Simultaneous Determination of 16 Kinds of Synthetic Cathinones in Human Urine Using a Magnetic Nanoparticle Solid-Phase Extraction Combined with Gas Chromatography-Mass Spectrometry

Abstract

A specific and sensitive approach using magnetic nanoparticle solid-phase extraction combined with gas chromatography-mass spectrometry (GC-MS) was carried out and applied in the simultaneous determination of 16 kinds of synthetic cathinones in human urine. The functionalized extraction material (Fe₃O₄/NH₂-MWCNTs) was synthesized and factors affecting the extraction efficiency were all tested. Under the optimized conditions of magnetic nanoparticle solid-phase extraction, the determination of synthetic cathinones in human urine was carried out with GC-MS. Good linear relationships of 16 kinds of synthetic cathinones were obtained in the range of 0.005–5.00 μg/mL with the correlation coefficients (r) ranging from 0.9901 to 0.9979, the limits of detection were in the range between 0.005 and 0.01 μg/mL, and the limits of quantitation were between 0.01 and 0.02 μg/mL. Furthermore, the average intra-day precisions were below 8.90%, the average inter-day precisions were less than 9.52%, and the average recoveries were between 87.03% and 99.13%, respectively. The results show the advantages of the approach in the determination of trace synthetic cathinones in complex matrixes, such as environmentally friendly, fast detection, high efficiency and sensitivity. The practical application indicated that this method could provide scientific basis for the determination of drugs of abuse in forensic laboratories.

1. Introduction

In recent years, drug abuse has not only affected human nature and caused numerous crimes, but also has caused a serious problem throughout the world. Nowadays, as the “third-generation drugs”, new psychoactive substances (NPSs) have become popular around the world instead of traditional drugs and psychoactive substances. As the main categories in NPSs, synthetic cathinones mainly include cathinone, methcathinones, ethcathinones, cathinones containing methylenedioxy group, cathinones containing pyridine ring and others [1], all of them are often sold as “bath salt” or “plant fertilizer” [2]. Synthetic cathinones are sympathetic nerve stimulants, which can directly act on the central nervous system. Due to their weak ability to enter the nerve center through the blood–brain barrier, abusers often intake large doses and continuously use them to obtain the expected excitement, which leads to serious brain damage [3].

The structures of synthetic cathinones are similar to amphetamine, but their effects on the human body are different [4]. Abusers who use by means of oral consumption, intravenous injection or nasal inhalation will initially experience feelings of euphoria and excitement, as well as anxiety and mania, before developing depression and suicidal tendencies, accompanied by side effects such as hallucinations, confusion, loss of appetite, weight loss, headache, dizziness, nausea and vomiting, and general pain [5]. Long-term abuse leads to psychological and physical dependence and eventually addiction, which may lead to a series of physical and psychological damages and even death [6]. While synthetic cathinones are metabolized in the liver, they would are mainly excreted in urine in free or combined form [7]. At present, the analysis methods of synthetic cathinones in different matrices include ultraviolet–visible spectrometry [8], gas Chromatography-Mass spectrometry (GC-MS) [9], high-performance liquid chromatography [10], liquid Chromatography-Mass spectrometry (LC-MS) [11], capillary electrophoresis [12] and electrochemical method [13]; most previous studies have only focused on the identification of a few kinds of synthetic cathinones, paying little attention to the application of novel sample pretreatment techniques [14].

Due to the complicated matrix and the low concentration of targeted analytes, it is difficult to obtain good sensitivity and low detection while the compounds in the complex matrix including urine and blood are directly analyzed; usually, the expected detection concentration in urine should be less than 10 ng/mL. Therefore, the sample preparation step is essential in the determination procedure. Generally, many extraction methods including liquid–liquid extraction [11] and solid-phase extraction [15] have been widely used in the sample preparation of different analytes in various matrices. As a new mode of solid-phase extraction method based on the use of magnetic nanoparticles, magnetic solid-phase extraction (MSPE) has attracted more attention in separation science because of the high extraction efficiency and convenient operation. The magnetic sorbents are directly dispersed in sample solution instead of packing in a cartridge, and then gathered with external magnetic field. This process is time saving, low cost and environmentally friendly [16]. MSPE has been widely applied in environmental pollutant analysis [17] and drug analysis [18], especially magnetic carbon nanoparticles as the adsorbent [19]. However, there are few reports on the application of MSPE in the determination of synthetic cathinones, especially in biological matrix.

