Dispersive solid-phase extraction for simultaneous determination of four amphetamines drugs in urine using gas chromatography–mass spectrometry

Article abstract of DOI:10.1007/s13738-021-02339-0

A sample preparation method based on modified Quick, Easy, Cheap Effective, Rugged, and Safe (QuEChERS) and GC–MS method was developed and validated for identification and quantification of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in human urine. Several derivatization parameters such as derivatization temperature, time, and volume of derivatizing agent were optimized. dSPE parameters were optimized. Under the optimized conditions, the method was validated with respect to selectivity, linearity, limits of detection and quantification, precision, accuracy, and recoveries. Linearity was obtained in the concentration range of 50–1200?ng/mL for all the drugs. The intra- and inter-day precision and accuracy were acceptable. The RSD values ranging from 0.08 to 3.43% and 0.23% to 3.0% for intra- and inter-day precision were obtained, respectively. Accuracy was within the required limits (± 15% and ± 20% at LOQ) for all analytes at three concentrations studied. Analyte recoveries that ranged from 87.8 to 95% were obtained. These figures resulted within the best recovery percentages as comparing with the previous studies. The method was successfully applied to the routine work of the Sudanese Forensic laboratories to detect and quantify the AP, MA, MDA, and MDMA in urine samples.

Full text of DOI:10.1007/s13738-021-02339-0

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-021-02339-0
ORIGINAL PAPER
Dispersive solid‑phase extraction for simultaneous determination
of four amphetamines drugs in urine using gas chromatography–mass
spectrometry
Mahir Z. Elboraie1 · Abdalla A. Elbashir1,2 · Eltayeb E. M. Eid3 · Hassan Y. Aboul‑Enein4
Received: 24 May 2021 / Accepted: 5 July 2021
© Iranian Chemical Society 2021
Abstract
A sample preparation method based on modifed Quick, Easy, Cheap Efective, Rugged, and Safe (QuEChERS) and GC–MS
method was developed and validated for identifcation and quantifcation of amphetamine (AP), methamphetamine (MA),
3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in human urine. Several
derivatization parameters such as derivatization temperature, time, and volume of derivatizing agent were optimized. dSPE
parameters were optimized. Under the optimized conditions, the method was validated with respect to selectivity, linear-
ity, limits of detection and quantifcation, precision, accuracy, and recoveries. Linearity was obtained in the concentration
range of 50–1200 ng/mL for all the drugs. The intra- and inter-day precision and accuracy were acceptable. The RSD values
ranging from 0.08 to 3.43% and 0.23% to 3.0% for intra- and inter-day precision were obtained, respectively. Accuracy was
within the required limits ( 15% and 20% at LOQ) for all analytes at three concentrations studied. Analyte recoveries that
ranged from 87.8 to 95% were obtained. These fgures resulted within the best recovery percentages as comparing with the
previous studies. The method was successfully applied to the routine work of the Sudanese Forensic laboratories to detect
and quantify the AP, MA, MDA, and MDMA in urine samples.
Keywords Drug abuse · QuEChERS · GC–MS · MSTFA · Human urine · Dispersive solid-phase extraction
Introduction
nervous system, acting on neurons in the brain to create feel-
ings of pleasure and well-being [2–4], they include a number
of therapeutic agents and drug abused (illicit drugs) [2, 3, 5].
The abuse of amphetamines is continuing to be listed among
the most commonly attending recreational drugs worldwide
especially among adolescent and youths for performing hard
activities like truck drivers [5–8], they are used most fre-
quently for doping during competitions [2, 9]. A new genera-
tion of synthetic amphetamines with modifed ring system,
which derived from illegal origin have been consumed as
a new drug of abused especially by young people [6, 10].
Since these drugs are synthesized in a clandestine labora-
tory, there would be no knowledge of actual composition
of the product and no documentation of ensuring safety, so
the abuse of these designer drugs poses some risk to the
abusers [6, 10].
The secondly most widely used drugs of abuse (DOA) are
amphetamines, opiates, and cocaine; they are second to
cannabis in most countries [1]. Amphetamine (AP) and its
related compounds are powerful stimulants of the central
* Abdalla A. Elbashir
aaeahmed@kuf.edu.sa
* Hassan Y. Aboul-Enein
haboulenein@yahoo.com
1
Department of Chemistry, Faculty of Science, University
of Khartoum, 11115 Khartoum, Sudan
2
Department of Chemistry College of Science, King Faisal
University, P.O.Box 400, Al-Ahsa 31982, Saudi Arabia
3
Department of Pharmaceutical Chemistry
A number of analytical methods for the determination
of amphetamines in biological samples have been reported
in the literature. These include gas chromatography–mass
spectrometry (GC–MS) [11–14], liquid chromatogra-
phy–mass spectrometry (LC–MS) [14–16], and capillary
and Pharmacognosy, Unaizah College of Pharmacy, Qassim
University, Buraydah, Saudi Arabia
4
Division of Pharmaceutical and Drug Industries Research
Division, Pharmaceutical and Medicinal Chemistry
Department, National Research Centre, Dokki, Cairo, Egypt
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
electrophoresis–mass spectrometry (CE-MS) [17]. Prior to
the GC analysis, derivatization is necessary to improve the
amphetamine analysis [18, 19], that is because the analysis
of free amphetamines by GC–MS is associated with difcul-
ties and results in poor peak resolution [18, 19].
