American Journal of Anal yt ical Chemistry, 2011, 2, 235-242
doi:10.4236/ajac.2011.22028 Published Online May 2011 (
Copyright © 2011 SciRes. AJAC
Liquid Chromatographic Determination of Scopolamine in
Hair with Suspended Drop Liquid Phase
Microextraction Technique
Mahboubeh Masrournia1*, Zarrin Es’haghi2, Mostafa Amini1
1Department of Chemistry, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran
2Department of Chemistry, Faculty of Sciences, Payam Noor University, Tehran, Iran
Received December 3, 2010; revised March 10, 2011; accepted March 14, 2011
Hair analysis is used in some branches of alternative medicine as a method of investigation to assist
diagnosis. It is very useful when a history of drug use is difficult or impossible to obtain. In this research
suspended droplet liquid phase microextraction (SDLME) coupled with high-performance liquid chroma-
tography and photodiode array detection (HPLC-DAD) was used for preconcentration and analysis of sco-
polamine in hair samples. Therefore scopolamine was extracted from 2.0 g hair sample incubated in metha-
nol (5 h, 50˚C) and adjusted to pH 7.4 with, Na2HPO4–H3PO4 buffer solution (donor phase, P1) into an or-
ganic phase (P2) 350 µl n-octanol and then back extracted into a micro drop of aqueous acceptor phase (P3),
adjusted at pH 3, with HCL. The extraction time, T1 (from P1 to P2) was 2 min and T2 (from P2 to P3) was 30
min. Optimum instrumental conditions were included; A C18 reverse phase column with water-acetonitrile-
methanol (80:10:10) as the mobile phase was used and wavelength for UV detection was 205 nm. The linear
range was 10 to 10000 ng·mL–1, enrichment factor, detection limit and relative standard deviation were 77,
0.1 ng·mL–1 and 5.4 respectively.
Keywords: Column Liquid Chromatography, Directly Suspended Droplet Liquid Phase Microextraction
(SDLME), Scopolamine, Hair Sample
1. Introduction
Tropane alkaloids such as atropine and scopolamine
(Figure 1) are muscarinic antagonists that block neu-
ron-transmission across muscarinic cholinergic receptors.
The toxicity of these drugs has been well known for
centuries and has been linked to poisoning and death,
usually due to heart or respiratory failure. Scopolamine
was shown toxic effects on central and peripheral nerv-
ous system [2]. Besides an increase of recreational abuse
[3], scopolamine has been occasionally used for its seda-
tive properties.
Tropane alkaloid determination in biological samples
such as serum, blood, urine and hair has a significant
importance in poisoning and forensic case [4]. Moreover
hair analysis is a new perspective in forensic toxicology
Hair may be considered for retrospective purposes
when blood and urine are no longer expected to contain a
particular contaminant, typically a year or less. Its most
widely accepted use is in the fields of forensic toxicology
and environmental toxicology. Several alternative medi-
cine fields also use various hair analysis for environ-
mental toxicology but these uses are controversial and not
standardized [7] But, drug determination in the human
Figure 1. Structure of the scopolamine and its pKa and log
Po/w values [1].
hair or biological fluids is often complicated by low
analyte concentration and the complex sample matrix.
Because of this, sample preparation is crucial in drug
analysis and includes both analyte pre-concentration and
sample clean-up [8].
Some methods for analyzing tropane alkaloids in bio-
logical samples have been published including HPLC-
MS-MS [4] and LC-MS-MS [5,9]. These methods re-
quire laborious sample preparation and expensive in-
Relatively recent three phase microextraction was de-
veloped to extract ionisable and chargeable compounds
from different aqueous samples [10,11]. Ma and
Cantwell used an organic solvent to separate two aque-
ous phase, donor phase and acceptor phase. The pH of
donor phase was adjusted to basic and the acceptor phase
was acidic (for basic compounds). An ionisable com-
pound was extracted from the donor phase into the or-
ganic phase, and then back extracted into the acceptor
phase. Compared to the supported liquid membrane
(SLM), suspended droplet liquid phase microextraction
(SDLME) uses an unsupported liquid organic membrane.
The thickness of the organic film is easier to control, and
because this organic layer is changed for every extraction,
no memory effect was observed together with long term
instability in SDLME [12].
In the present work, a simple SDLME device was
set-up to pre-concentrate scopolamine from hair samples
before HPLC analysis. We also investigated various as-
pects of the microextraction conditions including the
effect of organic solvent, composition of acceptor and
donor phase, extraction times in each step, and stirring
rates. The main aim of our work is to develop a precon-
centration SDLME technique for scopolamine extraction
to make its evaluation in hair samples with HPLC me-
thod feasible.
2. Experimental
2.1. Chemicals and Reagents
Scopolamine was purchased from Sigma-Aldrich (Saint
Louis, USA). HPLC grade water, methanol and acetone-
trile also were purchased from Merck (Darmstadt, Ger-
many) and 1-octhanol from Applichem (Darmstadt, Ger-
many). Main stock solution of the scopolamine (2000
µg·L–1) was prepared in methanol and stored at 4˚C. Fresh
sample solutions containing scopolamine at different con-
centrations were prepared from the main stock solution.