In this study, a specific and sensitive method for the analysis of synthetic cathinones using magnetic nanoparticle solid-phase extraction coupled with GC-MS was developed and applied in the simultaneous determination of synthetic cathinones in human urine. The factors which influenced the extraction efficiency were tested, respectively, and the qualitative and quantitative analysis of 16 kinds of synthetic cathinones in human urine were also carried out. The experimental results showed that this method could provide a scientific basis for the determination of drugs of abuse in forensic laboratories and strong evidence for detection and litigation of cases.

2. Materials and Methods

2.1. Chemicals and Reagent

All the standards of 16 kinds of synthetic cathinones, including cathinone, methcathinone (MC), 4-chloroethcathinone (4-CEC), 4-methylpentedrone (4-MPD), n-ethylhexedrone (NEH), 4-chlorobutylcathinone (4-CBC), 3,4-methylenedioxy-n-methylcathinone (MDMC), 4′-methyl-α-pyrrolidinopropiophenone (4-MePPP), 3,4-methylenedioxy-n-ethylcathinone (MDEC), 4-chloro-α-pyrrolidinopropiophenone (4-Cl-α-PPP), 4-fluoro-α-pyrrolidinopentiophenone (4-F-α-PHP), 4-chloro-α-pyrrolidinopentiophenone (4-Cl-α-PVP), 4-chloro-α-pyrrolidinohexanophenone (4-Cl-α-PHP), 3,4-methylenedioxypyrovalerone (MDPV), 3,4-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP), 2-pyrrolidin-1-yl-1-tetralin-6-yl-hexan-1-one (TH-PHP) were purchased from the Institute of Forensic Science of China (Beijing, China) and only used for research purposes; their molecular formula and chemical structures are listed in Table 1. N, N-Dimethylaniline, ethyl acetate, ammonia, Na₂CO₃ and NaHCO₃ were all of analytical grade and purchased from Guoyao Group Chemical Reagent Shenyang Co., Ltd. (Shenyang, China). FeCl₂·4H₂O and FeCl₃·6H₂O were both of analytical grade and purchased from Hengxing Chemical Reagent Tianjin Co., Ltd. (Tianjin, China). Amino modified multi-walled carbon nanotubes (NH₂-MWCNTs) were purchased from Nanjing XFNANO Materials Technology Co., Ltd. (Nanjing, China). Human drug-free urine was obtained from volunteers. The deionized water used in the experiments was purified from a Milli-Q system (Milford, MA, USA).

Table 1. Physical and chemical parameters of 16 kinds of synthetic cathinones.

No	Name	Abbreviate Name	Molecular Formula	Molecular Mass	Chemical Structure	pKa	logP	Boiling Point (°C)
1	Cathinone	-	C9H11NO	149.19		7.97	1.16	255.0
2	Methcathinone	MC	C10H13NO	163.22		7.14	1.39	254.8
3	4-chloroethcathinone	4-CEC	C11H14ClNO	211.69		7.14	2.61	317.9
4	4-methylpentedrone	4-MPD	C13H19NO	205.29		-	2.92	313.0
5	n-ethylhexedrone	NEH	C14H21NO	219.32		-	3.52	322.3
6	4-chlorobutylcathinone	4-CBC	C13H18ClNO	239.74		-	-	-
7	3,4-methylenedioxy-N-methylcathinone	MDMC	C11H13NO3	207.22		-	0.81	350.1
8	4′-methyl-α-pyrrolidinopropiophenone	4-MePPP	C14H19NO	217.31		-	-	-
9	3,4-methylenedioxy-N-ethylcathinone	MDEC	C12H15NO3	221.25		7.75	1.34	362.2
10	4-chloro-α-pyrrolidinopropiophenone	4-Cl-α-PPP	C13H16ClNO	237.73		-	-	-
11	4-fluoro-α-pyrrolidinopentiophenone	4-F-α-PHP;	C16H22FNO	263.35		-	-	-
12	4-chloro-α-pyrrolidinopentiophenone	4-Cl-α-PVP	C15H20ClNO	265.78		-	4.33	380.7
13	4-chloro-α-pyrrolidinohexanophenone	4-Cl-α-PHP	C16H22ClNO	279.81		-	-	-
14	3,4-methylenedioxypyrovalerone	MDPV	C16H21NO3	275.34		8.41	3.06	415.5
15	3,4-methylenedioxy-α-pyrrolidinohexanophenone	MDPHP	C17H23NO3	289.37		-	3.59	427.7
16	2-pyrrolidin-1-yl-1-tetralin-6-yl-hexan-1-one	TH-PHP	C20H29NO	299.45		-	-	-