unit 23, Markham ON L3R 4G7, Canada. The chemicals
methanol (MeOH), acetonitrile (ACN), sodium hydrox-
ide (NaOH), anhydrous magnesium sulfate (MgSO4), and
sodium chloride (NaCl) were bought from SDFCL, 315–317,
TV Industrial Estate, 248, Worli Road, Mumbai 30, India;
all chemicals were of analytical grade. Dispersive solid-
phase extraction (dSPE) tubes part number CUMC182CT
(2.0-mL dSPE tube) were supplied by UCT (Bristol, USA).
The standard drugs were mixed and diluted in methanol to
prepare stock solution at 10 µg/mL of (AP, MA, MDA, and
MDMA) and stored at −20 °C before use.
For obtaining successfully analysis of large numbers of
samples, forensic laboratories require rapid analysis meth-
ods, which cannot be achieved by using the conventional
sample preparation technique that is generally employed in
forensic laboratories for urine drug testing.
To perform a reliable and accurate chromatographic
analysis, sample preparation is important step to isolate the
target drug [20, 21]. Various sample preparation procedures
have been employed for the extraction and pre-concentration
of amphetamines from biological fuids, such as liquid–liq-
uid extraction (LLE) [22–24], liquid-phase microextraction
(LPME) [25, 26], dispersive liquid–liquid microextraction
(DLLME) [27, 28], solid-phase extraction (SPE) [29–32],
and solid-phase microextraction (SPME) [33, 34]. LLE and
SPE are widely used; however, LLE needs large amounts of
high-purity solvents that are expensive and toxic, whereas
SPE is time consuming and expensive [35].
Samples
Urine samples received to the Forensic Laboratories for
criminal analysis and blank urines collected from laboratory
staf member were stored at −20 °C before analysis. 2.0 mL
of the blank urine was spiked to 200 µL of the calibration
work standard drugs (each of the eight concentration levels)
and placed in a 10-mL test tube.
Sample preparation
QuEChERS (quick, easy, cheep, efective, rugged, and
safe) approach was commonly used for the analysis of pes-
ticide residues in fruits and vegetables [36], and acrylamide
in food [37]. This cleanup procedure has lately demonstrated
its efectiveness for the determination of drugs or toxic com-
pounds in human biofuids [1, 2] using LC–MS/MS and
CE-MS. This technique is simple, rapid, and therefore com-
patible for forensic analysis requirements.
The extraction of the drugs was performed using acetonitrile
organic solvent and purifed using Q-sep dispersive solid-
phase extraction (dSPE) tubes.
This method involves two steps—Step one (Extraction):
2.0 mL of acetonitrile (containing 200 mg of NaOH-appar-
ent pH 12.4) was added to the 2.0 mL of blank urine sam-
ple that is placed in the 10-mL test tube and spiked with
the 200 µL of the drugs. 400 mg of anhydrous MgSO4 and
100 mg of anhydrous NaCl were added to the test tube.
The test tube was vortexed and centrifuged at 2000 rpm for
10 min. Step two (cleanup): 1.0 mL of the supernatant was
transferred into a dSPE tube containing 150 mg of MgSO4,
and 50 mg of C18, the mixture was vortexed and centri-
fuged at 3000 rpm for 5 min, the supernatant was fltered
through 4.5-µm flter paper into a 5 mL vial, the vial content
was totally dried under steam of nitrogen and derivatized
using MSTFA reagent at optimum derivatization conditions
(80 °C/25 min) that are obtained step-by-step in this work.
Then, the extract and derivatized drugs underwent GC–MS
analysis. The recovery test was carried out by spiking the
drugs after the fltration step, at three concentration levels
with the calibration curve.
The present paper describes the development and valida-
tion of a GC–MS method based on QuEChERS extraction
for the simultaneous determination of the (AP, MA, MDA,
and MDMA) in human urine for forensic purposes. Basic
QuEChERS was modifed by optimizing the quantity of the
salts in order to obtain the high recovery of studied drugs.
Experimental
Chemicals and materials
Analytical standard of amphetamine (AP), methampheta-
mine (MA), 3,4-methylenedioxyamphetamine (MDA), and
3,4-methylenedioxymethamphetamine (MDMA) was pur-
chased from Lipomed Serviced to Health, Lipomed AG
Fabrikmattenweg 44,144 Arlesheim, Switzerland. The entire
standards were 1.0 mg/mL solution in methanol. The silylat-
ing reagent N-methyl(trimethyl-silyl) trifouroacetamide
[MSTFA] was obtained from Sigma-Aldrich, 3050 Spruce
street, St. Louis MO 63,103, USA. The kits for screening
immunoassay analysis (Rapid Response Multi-Drug Test
Panel) were delivered from BTNX Inc. 570 Hood Road,
Optimization of QuEChERS and dSPE process
In the QuEChERS and dSPE optimization process, the
amount of NaCl and anhydrous MgSO4 were studied. The
amount of NaCl and anhydrous MgSO4 was studied by
frst fxing the MgSO4 at 400 mg and varying the amounts
of NaCl added (50–200 mg). This method involves two
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Journal of the Iranian Chemical Society
steps—Step one (Extraction): 2.0 mL of acetonitrile (con-
taining 200 mg of NaOH-apparent pH 12.4) was added to
the 2.0 mL of blank urine sample that is placed in the four
10-mL test tubes and spiked with the 200 vµL of the drugs
followed by the addition of 400 mg of anhydrous MgSO4.
