Ultrasound-Assisted Low-Density Solvent Dispersive Liquid-Liquid Extraction for the Determination of Amphetamines in Biological Samples Using Gas Chromatography-Mass Spectrometry

Liang Meng; Wenwen Zhang; Pinjia Meng; Yuxian Liu

doi:10.4103/2349-5014.164654

ORIGINAL ARTICLE

Year : 2015 | Volume : 1 | Issue : 2 | Page : 114-118

Ultrasound-Assisted Low-Density Solvent Dispersive Liquid-Liquid Extraction for the Determination of Amphetamines in Biological Samples Using Gas Chromatography-Mass Spectrometry

Liang Meng¹, Wenwen Zhang², Pinjia Meng³, Yuxian Liu⁴
¹ Department of Forensic Science, Fujian Police College, Fuzhou 350007, PR, China
² Traffic Management Bureau, Beijing Municipal Public Security Bureau, Beijing 100037, PR, China
³ College of Forensic Science, People's Public Security University of , Beijing 100038, China
⁴ Legal Affairs Section, Hunan Provincial Public Security Department, Changsha 410001, China

Date of Web Publication

27-Nov-2015

Correspondence Address:
Liang Meng
Department of Forensic Science, Fujian Police College, 59, Shoushan Road, Cangshan District, Fuzhou 350007
China

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/2349-5014.164654

Abstract

In order to control drug crime effectively, it is necessary to develop selective analytical methods suitable for unambiguous identification and determination of drugs in illicit samples and biological matrices. A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction, (UA-LDS-DLLME) has been applied to the determination of four amphetamines (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyamphetamine) in urine samples by gas chromatography-mass spectrometry. The parameters affecting extraction efficiency have been investigated and optimized. UA-LDS-DLLME used ultrasound energy to assist in the emulsification process without any disperser solvent. Under the optimized conditions, linearity was observed for all analytes in the 0.15–10 μg/mL range with correlation coefficients (R) ranging from 0.9886 to 0.9894. The recoveries of 75.6–91.4% with relative standard deviations of 2.5–4.0% were obtained. The limits of detection (S/N = 3) were estimated to be in the 5–10 ng/mL range. The UA-LDS-DLLME technique had the advantages of shorter extraction time, suitability for simultaneous pretreatments of batches of samples, and the higher extraction efficiency. It was successfully applied to the analysis of amphetamines in real human urine samples.

Keywords: Amphetamines, biological samples, dispersive liquid-liquid microextraction, gas chromatography-mass spectrometry, ultrasonication

How to cite this article:
Meng L, Zhang W, Meng P, Liu Y. Ultrasound-Assisted Low-Density Solvent Dispersive Liquid-Liquid Extraction for the Determination of Amphetamines in Biological Samples Using Gas Chromatography-Mass Spectrometry. J Forensic Sci Med 2015;1:114-8

How to cite this URL:
Meng L, Zhang W, Meng P, Liu Y. Ultrasound-Assisted Low-Density Solvent Dispersive Liquid-Liquid Extraction for the Determination of Amphetamines in Biological Samples Using Gas Chromatography-Mass Spectrometry. J Forensic Sci Med [serial online] 2015 [cited 2022 May 20];1:114-8. Available from: https://www.jfsmonline.com/text.asp?2015/1/2/114/164654

Introduction

Amphetamines are potent synthetic central nervous system stimulants of the phenethylamine class that is used in the treatment of attention deficit hyperactivity disorder and narcolepsy. Taking amphetamines generate a feeling of excitement and vitality, and amphetamines are lesser addictive than other drugs. Moreover, ephedrine is easily obtained, and amphetamines are easy to synthesize. Thus, the abuse of this kind of drug has become severe during the last several decades.^[1] In order to control drug crime effectively, it is necessary to develop selective analytical methods suitable for unambiguous identification and determination of drugs in illicit samples and biological matrices. This has traditionally been carried out using gas chromatography (GC),^[2],[3] high-performance liquid chromatography (HPLC),^[4] capillary electrophoresis,^[5],[6],[7] and other methods.