2.2. Instruments and Equipment

The following instruments and equipment were used in the present study: POLARIS Q gas Chromatography-Mass spectrometer (Thermo Fisher, Austin, TX, USA); KQ-200VDE ultrasonic cleaner (Kun Shan Ultrasonic Instruments Co., Ltd., Suzhou, China); HJ-3 magnetic stirrer (Henan Yuhua Instrument Co., Ltd., Gongyi, China); XW-80A Vortex mixer (Shanghai Huxi Analysis Instrument Factory, Shanghai, China); pH-10/100 acidity meter (Shanghai Lichen Instrument Technology Co., Ltd., Shanghai, China); HITACHI-8100 and HITACHI-7800 electron microscope (Hitachi, Honshu, Japan); EV7 magnetometer (ADE, Milpitas, CA, USA); Nicolette 5DXB FT-IR (Thermo Fisher, Austin, TX, USA); HC-3018 centrifuge (Anhui USTC Zonkia Scientific Instruments Co., Ltd., Hefei, China); BSA 224S-CW electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd., Shanghai, China).

2.3. GC-MS Conditions

HP-5 MS capillary column (30 m × 0.25 mm i.d., 0.25 µm) was employed in GC-MS and the column oven temperature program started at an initial temperature of 80 °C, held for 1 min, and increased to a final temperature of 280 °C at a rate of 20 °C/min, and then held at 280 °C for 10 min. Helium was used as carrier gas for the chromatography and the constant flow rate was 1.0 mL/min. An aliquot of the sample of 1 μL was injected under a split ratio of 10:1. The temperatures of the injector and the transfer line were maintained at 250 °C and 280 °C, respectively. Electron ionization (EI) ion source was utilized in the mass spectrometer, under the energy of 70 eV and the ion source temperature at 250 °C. Acquisition modes were full scan within the range of m/z 40~m/z 400 and selective ion monitor (SIM) using the specific fragments of aiming compounds. Data acquisition and instrument control were performed using Xcalibur software (2.2.42, Thermo Fisher, Austin, TX, USA). The corresponding data analysis was performed with Microsoft Office Excel 2019.

2.4. Synthesis of Magnetic Amino Modified Multi-Walled Carbon Nanotubes

In this work, a chemical co-precipitation method was chosen to prepare magnetic amino modified multi-walled carbon nanotubes according to the reports in the literatures [20]. The process was as follows: FeCl₂·4H₂O (1.23 g) and FeCl₃·6H₂O (3.36 g) were suspended in 200 mL of deionized water and stirred to be dissolved. Then, 0.5 g of amino modified multi-walled carbon nanotubes were added in the solution which was obtained above. Then, these were uniformly mixed and vortexed for 4 min. Subsequently, the solution was adjusted to pH > 9 with ammonia water (5%), and stirred with a magnetic stirrer under a water bath of 80 °C for 30 min. The precipitate was isolated with a strong magnet, and the supernatant was discarded from the precipitate by decantation, and then the nanocomposite was washed several times with deionized water until the solution was neutral. Finally, the composite was dried at 80 °C for 24 h and magnetic carbon nanocomposite materials (Fe₃O₄/NH₂-MWCNTs) were obtained.

2.5. Characterization of Magnetic Amino Modified Multi-Walled Carbon Nanotubes

The magnetic carbon nanocomposite material (Fe₃O₄/NH₂-MWCNTs) was characterized by field emission scanning electron microscopy (FE-SEM), field emission transmission electron microscope (FE-TEM), and FT-IR.

FE-SEM was accomplished on a HITACHI-8100 electron microscope at an accelerating voltage of 1.0 kV, before the materials were dispersed into an appropriate concentration and cast onto a glass sheet at room temperature.

FE-TEM was performed on an HITACHI-7800 electron microscope at an accelerating voltage of 10.0 kV at a magnification of 8000.

FT-IR spectra of magnetic particles were recorded on KBr disk using a Nicolette 5DXB FT-IR in the 400–4000 cm⁻¹ region with a resolution of 4 cm⁻¹. Each spectrum was obtained by averaging 32 consecutive scans.

2.6. Sample Processing

The appropriate amounts of 16 kinds of synthetic cathinones standards and internal standard N, N-dimethylaniline were accurately weighed and dissolved in deionized water to prepare standard stock solutions with a concentration of 1.0 mg/mL, respectively. According to the calculation, standard stock solutions and human drug-free urine were mixed in a predetermined ratio to prepare a urine sample which contains 16 kinds of synthetic cathinones under the concentration required for the experiment.