The frst, second, third, and fourth tubes were added with
50,100, 150, and 200 mg of anhydrous NaCl, respectively,
and the mixture was vortexed and centrifuged at 2000 rpm
for 10 min. The effect of varying amounts of MgSO4
(350–600 mg) was studied at 100 mg NaCl (best amount).
Similar procedure as for NaCl was followed.
within 20% bias and below 20% relative standard devia-
tion (RSD), respectively, where the limit of detection (LOD)
is defned as the minimum detection ability for the system,
or as the amount of analyte in the sample below which the
spectrometer system cannot detect the analyte [22 and 41].
The LOD and LOQ were calculated based on the approach
of “Standard Deviation of the Response and the Slope”
whereby the LOD is expressed as LOD = 3.3 s/S and the
LOQ is expressed as LOQ=10 s/S, where s is the standard
deviation of the linear regression line and S is the slope of
the curve [38, 39]. The recovery was determined by com-
paring the response from the drug spiked to the blank urine
samples and then extracted, with the response from already
extracted blank urine samples and then spiked to the same
analyte at the equivalent concentration level. The recovery
was expressed as percentage. Analytical recovery between
80 and 120% was considered acceptable [4, 38].
Optimization of the derivatization procedure
A systematic optimization of the derivatization experimental
procedure was deemed necessary to achieve higher yields
and enhance method sensitivity. Parameters including deri-
vatization time, temperature, and volume of the derivatizing
agent were step-by-step studied and optimized.
GC–MS analysis
Method validation
GC–MS analysis was performed using Shimadzu
GC–MS-TQ8040, coupled to an AOC-5000 Autosampler.
Analytic chromatographic separation was achieved using
a capillary column (RTx-5MS, 30 m–0.25 mm i.d- and
0.25 um thickness). Carrier gas (helium) was maintained at
a total fow rate of 50 mL/min, all injections were in splitless
mode with a column fow rate of 1.4 mL/min, the injection
port temperature was 250 °C. The ion source and interface
temperature were 200, 250 °C, respectively, mass cut time
was 3 min. The mass spectrometer was operated at 70 eV in
the electron impact (EI) mode with the types of ionization
mode (scan ionization mode and selected ion monitoring
mode). In scan ionization mode, the m/z ions used were in
the range of 35 m/z to 400 m/z, where in selected ion moni-
toring (SIM) mode, only specifc ions were used, where one
ion was used as a quantifer, while two other ions were used
as qualifers for the analysis of the target compounds.
The developed method was validated in terms of linearity,
selectivity, accuracies, precisions, limits, and recovery. In
order to obtain these validation data, calibration curves were
constructed based on peak area obtained with the drugs at
eight diferent concentration levels (50, 100, 200, 500, 800,
1000, and 1200 ng/mL) that spiked to blank urine samples
and three analyses were made for each point level. The range
of linearity was detected. Coefcient of determination (r2)
was used to express the linearity of the regression line. Lin-
earity was accepted if r2≥0.98 [5]. Chromatographic selec-
tivity was evaluated by the presence or absence of co-eluting
peaks at the retention times of the analytes [38]. The accu-
racy of the method was evaluated by spiking blank matrix at
three diferent concentration levels (low, 50; medium, 500;
and high, 1200 ng/mL) and through the calculation of the
percentage deviation between the calculated value and the
nominal value [accuracy (%) = (experimental concentra-
tion/theoretical concentration) *100] [5, 38, 39]. Precision
is defned as the degree of agreement between independent
results obtained under specifc circumstances [39]. Precision
data were quantifed by analyzing the peak areas of three
replicates of three concentrations levels within the calibra-
tion curves and calculating the RSD. Within-day precision
and time-diferent intermediate precision were calculated
as the relative standard deviation (RSD)=s/×100% where
RSD values below 15% and at LOQ below 20% were accept-
able for quantitative analysis [5, 39, and 40]. The Limit of
quantitation (LOQ) is defned as the lowest amount of ana-
lyte in a sample which can be quantitatively determined with
suitable precision and accuracy [39], where at this level,
the analytes should have accuracy and precision values
Results and discussion
Optimization of amount of NaCl and anhydrous
MgSO4 added in QuEChERS
It has been reported that the quantity of the salt added afects
recovery of the dSPE extraction [1]. Therefore, the opti-
mum amount of salt combination (MgSO4 and NaCl) added
was investigated by fxing the amount of anhydrous MgSO4
(400 mg) and varying the amounts of NaCl added (50,100,
150, and 200 mg). The optimum peak areas of AP, MA,
MDA, MDMA were obtained by adding 100 mg of NaCl and
were used in subsequent experiments. The efect of vary-
ing the amount of anhydrous MgSO4 salt (350–600 mg) at
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Journal of the Iranian Chemical Society
100 mg of NaCl was then studied. The optimum peak areas
of AP, MA, MDA, MDMA were obtained with 400 mg
anhydrous MgSO4. The role of anhydrous MgSO4 is to
enhance the extraction efciency by drying the extraction
solvent (acetonitrile) to achieve the maximum amount of
analytes extracted. The role of anhydrous NaCl is to enhance
the extraction efciency by separating the two layers com-
pletely, the solvent layer and the aqueous layer (urine) with
no emulsion in between [37].