In law enforcement, drug inspection in biological samples is becoming more routine. However, a biological sample matrix is complex, containing trace levels of the target analyte. Therefore, it is essential to use an effective extraction and purification technology for the analysis of trace amounts of analytes in biological samples. Recently, a miniaturized modification of the traditional extraction methods termed dispersive liquid-liquid microextraction (DLLME),^[8] has been developed. DLLME is based on extraction of cloudy solutions, such as cloud point extraction and many different forms have been developed. Ultrasound-assisted low-density solvent DLLME (UA-LDS-DLLME)^[9] uses ultrasound energy to assist in the emulsification process without any disperser solvent. This prevents extraction solvent loss and reduces organic solvent consumption significantly, which improves the extraction efficiency. Moreover, extraction solvents that are lesser dense than water are easily collected after de-emulsification. The outstanding advantages of DLLME are its simplicity, rapidity, and inexpensiveness,^[8] and DLLME has been widely used in environmental, food, fragrance, flavor, forensic, pharmaceutical, and biological analyses.^[8],[10]

In the present work, UA-LDS-DLLME was developed for the determination of four amphetamines (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyamphetamine) in urine samples by GC-mass spectrometry (GC-MS). The different parameters affecting the extraction process such as extraction solvent, solvent volume, pH of sample, extraction time, and ionic strength were studied and optimized in detail. The advantages and disadvantages of this method are also discussed. The recommended method was successfully employed to determine trace levels of target analytes in human urine samples.

Experimental

Reagents and materials

Methamphetamine hydrochloride (MA), amphetamine sulfate (AM), 3,4-methylenedioxymethamphetamine hydrochloride (MDMA), and 3,4-methylenedioxyamphetamine hydrochloride (MDA) standards were purchased from the Institute of Forensic Science (99%, Ministry of Public Security, Beijing, China). The analytical grade (AR) toluene, benzene, o-xylene, cyclohexanone, ethyl acetate, butyl acetate, octyl acetate, n-hexane, and cyclohexane were purchased from Sinopharm (Beijing, China) and redistilled in a glass distillation system to remove trace impurities. All other AR reagents used for experiments were purchased from Sinopharm. All standard solutions were diluted with double distilled water and stored in the refrigerator at 4°C prior to use.

The blank urine samples were obtained from volunteers at our college (n = 10), which were confirmed drug-free. A mixed stock solution containing MA, AM, MDMA, and MDA at 1000 μg/mL was prepared in HPLC-grade methanol. A series of standard samples was prepared by mixing appropriate amounts of the stock solution with blank urine in 10 mL volumetric flasks. The mimic samples were prepared daily. All standard solutions were stored at 4°C.

Instrumentation

The analyses were performed using an Agilent 6890A gas chromatograph coupled to a single quadrupole 5973 GC\MS instrument (CA, USA). The separation of the extracted compounds was carried out on a J and W DB-5MS capillary column (30 m × 0.32 mm ID and 0.25 μm film thickness, CA, USA). The column was initially maintained for 2 min at 100°C; subsequently, the temperature was increased to 300°C at a rate of 20°C/min and held for 10 min. The total run time was 22 min. Helium (99.99%) was used as a carrier gas with a constant flow rate of 1.2 mL/min. The injection was performed in the splitless mode at 260°C. The injection volume was 1 μL. The electron ionization (EI, 70 eV) source temperature was 230°C, and the temperature of the quadrupole mass analyzer was 150°C. The solvent delay time was 3.5 min. The instrument was operated in the scan mode for qualitative analysis and selected ion monitoring (SIM) mode for quantitative analysis. The retention times and m/z ratios of characteristic ions in the mass spectra were selected for quantification (AM: 44, 65, and 91 in a time period of 3.5–4.5 min; MA: 58, 91, and 148 in a time period of 4.5–6.5 min; MDA: 44, 135, and 179 in a time period of 6.5–7.2 min; MDMA: 58, 77, and 135 in a time period longer than 7.2 min).