2.7. Magnetic Solid-Phase Extraction Procedures

In magnetic solid-phase extraction procedures, many factors including the kind of magnetic adsorbents, pH of the solution, adsorption mode and time, species and amounts of eluents, desorption mode and time influence the extraction efficiency. These were optimized via comparing the peak area ratio of MC and the internal standard in human urine obtained with GC-MS under different levels in this article and the satisfactory choice was obtained as follows: firstly, 40 mg Fe₃O₄/NH₂-MWCNTs was added into 5 mL of human urine solution in a centrifuge tube. The mixture was vortexed for 4 min and keep stand for 1 min. Secondly, a strong magnet was deposited at the bottom of the centrifuge tube, and the magnetic nanocomposite was isolated from the suspension. Approximately 2 min later, the suspension was decanted when it became limpid. Finally, 1 mL ethyl acetate was added to the centrifuge tube and ultrasonicated for 30 s. After the suspension was separated from the magnetic nanocomposites with a strong magnet, the suspension was collected and evaporated to 0.1 mL, 1 µL was injected for the analysis of GC-MS. The schematic procedure for MSPE and the analysis of 16 kinds of synthetic cathinones in human urine with GC-MS is shown in Figure 1.

Figure 1. Schematic procedure for MSPE and the analysis of 16 kinds of synthetic cathinones in human urine with GC-MS.

2.8. Liquid–Liquid Extraction and Solid-Phase Extraction Procedures

Liquid–liquid extraction: 5 mL of human urine samples spiked with 5.0 μg/mL of MC was added into a centrifuge tube, 5 μL internal standard solution, 0.5 mL Na₂CO₃–NaHCO₃ buffer and 20 mg NaCl were added into the solution, respectively, and then 3 mL ethyl acetate was added and mixed, the mixture was vortexed for 2 min and centrifuged at 10,000 prm for 3 min, the suspension was collected and directly analyzed via GC-MS.

Solid-phase extraction: a solid-phase extraction column of 500 mg C₁₈ was preconditioned by 2 mL methanol followed by 2 mL deionized water. Aqueous human urine samples spiked with 5.0 μg/mL of MC was added with 5 μL internal standard solution and 0.5 mL Na₂CO₃–NaHCO₃ buffer, and then mixing, and the mixture was passed through the solid-phase extraction column and washed by 2 mL deionized water. The analyte was then eluted with 3 mL methanol and directly collected for the analysis with GC-MS.

3. Results

3.1. Selection of Magnetic Carbon Nanotube

According to references [21], different kinds of carbon nanotubes could be applied as MSPE materials, including single-walled carbon nanotubes, multi-walled carbon nanotubes, hydroxylated carbon nanotubes, amino modified carbon nanotubes and carboxylic carbon nanotubes. According to the structural characteristics of synthetic cathinones, all of the carbon nanotubes mentioned above have potential in the pretreatment of synthetic cathinones in complex matrix, so MSPE based on the different adsorbents of 5 types carbon nanotubes was performed during the pretreatment of MC in human urine. As shown in Figure 2, the compared results show that the adsorption performance and extraction efficiency of amino modified carbon nanotubes were better than others. Due to that, magnetic amino modified carbon nanotubes were selected for the extraction and enrichment of synthetic cathinones in human urine, and the synthesis and characterization of this kind of MSPE material were discussed in detail.

Figure 2. The comparison of absorption performance on 5 kinds of absorptions.

3.2. Characterization of Fe₃O₄/NH₂-MWCNTs

In this study, the structure of the resulting magnetic nanocomposites was first characterized by electron microscope. Scanning electron micrographs (SEMs) and transmission electron microscopy (TEM) results were obtained as shown in Figure 3. The SEM image (Figure 3a) revealed that the aminated magnetic carbon nanotube material was composed of uniform particles with the size distribution in the range of 10–20 nm, and Fe₃O₄ particles attached to the surface of the carbon nanotubes to make them magnetic (Figure 3b). Fourier transform infrared spectrum (FT-IR) of Fe₃O₄/NH₂-MWCNTs is shown in Figure 3c. There are three strong adsorption bands located at 566 cm⁻¹, and it is ascribed to the Fe–O–Fe stretching vibration of Fe₃O₄. The peaks appeared at 3416, 2359 and 1626 cm⁻¹, which belonged to the stretching vibration of N–H, the stretching vibration of CH₂ and the stretching vibration of the C–C bond, respectively. As shown in Figure 3d, the magnetization hysteresis loop of the material was S-like, which indicated that the composite was superparamagnetic. The saturation magnetization value of Fe₃O₄/NH₂-MWCNTs was calculated to be 35 emu/g, which was sufficient to ensure the easy and quick separation of the compounds from solutions.