To study the efect of time on the derivatization efciency,
the temperature was set at 80 °C and diferent derivatization
reaction times (15, 20, 25, and 30 min) were studied. The best
results were obtained at the derivatization reaction time 25 min.
The volume of the derivatizing agent was also studied:
50, 100, 150, and 200 µL MSTFA were added in the resi-
due. Best results were obtained with 50 µL, and thus, this
volume was selected for further experiments. The fnally
selected derivatization parameters were as follows: 50 µL of
MSTFA was added to the evaporated drug and derivatized
for 25 min at 80 °C and then injected at the GC–MS system.
The optimized derivatization parameters were used in all
experimental work in this study.
Optimization of the derivatization procedure
The frst parameter studied was the temperature of derivat-
izing reaction. 100 µL of 500 ng/mL amphetamine was totally
evaporated and dried under steam of nitrogen, and then 100 µL
of MSTFA was added in the residue and the time of heat was
set at 20 min, the derivatization temperature was ranged from
60, 70, 80, and 90 °C. The best result was obtained at 80 °C
and thus it was selected for the following experiment.
Chromatograms for the simultaneous separation of the
amphetamines drugs AP, MA, MDA, and MDEA with and
without derivatization are shown in Fig. 1a and b, respec-
tively. Without derivatization, the peak area values were very
low, because the drug is volatile and some of the underivat-
ized drug is lost by evaporation [13, 14].
Fig. 1 The chromatograms of mixture of standard of AP, MA, MDA, and MDMA a underivatized b derivatized
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Journal of the Iranian Chemical Society
Unlike the standard drugs, the extracted and derivatized
urinal drugs were GC–MS identifed and quantifed using
the selected ion monitoring (SIM), where one peak (the
highest) was used for quantifcation for each extracted drug.
Figures 2 shows the chromatogram of the urinal extracted
derivatized amphetamines drugs using (SIM) mode.
The most relative abundance masses m/z and the reten-
tion times of the derivatized standard amphetamines drugs
are shown in Table 1. Their masses and retention times
were used to identify the corresponding ones that urinary
extracted, using the methodology mentioned above then
derivatized and GC–MS analyzed. The retention times
reported here are shorter than those reported by Jurado et al.,
10.9 min [4], Nishida et al., 11.3 min [3], and by Villamor
et al., 12.8 [6].
MDEA, parameters such as linearity, limit of detection
(LOD), limit of quantification (LOQ), precision (repeat-
ability and reproducibility) (RSD%), and accuracy
(recovery) were evaluated at optimum extraction condi-
tions using 2.0 mL of acetonitrile (containing 200 mg
of NaOH-apparent pH 12.4, 400 mg anhydrous MgSO4,
100 mg NaCl) and optimum derivatization condition
using 50 µL of MSTFA reagent at 80 °C temperature for
25 min.
Selectivity
Eight diferent urine samples obtained from laboratory staf
(drug non-abuser) were tested for selectivity test. There was
no interfering peak at the retention times of the analytes.
Representative chromatograms obtained from blank urine
sample, spiked urine sample, and urine positive sample are
shown in Fig. 3a, b, and c, respectively.
Method validation
To validate the performance of the proposed modified
QuEChERS with dSPE method for AP, AM, MDA, and
Fig. 2 Urinal extracted derivatized peaks using selective ion monitoring (SIM) mode. a AP b MA c MDA d MDMA
Table 1 Retention times and
Analyte
Retention time
Identifed mass ions
Quanti-
fed mass
ion
mass ions the simultaneous
GC–MS analysis of the
derivatized standard
amphetamines drugs
Derivatized methamphetamine
Amphetamine-TMS derivative
Derivatized MDA
3.545
4.085
4.930
5.140
58, 65, 91
44,73,116
44,136
58
116
44
Derivatized MDMA
58, 77
58
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Journal of the Iranian Chemical Society
Linearity, limit of detections (LODs), and limit
of quantifcations (LOQs)
Jurado et al., using SPME coupled with GC–MS for analysis
of the same drugs in urine samples [4]. However, the values
obtained by our methods are lower than those obtained by
Gentili et al., using HS-SPME [40]. The results of the linear-
ity experiments and LODs and LOQs are shown in Table 2.