Extraction procedure

A 1.0 mL urine sample was placed in a 2 mL centrifuge tube, and the pH was adjusted to 0.1 mL of ammonia. Then, 100 μL of toluene as the extractant was added to the sample solution. The mixture was sonicated vigorously in an ultrasonic bath (Kunshan, Shanghai) for 3 min with occasional manual shaking to form a cloudy suspension, facilitating the mass transfer of target analytes into the extractant. Subsequently, the tube was centrifuged for 3 min at 10000 rpm. Finally, the upper layer consisting of the low-density extractant was withdrawn and injected into the GC-MS instrument for analysis.

Results and Discussion

Optimization of ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction

In order to obtain the best extraction performance, different parameters affecting the extraction process such as the type and volume of extraction solvent, pH of the sample solution, extraction time, and ionic strength were studied and optimized using the mimic urine samples spiked with amphetamine. Each extraction condition was repeated 5 times within 1-day.

Selection of the extraction solvent

During the ultrasonication process, the extraction solvent is in intense contact with the sample solution, resulting in its dispersion throughout the aqueous phase while an emulsion is formed without any disperser solvent; the analyte is then extracted into the organic phase. Toluene, benzene, o-xylene, cyclohexanone, ethyl acetate, butyl acetate, octyl acetate, n-hexane, and cyclohexane were used as extraction solvents to analyze the effect of the solvent on extraction efficiency. As shown in [Figure 1], toluene, o-xylene, ethyl acetate, and butyl acetate showed superior extraction efficiencies. However, when o-xylene was the extraction solvent, the chromatographic peak shapes of MDA and MDMA became abnormal. Butyl acetate also showed good extraction efficiency for the impurities in biological samples. Therefore, toluene was selected as the extraction solvent in subsequent experiments.

Figure 1: Effects of different organic solvents on extraction efficiency. Toluene displayed the best extraction performance. Extraction conditions: Sample solution, 1.0 mL at pH 13.0; ultrasonication time, 3 min; room temperature. Spiked analyte concentration 500 ng/mL

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Effect of the pH of the sample solution

The sample solution must be adjusted to a desired pH at which the analytes are uncharged; thus, the uncharged molecular forms of the analytes could be extracted into the extraction solvent effectively. The effect of sample pH was tested in the 10.0–14.0 pH range. All analytes are alkaline compounds and have pKa values above 7.0.^[11] The extraction efficiency may have increased with increasing sample pH. However, as shown in [Figure 2], the peak areas reached a maximum at pH 13.0 and then decreased slightly. This may be because basic interferents in biological samples were coextracted at high pH by the extraction solvent and competed with the target analytes. Therefore, a pH of 13.0 was selected for the sample solutions.

Figure 2: Effect of sample pH on extraction efficiency. The best extraction efficiency was obtained at pH 13.0. Extraction conditions: Sample solution, 1.0 mL; extraction solvent, toluene (100 μL); ultrasonication time, 3 min; room temperature. Spiked analyte concentration 500 ng/mL

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Effect of the volume of the extraction solvent

The effects of using 25, 50, 100, 150, and 250 μL of the extraction solvent on the extraction efficiency were investigated. [Figure 3] shows the extraction efficiency versus the volume of extraction solvent. It is clear that as the volume of toluene increased, the relative peak areas of the analytes increased initially, reached a maximum at 100 μL, and then decreased. This was probably because loss of extraction solvent may have occurred when the volume of extraction solvent was lower, and therefore, the extraction solvent could not be dispersed effectively in the sample solution, leading to the low extraction efficiency. When the extraction solvent was increased to a certain volume, the extraction efficiency reached a maximum. Subsequently, the concentration of analytes in the extraction solvent would decrease as the volume of extraction solvent increased further. Thus, 100 μL of toluene was selected as the optimal volume of extraction solvent.