Figure 3. SEM image (a), TEM image (b), FT-IR spectrum (c) and magnetization curve (d) of Fe₃O₄/NH₂-MWCNTs.

3.3. Optimization of MSPE Conditions

The magnetic nanocomposites were applied in the MSPE of 16 kinds of synthetic cathinones in human urine. Because a series of experimental parameters may influence the purified, extracted and enriched analytes, on the other hand, the chemical structures and physicochemical properties of these 16 kinds of compounds were similar, and MC was chosen as the analyte and 5 μg/mL MC human urine solution was applied in the optimization of MSPE conditions to improve the extraction performance of MSPE. Several parameters including adsorbent amounts, pH of solution, adsorption mode and time, species and amounts of eluents, desorption mode and time were all tested in detail based on the peak area ratio of MC with the internal standard.

In the MSPE procedures, the amounts of magnetic adsorbents influenced the extraction efficiency. In this study, the amounts of magnetic adsorbent were tested at six levels. As shown in Figure 4a, the peak area ratio particularly increased when the amount of magnetic adsorbent increased from 5 to 60 mg, and then no significant difference appeared while the amount of extractant reached 40 mg, which was enough for the extraction. Ultimately, 40 mg was chosen as the optimized adsorbent amount in this work.

Figure 4. Effects of the amount of extractant (a), extraction mode (b), adsorption time (c), pH of solution (d), species of eluent (e), amount of eluent (f), desorption mode (g) and time (h) on the extraction efficiency (n = 3).

In order to make the magnetic adsorbent come into sufficient contact with the solution to quickly achieve adsorption equilibrium, three commonly extraction modes including shake, vortex, and ultrasonic were investigated in this work. The results are shown in Figure 4b. It can be seen that the extraction efficiency of three modes was significantly different, and vortex was the best choice for the MSPE extraction in this article.

Simultaneously, the adsorption time was another important factor affecting the adsorption course and extraction efficiency. The effects of vortex time from 1 to 5 min were tested and the results are shown in Figure 4c. It can be seen that the peak area ratio continuously increased with an extending time from 1 to 4 min, and remained constant with the further prolongation of extraction time. As a result, the adsorption time was designed as 4 min.

The pH of solution is also a key factor which can influence the extraction efficiency in the MSPE procedure and determine the present form of the analytes. In this study, it was carried out in the range of 7–13 via adding different volumes of Na₂CO₃–NaHCO₃ buffer into human urine samples, and the comparing analysis results are shown in Figure 4d. The results showed that the peak area ratio increased with pH in the range of 7–10, and decreased in the range 11–13. This phenomenon was determined with the pKa and closely related to the chemical properties of the analytes as shown in Table 1. As a result, the pH value of the samples was adjusted to 10 with Na₂CO₃–NaHCO₃ buffer in all subsequent experiments.

The desorption course also plays an important role in the MSPE procedure. During the desorption course, the species and amount of eluent, desorption method and time were required to be optimized. The desorption capacity of the elution solvent to the target product determines the extraction efficiency. In this study, solvents, namely toluene, cyclohexane, dichloromethane, chloroform, ethyl acetate, n-hexane and acetone were selected for the elution solvents to test the desorption capacity and the results are shown in Figure 4e. These six solvents all have desorption capacity in the MSPE procedure, and the desorption performance of ethyl acetate was the best, which was employed as the elution solvent.

The amount of eluent was another factor which needed to be optimized in the extraction process. A series of experiments with different amounts of ethyl acetate were performed, and the results are shown in Figure 4f. The result demonstrated that a significant increase in the peak area ratio occurred as the amount of eluent increased from 0.5 mL to 1 mL, and then there were no changes while more eluents were used. As a result, the amount of eluent was chosen to be 1 mL in the experiments.

In order to obtain an excellent extraction efficient, the desorption method, a crucial factor influencing the extraction efficiency, should also be optimized. Three common desorption modes including ultrasonic, vortex and shaking were investigated in this work, and the results are shown in Figure 4g. Based on the comparing results, ultrasonic was chosen as the desorption method for MSPE extraction.