The linearity of the method was investigated at eight difer-
ent concentration levels of AP, AM, MDA, and MDEA rang-
ing from 50 to 1200 ng/mL. Good linearity was obtained
for all analytes with coefcient of determination, (r2) in the
range of 0.984–0.997.
Precision and accuracy
The LOD and LOQ of this method were calculated using
the calibration curves. The LOD was established using
LOD=3.3 x (s/S) and the LOQ=10 x (s/S), where s is the
standard deviation of the intercept and S is the slope of the
curve. The LOQ and LOD were found to be 42 and 13 ng/
mL, respectively, which was higher than those obtained by
The results for intraday and inter-day precision and accu-
racy are shown in Tables 3 and 4, respectively. The intra-
precision and inter-precision (as RSD) were within the
required limits for all analytes. The within-day precision
was measured as percentage standard deviation at three
replicate analyses at three concentrations level 50, 500,
Fig. 3 Chromatograms of a blank urine sample, b spiked urine sample, and c positive urine sample
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Journal of the Iranian Chemical Society
Table 2 Correlation coefcient, concentration linear range for the
urine linear regression simultaneous analysis of urinal extracted
amphetamines and drugs (in mixture) and their LOQ and LOD
values ranging from 0.08 to 3.43%. The intraday accu-
racy for AP, MA, MDA, and MDMA was found to be in
the range of 92.2–106.1%, 85.2–95.6%, 92.6–116.6%, and
87.8–117.4%, respectively, at three concentration levels
Table 3.
Drug analyte
Determination
LOQ (ng/mL)
LOD (ng/mL)
coefcient (R2)
Similarly, the diferent-day precision was assessed,
the method exhibits precision within % RSD values rang-
ing from 0.20 to 3.0%, Table 4. Accuracy was within the
required limits ( 15% and 20% at LOQ) for all analytes
at three concentrations studied. The inter-day accuracy for
AP, MA, MDA, and MDMA was found to be in range of
90.8–113.4%, 92.2–104%, 85.2–107.6%, and 87.6–114.8%,
respectively, at three concentration levels Table 4.
AP
MA
MDA
MDMA
0.984
0.992
0.997
0.993
43.17
43.02
42.93
43.00
12.95
12.91
12.88
12.90
and 1500 ng/mL. Based on the results obtained from
Table 3, the method exhibits precision within % RSD
Table 3 Intra-assay precision (a day precision) and accuracy at diferent concentration levels (low, intermediate, and high) for amphetamines
drugs
Analyte
Theoretical concen-
tration (ng/mL)
Mean peak area SD
%CV (RSD) (%)
Experimental concen- Accuracy %
tration (ng/mL)
aAccu-
racy bias
(%)
(n=3)
AP
50
49,207
49,896
50,992
43,186
43,677
44,456
32,253
32,810
33,686
28,393
28,769
29,722
62.5
39
106.8
39
77.1
57
1105
829
275
24.4
46.5
338.3
0.13
0.08
0.21
0.1
46.1
520
92.2
−7.8
4.0
6.16
−1.5
−6.08
−4.75
−7.4
14.4
16.58
12.0
500
1200
50
500
1200
50
500
1200
50
500
1200
104%
106.1%
85.2%
93.9%
95.6%
92.6%
114.4
116.6
112%
87.8%
117.4%
1274
42.5
469.6
1143
46.3
572
1399
56
439
MA
0.2
0.13
3.43
2.53
0.82
0.1
0.16%
1.14%
MDA
MDMA
−12.2
17.72
1409
aAccuracy: [(found-added/added)]×100
Table 4 Inter-assay precision and accuracy at diferent concentration levels (high, intermediate, and low) in fve diferent days for amphetamines
drugs
Analyte
AP
Theoretical concen- Mean peak area SD
%CV (RSD) (%)
Experimental concen- Accuracy %
tration (ng/mL)
aAccuracy
bias (%)
tration (ng/mL)
(n=15)
50
49,215
49,965
50,723
43,190
43,735
44,462
32,251
32,774
33,286
28,381
28,825
29,691
380.3
608
0.77
1.22
1.23
0.23
0.29
0.20
3.0
2.7
2.0
0.64
0.29
2.1
51.6
567
1089
46.1
520
103.2
113.4
90.8
92.2
104
3.2
13.4
−9.25
−7.8
4.0
−4.1
−11.2
7.6
−14.8
−12.4
−0.8
14.8
500
1200
50
500
1200
50
220
101
126.6
89.1
953
MA
1151
44.4
538
1022
43.8
496
96
MDA
MDMA
88.8
107.6
85.2
87.6
99
500
1200
50
500
1200
873
667
181
84.5
615.4
1378
114.8
aAccuracy: [(found-added/added)]×100
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Journal of the Iranian Chemical Society
Table 5 Concentrations of amphetamine and related compounds
in 17 immunoassay amphetamine positive out of 21 urine samples
received to the forensic laboratories
20 min for sample preparation and cleanup, and 12 min for
the GC–MS analysis. The method developed was success-
fully applied for the analysis of urine samples, and out of
21 samples, 17 were tested positive for amphetamines. This
method has proven to be suitable for application in the ana-
lytical routine analysis of forensic laboratory.