Figure 3: Effect of extraction solvent volume on extraction efficiency. A 100 μL of toluene was selected as the optimal volume of extraction solvent. Extraction conditions: Sample solution, 1.0 mL at pH 13.0; extraction solvent, toluene; ultrasonication time, 3 min; room temperature. Spiked analyte concentration 500 ng/mL

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Effect ofultrasonicationtime

The effect of ultrasonication times ranging from 1 to 10 min on the extraction efficiency was examined. Ultrasonication resulted in a significant increase in the extraction efficiency because ultrasound energy improves the dispersion of the extraction solvent in the aqueous solution and results in an excellent cloudy solution.^[12] However, at the beginning of the ultrasonication process, the emulsification was incomplete. Occasional manual shaking improved to enable the formation of a cloudy suspension effectively without any disperser solvent, and increased the extraction efficiency and lowered the ultrasonic extraction time. The results showed that the highest efficiency was obtained after sonication for 3 min. The reason may be that if the ultrasonic extraction time was too short, the contact between the extraction solvent and the sample solution would be insufficient, leading to low extraction efficiency. On the other hand, if the ultrasonic extraction time was too long, thermal effects could lead to the volatilization of the extraction solvent and the loss of analytes.

Effect of ionic strength

For DLLME, it can be seen that the best extraction efficiencies for all of the analytes were obtained without the addition of NaCl. In view of mass transfer in the DLLME procedure is not the primary influence, the possible increase in the distribution factors was compensated or even canceled by the decrease in the phase ratio.^[13] Thus, NaCl was not added in all subsequent experiments.

Evaluation of the dispersive liquid-liquid microextraction method

Typical chromatograms obtained for the spiked biological samples after DLLME extraction are shown in [Figure 4]. Linearity, limit of detection (LOD), and repeatability were investigated under the optimized conditions using the biological samples spiked with analytes. Each set of extraction data was obtained five times within 1-day. The performance of the developed procedure is summarized in [Table 1]. The calibration curves were linear for the concentrations of all analytes in the 0.15–10 µg/mL range with correlation coefficients (r) ranging from 0.9886 to 0.9894. The LOD (S/N = 3) was estimated to be in the 5–10 ng/mL range. Such detection sensitivity suggests high potential for monitoring amphetamines in biological samples. In the analyses of the spiked samples, recoveries of 75.6–91.4% with relative standard deviations (RSDs) of 2.5–4.0% were obtained.

Figure 4: Typical chromatograms obtained for the spiked urine samples after dispersive liquid-liquid microextraction. (a: Scan mode (I), b: Selected ion monitoring mode (II)). Extraction conditions: Sample solution, 1.0 mL at pH 13.0; extraction solvent, toluene (100 μL); ultrasonication time, 3 min; room temperature. Spiked analyte concentration 500 ng/mL

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Table 1: Analytical performance data for amphetamines by the DLLME method

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Analysis of real biological samples

The proposed UA-LDS-DLLME procedure described above was applied to the determination of drugs of abuse in real human urine samples. [Figure 5] depicts typical chromatograms (SIM mode) of real samples obtained after DLLME. The extraction was repeated 3 times within 1-day. Methamphetamine and amphetamine were found in the samples with concentrations of 243 ng/mL and 52 ng/mL, respectively.

Figure 5: Typical chromatograms (selected ion monitoring mode) of real urine samples after dispersive liquid-liquid microextraction. Extraction conditions: Sample solution, 1.0 mL at pH 13.0; extraction solvent, toluene (100 μL); ultrasonication time, 3 min; room temperature

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Conclusion

The UA-LDS-DLLME method was very simple, rapid, and convenient, consumed microliter amounts of solvent and was successfully applied to the determination of multiple drugs of abuse in human urine samples. The UA-LDS-DLLME technique had the advantages of shorter extraction time and suitability for multiple simultaneous sample pretreatments. In the analyses of the spiked samples, recoveries of 75.6–91.4% with RSDs of 2.5–4.0% were obtained. The LOD (S/N = 3) was estimated to be in the 5–10 ng/mL range. The experimental results revealed that the UA-LDS-DLLME technique provided good analytical performances for biological samples and has the potential to be a powerful tool for the analysis of drugs of abuse in forensic investigations.

Acknowledgements

Financial support from the Technology Project of Education Department in Fujian: (JA14336) is gratefully acknowledged.

Financial support and sponsorship

The work was supported by Technology Project of Education Department in Fujian: A (JA14336).

Conflicts of interest

There are no conflicts of interest.

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