The desorption time was another parameter needed to be explored. A variety of desorption times ranging from 10 to 60 s were studied and the results are shown in Figure 4h. As shown in the figure, 30 s was enough to elute the analyte absorbed by adsorbents. Upon prolonging to 60 s, no obvious increase occurred. Additionally, continuous ultrasonication for a long time may cause the structural destruction of the magnetic adsorbents. As a result, 30 s was accepted as the desorption time.

In a word, during the whole MSPE procedures, absorbents need 40 mg; vortex adsorption needs 4 min; ethyl acetate must be chosen to be the eluent, ultrasonication is to be performed for desorption and the desorption time must be 30 s. Those parameters were selected as the optimal conditions of MSPE procedures of synthetic cathinones in human urine for further work, while the pH value was adjusted to 10 with Na₂CO₃–NaHCO₃ buffer.

3.4. Comparison with Liquid–Liquid Extraction and Solid-Phase Extraction

In order to evaluate the extraction efficiency of the MSPE technique developed in this work, a comparison with two kinds of widely applied pretreatment methods in forensic science including liquid–liquid extraction and solid-phase extraction were accomplished. Liquid–liquid extraction and solid-phase extraction experiments were performed by using 3.0 mL of ethyl acetate and a C₁₈ solid-phase sample-preparation column to extract 5.0 mL human urine samples spiked with 5.0 μg/mL of MC, respectively. The results are shown in Table 2, which revealed that the results of these three methods were closer while the extraction ratio obtained with MSPE was a little higher than the solid-phase extraction and a little bit lower than liquid–liquid extraction. The experimental costs and the solvent usages of the MSPE approach mentioned in this work were lower than those of the liquid–liquid extraction and solid-phase extraction.

Table 2. Comparison of MSPE with liquid–liquid extraction and solid-phase extraction.

Method	Detection Result (μg/mL)	Extraction Ratio (%)
MSPE	4.78	95.6
Liquid–liquid extraction	4.85	97.0
Solid-phase extraction	4.54	90.8

3.5. Method Validations

Under the optimized MSPE conditions, combined with gas Chromatography-Mass spectrometry, a determination approach was developed and applied in the analysis of 16 kinds of synthetic cathinones in human urine. Method validations including GC-MS experimental conditions, retention times and characteristic ions, linearity equations, coefficients, linearity ranges, the limits of detection (LODs) and the limits of quantitation (LOQs), precisions and recoveries of the analytes were all studied.

According to the relevant literature reports [22], the HP-5 MS column was chosen for the analysis of 16 kinds of synthetic cathinones by GC-MS in this work, and the parameters of temperature programming were optimized by comparing the retention times and resolutions of the target substances. Under the optimized GC-MS experimental conditions, the total ions chromatogram of 16 kinds of synthetic cathinones were obtained as shown in Figure 5, and the retention times of these analytes are listed in Table 3. As shown in Figure 5 and Table 3, 16 kinds of synthetic cathinones could be separated well and reach the requirements of qualitative and quantitative analysis. Furthermore, the mass spectra and characteristic ions of qualitative and quantitative analysis were obtained and shown in Table 3, respectively.

Figure 5. Total ions chromatogram of 16 kinds of synthetic cathinones with GC-MS. Peaks: (1) Cathinone; (2) MC; (3) 4-CEC; (4) 4-MPD; (5) NEH; (6) 4-CBC; (7) MDMC; (8) 4-MePPP; (9) MDEC; (10) 4-Cl-α-PPP; (11) 4-F-α-PHP; (12) 4-Cl-α-PVP; (13) 4-Cl-α-PHP; (14) MDPV; (15) MDPHP; (16) TH-PHP.

Table 3. Retention times and mass to charge of characteristic ions for 16 kinds of synthetic cathinones.

No	Name	Retention Time (min)	Qualitative Ions (m/z)	Quantitative Ion (m/z)
1	Cathinone	5.91	44, 77, 51	44
2	MC	6.09	58, 77, 51	58
3	4-CEC	7.60	72, 44, 111	72
4	4-MPD	7.85	86, 44, 91	86
5	NEH	7.97	114, 77, 58	114
6	4-CBC	8.43	100, 58, 111	100
7	MDMC	8.50	58, 149, 121	58
8	4-MePPP	8.73	98, 56, 119	98
9	MDEC	8.80	72, 44, 149	72
10	4-Cl-α-PPP	9.02	98, 56, 111	98
11	4-F-α-PHP	9.13	140, 84, 123	140
12	4-Cl-α-PVP	9.73	126, 84, 98	126
13	4-Cl-α-PHP	10.18	140, 84, 98	140
14	MDPV	10.78	126, 84, 149	126
15	MDPHP	11.18	140, 84, 98	140
16	TH-PHP	11.26	112, 70, 91	112