Sample Number/
analyte
Concentration (ng/mL)
MDMA
MDA
MA
AP
1
2
3
4
5
6
7
8
1226
1103
1099
1080
1068
1056
969
760
753
720
538
517
517
513
499
491
456
865
904
865
423
830
569
546
537
865
482
530
448
482
318
320
x
271
258
x
262
227
255
x
189
126
174
189
x
246
253
x
241
166
188
136
x
x
154
x
x
x
Author’s contributions All the authors contributed equality to this
manuscript.
Funding Not applicable.
Data availability Available upon request from the corresponding
author.
9
Declarations
10
11
12
13
14
15
16
17
Conflict of interest The authors declare no confict of interest.
Ethical approval An ethical approval for using urine samples was
received from Sudan Police Headquarters, General Administration of
Forensic Evidence, Forensic Laboratories, Khartoum, Sudan.
x
x
x
x
x
x
x
x
Human and animal rights Not applicable.
Consent to participate Not applicable.
x
x
x: absence of the target analytes
References
Application of the method
1. S. Dulaurent, S. El Balkhi, L. Poncelet, J.M. Gaulier, P. Mar-
quet, F. Saint-Marcoux, QuEChERS sample preparation prior
to LC-MS/MS determination of opiates, amphetamines, and
cocaine metabolites in whole blood. Anal. Bioanal. Chem.
408(5), 1467–1474 (2016)
From 21 urine samples that received to the Sudanese
Forensic Laboratories, 17 urine samples were found to be
amphetamine screening positive. In overall, 17 urine sam-
ples were tested positive for amphetamine with an average
concentration of 758.7 ng/mL (one sample was found to
be over the linear concentration), 15 samples were tested
positive for methamphetamine with an average concen-
tration of 603 ng/mL, 9 samples were tested positive for
MDA with average concentration of 216.8 ng/mL and 7
samples were tested positive for MDMA with average con-
centration of 197.7 ng/mL. The results are summarized in
Table 5.
2. B.D.S. Vagner, L.D.L. Claudimir, and D. Daniel; Determina-
tion of Amphetamine and Derivatives in Urine Using a Modi-
fed QuEChERS and Capillary Electrophoresis Tandem Mass
Spectrometry Analysis, Agilent Technologies, Inc., (2016)
5991–7019.
3. M. Nishida, A. Namera, M. Yashiki, M. Kojima, Routine analysis
of amphetamine and methamphetamine in biological materials by
gas chromatography-mass spectrometry and on-column derivati-
zation. J. Chromatogra. B 789, 65–71 (2003)
4. C. Jurado, M.P. Giménez, T. Soriano, M. Menéndez, M. Repetto,
Rapid analysis of amphetamine, methamphetamine, MDA, and
MDMA in urine using solid-phase microextraction, direct on-
fber derivatization, and analysis by GC-MS. J. Anal. Toxicol.
24, 11–16 (2000)
Conclusion
5. E. Miranda-G, M. Sorda, A.M. Salazar, C. Contras, L. Bau-
tista, A.E. Rojas Gaecia, P. Ostrosky-Wegman, Determination
of amphetamine, methamphetamine and hydroxyamphetamine
derivatives in urine by gas chromatography-mass spectrometry
and its relation to CYP2D6 phenotype drug users. J. Anal. Toxi-
col. 31, 31–36 (2007)
A fast GC–MS method based on QuEChERS extraction for
the simultaneous determination of the AP, MA, MDA, and
MDMA in human urine for forensic purposes was devel-
oped and validated. The study involves the optimization of
derivatization procedure and the quantity of the partition
salts in the QuEChERS cleanup procedure. The optimiza-
tion of the quantity of the partition was found to enhance
extraction recovery. The method is considered to be fast and
needs about half an hour to complete the sample analysis,
6. J.L. Villamor, A.M. Bermejo, P. Fernández, M.J. Tabernero, A
new GC-MS method for the determination of fve amphetamines
in human hair. J. Anal. Toxicol. 29, 135–139 (2005)
7. B. Moslah, M. Araoud, M.A. Nouioui, S. Najjar, D. Amira, N.
Ben Salah, A. Hedhili, Fast screening tests for the simultaneous
detection of 11 drugs of abuse in urine specimens. A forensic
1 3

Journal of the Iranian Chemical Society
chromatography-chemical ionisation mass spectrometry. J. Chro-
matogr. B Anal. Technol. Biomed. Life Sci. 769, 243–251 (2002)
24. D. Peroni, W. Van Egmond, W.T. Kok, H.G. Janssen, Advanc-
ing liquid/liquid extraction through a novel microfuidic device:
theory, instrumentation and applications in gas chromatography.
J. Chromatogr. A 1226, 77–86 (2012)
epidemiology study of 28,298 cases in Tunisia. Forensic Sci. Int.
