallelism, Oasis® MCX has the best extraction effect of TFMPP and mCPP in urine.
Figure 18: The comparison of recovery rates of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in urine using solid-phase extraction

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Extraction and detection of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood

The optimization of liquid-liquid extraction method

The comparison of extraction rate in different extractants

TFMPP and mCPP are soluble in organic solvents such as acetone, ethanol, diethyl ether, benzene, hexamethylene, trichloromethane, and so on. Due to many impurities in blood such as proteins and fats, ethyl acetate, the extraction effects of hexamethylene, ethyl acetate/hexamethylene (2/1, V/V), diethyl ether, benzene, trichloromethane, trichloromethane/isopropanol (4/1, V/V) were tested. The experiment was conducted in accordance with “section LLE”, and the results are shown in [Figure 19]. The extracted supernatant appears yellow as it is fat-soluble, and it has serious interference in chromatograms. The mixed solution of trichloromethane/isopropanol (4/1, V/V) was selected as extractant because alkaloid dissolves easily in trichloromethane and the protein in blood sample is precipitated by isopropanol.
Figure 19: The extraction rate of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in blood using different extractants

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The impacts of solution pH value on extraction rate

Compounds TFMPP and mCPP present weak alkalinity, so in this paper, the extraction efficiency under different pH value is studied, such as pH 3, pH 5, pH 8, pH 9, pH 10, pH 11, and pH 12. The experiment was conducted in accordance with section LLE, and the results are shown in [Figure 20]. The results indicate that the recovery is the best in pH = 11.0; however, the compounds cannot be detected in pH = 3.0 and 5.0. It seems that pH = 11.0 is the best condition.
Figure 20: The recovery rates of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in blood under different pH values

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The comparison of extraction and purification effect of 1-(3-trifluoromethylphenyl) piperazine and 1-(3-chlorophenyl) piperazine in blood using solid-phase extraction

This paper inspects the purification effect of SPE cartridge such as Oasis® SCX, Oasis® MCX, Oasis® PCX, Oasis® HLB, Bond Elute certify. The main process of Oasis® SCX, Oasis® MCX, Oasis® PCX, and Bond Elute certify is as follows: added 4 mL borate solution to 1 mL treated sample (from section sample pretreatment) to adjust pH = 6.0 and centrifuged for 15 min at a speed of 8000 r/min after 10 min oscillation. Used 1 mL water and 1 mL methanol, 1 mL deionized water, and 1 mL borate solution (pH = 6.0) to activate it in turn and used 3 mL water and 3 mL methanol in turn to leach it, then SPE cartridge was centrifuged to remove the leachate. Finally, collected the eluent after treated it using 5 mL 5% ammonium methanol. The eluent should be dried under nitrogen at room temperature. Diluted with methanol to 100 μL and moved it to sample vial for detection.

From [Figure 21], it can be seen that the eluent from PCX was brown, that from Bond Elute certify and SCX are clear, that from MCX is light yellow, but all of these have low recovery rates. For good spikes, recovery rate, and parallelism, HLB has the best extraction effect of TFMPP and mCPP in blood.
Figure 21: The recovery rates of 1.(3.trifluoromethylphenyl) piperazine and 1.(3.chlorophenyl) piperazine in blood using solid-phase extraction

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  Conclusion Top


This paper is on the research of the prior treatment methods (LLE, solid-phase extraction) and GC/NPD detection for TFMPP and mCPP in biological fluids (urine and blood) at the blankness of the test and regulation at present. This method possesses the practical value for its advantages of simple operation, high recovery, high sensitivity, and low detection limit, while the instruments are already available in laboratory. It is a simple, reproducible, and specific method to detect the piperazine-drugs in forensic science.

Acknowledgment

We are grateful to Fundamental research fund of Institute of Forensic Science, Ministry of Public Security(The detection of new drugs analysis in biological samples. No.2012JB005), Academician Foundation of the Ministry of Public Security of the People's Republic of China (No. 2011-23214203, 23215243, 23317015), Beijing Municipal Education Commission University Science and Technology Park Construction Project (2011-08111603) and Program for Young Innovative Research Team in China University of Political Science and Law (2014CXTD04,16CXTD05) the opening project fund of Shanghai Key Laboratory of Criminal Scene Evidence(2011-23417049) for their financial supports.

Financial support and sponsorship

This study was supported by Fundamental research fund of Institute of Forensic Science, Ministry of Public Security(The detection of new drugs analysis in biological samples. No.2012JB005), Academician Foundation of the Ministry of Public Security of the People's Republic of China (No. 2011-23214203, 23215243, 23317015), Beijing Municipal Education Commission University Science and Technology Park Construction Project (2011-08111603) and Program for Young Innovative Research Team in China University of Political Science and Law (2014CXTD04,16CXTD05) the opening project fund of Shanghai Key Laboratory of Criminal Scene Evidence(2011-23417049).

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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