In order to investigate the parameters of the qualitative and quantitative analysis of 16 kinds of synthetic cathinones with MSPE combined with GC-MS, 16 kinds of synthetic cathinones were spiked into a blank sample of human urine at nine concentration levels of 0, 0.01, 0.02, 0.05, 0.10, 0.50, 1.00, 2.00 and 5.00 µg/mL; the internal standard curve method was chosen as quantitative analysis method. The magnetic solid-phase extraction operation was carried out according to the optimal extraction conditions and analyzed with GC-MS. The obtained calibration curves of 16 kinds of synthetic cathinones all exhibited good linearity in the corresponding ranges, the linearity equations, coefficients and linearity ranges, and all of them are shown in Table 4. The LODs and LOQs were evaluated considering the accuracy, precision and the calculation based on the signal-to-noise (S/N) value of S/N = 3 and 10, respectively (Table 5).

Table 4. Linearity equations, coefficients, linearity ranges, LODs and LOQs of 16 kinds of synthetic cathinones in human urine.

No	Name	Linearity Equations	Coefficient (R2)	Linearity Ranges (µg/mL)	LOD (µg/mL)	LOQ (µg/mL)
1	Cathinone	Y = 0.077X + 0.070	0.9901	0.02–5.00	0.01	0.02
2	MC	Y = 0.067X + 0.052	0.9957	0.02–5.00	0.01	0.02
3	4-CEC	Y = 0.76X + 0.16	0.992	0.02–5.00	0.01	0.02
4	4-MPD	Y = 1.58X + 0.44	0.9949	0.01–5.00	0.005	0.01
5	NEH	Y = 5.30X + 1.32	0.9901	0.005–5.00	0.002	0.005
6	4-CBC	Y = 2.14X + 0.33	0.9922	0.01–5.00	0.005	0.01
7	MDMC	Y = 0.088X + 0.039	0.9906	0.02–5.00	0.01	0.02
8	4-MePPP	Y = 7.24X + 0.95	0.9932	0.01–5.00	0.005	0.01
9	MDEC	Y = 0.76X + 0.054	0.9945	0.02–5.00	0.01	0.02
10	4-Cl-α-PPP	Y = 6.45X + 1.12	0.9938	0.01–5.00	0.005	0.01
11	4-F-α-PHP	Y = 19.44X + 0.72	0.9973	0.01–5.00	0.005	0.01
12	4-Cl-α-PVP	Y = 1.56X + 0.49	0.9939	0.01–5.00	0.005	0.01
13	4-Cl-α-PHP	Y = 14.82X + 1.40	0.9914	0.005–5.00	0.002	0.005
14	MDPV	Y = 11.30X + 2.41	0.9911	0.005–5.00	0.002	0.005
15	MDPHP	Y = 6.95X + 0.57	0.9979	0.01–5.00	0.005	0.01
16	TH-PHP	Y = 9.64X + 0.64	0.9974	0.01–5.00	0.005	0.01

Table 5. Precisions and recoveries of 16 kinds of synthetic cathinones in human urine.