283, 35–40 (2018)
8. L. Guo, Z. Lin, Z. Huang, H. Liang, Y. Jiang, Y. Ye, Z. Wu,
R. Zhang, Y. Zhang, Y. Rao, Simple and rapid analysis of four
amphetamines in human whole blood and urine using liquid–liq-
uid extraction without evaporation/derivatization and gas chro-
matography–mass spectrometry. Forensic Toxicol. 33, 104–111
(2015)
25. M.K. Woźniak, M. Wiergowski, J. Aszyk, P. Kubica, J. Namieśnik,
M. Biziuk, Application of gas chromatography–tandem mass
spectrometry for the determination of amphetamine-type stimu-
lants in blood and urine. J. Pharm. Biomed. Anal. 148, 58–64
(2018)
9. M. Thevis, A. Thomas, W. Schanzer, Current role of LC-MS-(MS)
in doping control". Anal. Bioanal. Chem. 401, 405–420 (2011)
10. J.Y. Kim, S. Suh, J. Park, M.K. In, Simultaneous determination
of amphetamine-related new psychoactive substances in urine by
gas chromatography-mass spectrometry. Analytical Toxicol. 42,
605–616 (2018)
26. R. Wang, X. Qi, L. Zhao, S. Liu, S. Gao, X. Ma, Y. Deng, Ionic-
liquid-based dispersive liquid–liquid microextraction coupled
with high-performance liquid chromatography for the forensic
determination of methamphetamine in human urine. J. Sep. Sci.
39, 2444–2450 (2016)
11. T. Saito, H. Mase, S. Takeichi, S. Inokuchi, Rapid simultaneous
determination of ephedrines, amphetamines, cocaine, cocaine
metabolites, and opiates in human urine by GC-MS. J. Pharm.
Biomed. Anal. 43, 358–363 (2007)
27. S.C. Franco de Oliveira, M. Yonamine, Measurement uncertainty
for the determination of amphetamines in urine by liquid-phase
microextraction and gas chromatography-mass spectrometry.
Forensic Sci. Int. 265, 81–88 (2016)
12. G. Merola, S. Gentili, F. Tagliaro, T. Macchia, Determination of
diferent recreational drugs in hair by HS-SPME and GC/MS.
Anal. Bioanal. Chem. 397, 2987–2995 (2010)
28. D. Djozan, M.A. Farajzadeh, S.M. Sorouraddin, T. Baheri, Molec-
ularly imprinted solid phase extraction combined with simultane-
ous derivatization and dispersive liquid-liquid microextraction for
selective extraction and preconcentration of methamphetamine
and ecstasy from urine samples followed by gas chromatography.
J. Chromatogr. A 1248, 24–31 (2012)
13. S. Mohr, J.A. Wei, J. Spreitz, M.G. Schmid, Chiral separation of
new cathinone- and amphetamine-related designer drugs by gas
chromatography-mass spectrometry using trifuoroacetyll-prolyl
chloride as chiral derivatization reagent. J. Chromatogr. A 1269,
352–359 (2012)
29. T. Ahmadi-Jouibari, N. Fattahi, M. Shamsipur, Rapid extraction
and determination of amphetamines in human urine samples using
dispersive liquid-liquid microextraction and solidifcation of foat-
ing organic drop followed by high performance liquid chromatog-
raphy. J. Pharm. Biomed. Anal. 94, 145–151 (2014)
14. S. Malaca, T. Rosado, J. Restolho, J.M. Rodilla, P.M.M. Rochae,
L.S.C. Margalho, M. Barroso, E. Gallardo, Determination of
amphetamine-type stimulants in urine samples using microextrac-
tion by packed sorbent and gas chromatography-mass spectrom-
etry”. J. Chromatogr. B 1120, 41–50 (2019)
30. L.W. Chung, G.J. Liu, Z.G. Li, Y.Z. Chang, M.R. Lee, Solvent-
enhanced microwave assisted derivatization following solid-phase
extraction combined with gas chromatography-mass spectrometry
for determination of amphetamines in urine. J. Chromatogr. B
Anal. Technol. Biomed. Life Sci. 874, 115–118 (2008)
31. F. Wei, Y. Fan, M. Zhang, Y.Q. Feng, Poly(methacrylic acid-eth-
ylene glycol dimethacrylate) monolith in-tube solid-phase micro-
extraction applied to simultaneous analysis of some amphetamine
derivatives in urine by capillary zone electrophoresis. Electropho-
resis 26, 3141–3150 (2005)
15. R. Kronstrand, I. Nystrom, J. Strandberg, H. Druid, Screening for
drugs of abuse in hair with ion spray LC-MS-MS. Forensic Sci.
Int. 145, 183–190 (2004)
16. J. Wang, Z. Yang, J. Lechago, Rapid and simultaneous deter-
mination of multiple classes of abused drugs and metabolites in
human urine by a robust LC-MS/MS method-application to urine
drug testing in pain clinics. Biomed. Chromatogr. 27, 1463–1480
(2013)
17. P.M. O’Byrne, P.V. Kavanagh, S.M. Mcnamara, S.M. Stokes,
Screening of stimulants including designer drugs in urine using a
liquid chromatography tandem mass spectrometry system. J. Anal.