No	Name	Spiked Concentration (µg/mL)	Precision (RSD, %)		Accuracy (Bias)		Recovery (%)
			Intra-Day	Inter-Day	Intra-Day	Inter-Day
1	Cathinone	0.05	6.02	7.30	0.64	0.53	89.73
		0.20	6.74	7.58	0.42	0.73	87.03
		5.00	3.96	5.61	0.83	0.68	95.14
2	MC	0.05	4.21	6.03	0.91	0.29	90.00
		0.20	3.28	5.91	0.32	0.65	93.75
		5.00	3.03	5.37	0.28	0.69	96.60
3	4-CEC	0.05	3.06	7.68	0.31	0.51	90.97
		0.20	3.33	5.75	0.21	0.85	92.88
		5.00	1.62	3.84	0.32	0.52	98.34
4	4-MPD	0.05	2.85	4.57	0.86	0.27	92.23
		0.20	3.36	4.37	0.51	0.36	91.64
		5.00	2.50	3.56	0.53	0.47	97.43
5	NEH	0.05	6.05	7.86	0.91	0.62	91.08
		0.20	5.47	9.52	0.25	0.29	96.23
		5.00	5.78	7.09	0.66	0.72	93.56
6	4-CBC	0.05	3.42	5.49	0.35	0.52	94.23
		0.20	0.62	1.70	0.54	0.45	98.88
		5.00	2.43	3.14	0.36	0.43	97.43
7	MDMC	0.05	4.78	6.49	0.86	0.59	88.26
		0.20	4.87	6.18	0.66	0.42	90.63
		5.00	0.81	2.28	0.74	0.27	99.13
8	4-MePPP	0.05	5.10	6.23	0.57	0.44	91.88
		0.20	1.91	4.83	0.46	0.86	96.90
		5.00	2.91	5.35	0.22	0.31	96.40
9	MDEC	0.05	8.16	9.77	0.81	0.58	90.45
		0.20	6.50	6.85	0.18	0.25	90.56
		5.00	8.90	9.08	0.28	0.48	90.09
10	4-Cl-α-PPP	0.05	2.59	7.80	0.61	0.88	88.13
		0.20	1.34	4.60	0.63	0.58	91.02
		5.00	2.62	5.32	0.35	0.76	94.78
11	4-F-α-PHP	0.05	1.65	3.20	0.78	0.45	97.17
		0.20	4.80	6.41	0.81	0.49	94.03
		5.00	1.47	2.35	0.42	0.42	98.55
12	4-Cl-α-PVP	0.05	4.13	5.46	0.67	0.23	88.36
		0.20	3.91	4.10	0.19	0.13	89.58
		5.00	4.04	5.14	0.57	0.31	95.87
13	4-Cl-α-PHP	0.05	4.82	5.17	0.19	0.45	96.58
		0.20	2.75	4.52	0.89	0.36	95.84
		5.00	4.19	5.20	0.53	0.36	95.54
14	MDPV	0.05	5.06	8.25	0.59	0.29	89.34
		0.20	4.44	6.42	0.68	0.34	91.21
		5.00	5.38	6.63	0.63	0.25	94.08
15	MDPHP	0.05	5.69	6.72	0.52	0.28	93.25
		0.20	3.47	4.64	0.59	0.62	94.94
		5.00	5.91	7.75	0.87	0.38	93.62
16	TH-PHP	0.05	3.52	8.53	0.51	0.35	92.10
		0.20	3.68	6.60	0.45	0.26	94.92
		5.00	5.26	7.18	0.66	0.44	94.94

Accuracy and precision experiments were accomplished with different concentrations set to low, medium and high levels as 0.05, 0.20 and 5.00 µg/mL for 16 kinds of synthetic cathinones. The relative standard deviation (RSD) and bias were determined by analyzing spiked samples’ RSDs and the biases of the intra-day precision (n = 3) and the inter-day precision (three consecutive days, n = 9) as shown in Table 5. The RSDs were less than 8.90% and 9.52%, and the biases were less than 0.91 and 0.88, respectively. The results indicate that the method has good precision and repeatability in the quantitative analysis of synthetic cathinones in human urine using MSPE combined with GC-MS.

The recovery experiments were also performed, and the recovery rate varied from 87.03% to 99.13% at the three concentration levels as shown in Table 5. The results not only demonstrate that this approach provides good accuracy in the analysis of synthetic cathinones in human urine, but also show the potential advantages of the proposed method based on combining the MSPE technique with GC-MS in the identification of abused drugs.

3.6. Application in Real Case

In order to verify the accuracy of the proposed method, suspicious colorless liquid and urine from drug abusers, with the support of the Institute of Forensic Science of Criminal Investigation of the Police University of China, were analyzed with the proposed method. MC was detected in suspicious colorless liquid, and MC and cathinone were identified in urine. The obtained results testified that the proposed method could be applied in the testing of drug abuse.

4. Conclusions

In this work, a specific and sensitive method using MSPE in combination with GC-MS for the analysis of synthetic cathinones in human urine was developed. The MSPE technique is not only a cheap, convenient and environmentally friendly sample pretreatment technique, but it also reduces the exposure danger to the toxic solvents used in conventional extraction procedures such as liquid–liquid extraction. The experimental results and application in relevant case evidence inspection revealed its low limit of detection, good linearity and high repeatability and sensitivity. This work provides the scientific basis for case physical evidence identification, as well as provides strong evidence for case detection and litigation based on their higher sensitivity, efficiency and rapidity. In the future, the analysis of metabolites of these new psychoactive substances in human urine or blood may be performed with the same strategy, and the application in the identification of traces of forensic evidence in cases will also provide a tool to combat drug crime.