Toxicol. 37, 64–73 (2013)
32. A. Namera, A. Nakamoto, M. Nishida, T. Saito, I. Kishiyama,
S. Miyazaki, M. Yahata, M. Yashiki, M. Nagao, Extraction of
amphetamines and methylenedioxyamphetamines from urine
using a monolithic silica disk-packed spin column and high-per-
formance liquid chromatography-diode array detection. J. Chro-
matogr. A 1208, 71–75 (2008)
18. I. Kohler, J. Schappler, S. Rudaz, Highly sensitive capillary elec-
trophoresis-mass spectrometry for rapid screening and accurate
quantitation of drugs of abuse in urine. Anal. Chim. Acta 780,
101–109 (2013)
33. S.A. Haeri, S. Abbasi, S. Sajjadifar, Bio-dispersive liquid liquid
microextraction based on nano rhamino lipid aggregates combined
with magnetic solid phase extraction using Fe3O4@PPy mag-
netic nanoparticles for the determination of methamphetamine in
human urine, J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
1063 (2017) 101–106,
19. J.M. Holler, S.P. Vorce, T.Z. Bosy, A. Jacobs, Quantitative and
isomeric determination of amphetamine and methamphetamine
from urine using a nonprotic elution solvent and R-(-)-α-methoxy-
α-trifouromethlphenylacetic acid chlouride derivatization. J.
Anal. Toxicol. 29(7), 652–657 (2005)
20. A. Orfanidis, O. Mastrogianni, A. Koukou, G. Psarros, H. Gika, G.
Theodoridis, N. Raikos, A GC–MS method for the detection and
quantitation of ten majordrugs of abuse in human hair samples. J.
Chromatogr. B 1047, 141–150 (2017)
34. J. Zeng, J. Chen, M. Li, F. Subhan, F. Chong, C. Wen, J. Yu,
B. Cui, X. Chen, Determination of amphetamines in biological
samples using electro enhanced solid phase microextraction-gas
chromatography. J. Chromatogr. B Anal. Technol. Biomed. Life
Sci. 1000, 169–175 (2015)
21. N.H. Kwon, Y.R. Lee, H.S. Kim, J.C. Cheong, J.Y. Kim, Hybrid
solid-phase extraction for selective determination of methamphet-
amine and amphetamine in dyed hair by using gas chromatogra-
phy-mass spectrometry. Molecules 24, 2501 (2019)
35. I. Racamonde, R. Rodil, J.B. Quintana, R. Cela, In-sample derivat-
ization-solid-phase microextraction of amphetamines and ecstasy
related stimulants from water and urine. Anal. Chim. Acta 770,
75–84 (2013)
22. D.E. Raynie, Modern extraction techniques. Anal. Chem. 78, 3997
(2006)
36. M. Barroso, I. Moreno, B. da Fonseca, J.A. Queiroz, E. Gallardo,
Role of microextraction sampling procedures in forensic toxicol-
ogy. Bioanalysis 4, 1805–1826 (2012)
23. M. Pellegrini, F. Rosati, R. Pacifici, R. Zuccaro, F.S.
Romolo, A. Lopez, Rapid screening method for determina-
tion of Ecstasy and amphetamines in urine samples using gas
1 3

Journal of the Iranian Chemical Society
37. M. Anastassiades, S.J. Lehotay, D. Stajnbaher, F.J. Schenck, Fast
and easy multiresidue method employing acetonitrile extraction/
partitioning and dispersive solid-phase extraction for the determi-
nation of pesticide residues in produce. J. AOAC Int. 86, 412–431
(2003)
Forensic and Police Science; Barry A. J. Fisher, Editor; New
York. Amsterdam. Oxford.
40. C.A. Gouveia, A. Oliveira, S. Pinho, C. Vasconcelos, F. Carvalho,
R.F. Moreira, R.J. Dinis-Oliveira, “Simultaneous quantifcation
of morphine and cocaine in hair samples from drug addicts by
GC–EI/M. Biomed. Chromatogr. 26, 1041 (2012)
38. M.M.A. Omar, W.A. Wan Ibrahim, A.A. Elbashir, Sol–gel hybrid
methyltrimethoxysilane–tetraethoxysilane as a new dispersive
solid-phase extraction material for acrylamide determination in
food with direct gas chromatography–mass spectrometry analysis.
Food Chem. 158, 302–309 (2014)
41. S. Gentili, C. Mortali, L. Mastrobattista, P. Berretta, S. Zaami,
Determination of diferent recreational drugs in sweat by head-
space solid-phase microextraction gas chromatography mass spec-
trometry (HS-SPME GC/MS): Application to drugged drivers. J.
Pharm. Biomed. Anal. 129, 282–287 (2016)
39. T. Mills, W. Price, P. T. Price, and C. Roberson; “INSTROMEN-
TAL DATA FOR DRUG ANALYSIS”; ELSEVIER Series in
1 3