Hair Samples and Working Solutions
A bulk of blank hair, necessary for method development
and validation, was obtained from a men hairdresser’s
shop. The absence of scopolamine was verified. Hair
samples were obtained from addiction therapeutic center
of Azadshahr, Mashhad, Iran.
A standard of hair about 5 mm in diameter was cut
from close to the scalp at the vertex posterior area. Sam-
ples 2 - 4 cm long were selected for analysis.
2.0 grams of the hair samples were washed with dif-
ferent solvents as follow: 20 mL dichloromethane, 15
mL acetone, 15 mL methanol, 10mL methanol, at room
temperature for 5 min and then it was dried. The washed
and dried hair was finally cut into approximately 1 mm
pieces and digested by the following procedure: 2 ml
methanol as an extracting solvent was added to 40 mg of
hair. The pH was adjusted to 7.4 by phosphate buffer
solution. The samples were incubated at 50˚C for 5 h
[13]. In case of a remaining solid matrix, extracts were
filtered. The remaining was rinsed with 1 mL ethanol
and it was added to the extracted solution.
Stock solution containing 2.0 mg·mL–1 of scopolamine
was prepared, in methanol and stored at 4˚C. Standard
solutions were obtained by adding calculated amounts of
the stock solution into the blank hair solutions which
were prepared .These working samples were used for
optimization experimental and calibration curve. Limit of
detection (LOD) and limit of quantification (LOQ) of the
analyte were determined by decreasing concentrations of
spiked samples until signal to noise ratio (S/N) of 3 and
10 were obtained, respectively. All solutions were stored
at 4˚C and protected from light.
2.2. Instrumentation
2.2.1. HPLC System
The HPLC system used in this work was a Knauer (d-
14163, Germany) and consisted of a photodiode Knauer
(S2600) tunable absorbance detector and a 100/5-RP-18
column (4.6 mm diameter, 250 mm length) from Knauer
(Germany), was used for separation. A RP-18 guard
column was fitted upstream of the analytical column.
The mobile phase was water- methanol–acetonitrile op-
timized on (80:10:10 V/V) and delivered by two Knauer
S-1000 HPLC pumps. The flow rate of the mobile phase
was: 1 mL·min–1 and the UV detection wavelength were
monitored at 205 nm.
2.2.2. Suspended Droplet Liquid Phase
Microextraction (SDLME Procedure)
The microextraction device is shown in Figure 2. The
sample solution 5 ml adjusted to pH 10 with NaOH 0.1
M) was placed in a 6 mL vial. Then 350 ml organic sol-
vent was added and a stirring bar was (2 mm × 7 mm)
placed in the solution. An aluminum foil was used to
cover the lid of the vial during extraction to prevent the
Copyright © 2011 SciRes. AJAC
Figure 2. Illustration of the microextraction procedure for
directly suspended droplet. SDLME: (a) addition of the or-
ganic solvent to the aqueous sample solution, magnetic stir-
rer is off; (b) the mixture is being agitated, extraction pro-
cedure; (c) separation of the tiny drop of the organic solvent
and aqueous sample solution and then addition of the ac-
ceptor phase in to the organic solvent, magnetic stirrer is off;
(d) back-extraction procedure, magnetic stirrer is on [18].
evaporation of the organic phase. Then the mixture was
put on a Yellow line (USA) heater and magnetic stirrer
and was agitated for 5 min at 500 rpm. In the later step a
100 µL flat-cut HPLC microsyringe (Knauer, Germany)
was used to introduce the acceptor phase (10 µL droplet
of de-ionized water adjusted to pH 5 with HCl 0.1 M) to
the top center position of the immiscible organic solvent.
The mixture was stirred at 600 rpm for 20 min to cause
back-extraction. After this period the microdroplet was
picked up by the same HPLC microsyringe and was in-
jected into the HPLC system.
3. Results and Discussion
3.1. Theory of SDLME
SDLME consists of two processes and three phases: ex-
traction from donor phase (P1) into an organic solvent
(P2), and finally back-extraction from the organic phase
into an aqueous acceptor phase (P3). In such cases, the
pH of the sample is adjusted to make the analyte neutral
and thus extractable into the organic solvent. After
reaching the equilibrium of phase separation, the analyte
that are mostly transferred into the organic phase are
back-extracted into the second aqueous phase (acceptor)
set to a pH at which, the analyte are charged. This back-
extraction step introduces extra selectivity since neutral
compounds will preferably stay in the organic phase [14,
15]. The theory of the method is well defined by the oth-
ers [16,17].
3.2. Optimization Procedure
To obtain the optimal extraction conditions for the best
efficiency, various parameters like organic solvent, ex-
traction and back-extraction times, different volumes of
phases, stirring speed, pH, this can be discussed as fol-
3.2.1. Organic Solvent Selection
In SDLME, the type of the organic solvent is an essential
factor for achieving the efficient analyte preconcentra-
tion. There are several requirements for obtaining the
selected organic solvent. The organic phase serves to
separate the aqueous acceptor phase from the aqueous
donor phase. The organic phase must, therefore, be im-
miscible with both the acceptor and the donor phase. The
solubility of the analyte should be higher in the organic
phase than the donor phase to promote the extraction of
the analyte. On the other hand, the solubility of the ana-
lyte should be lower in the organic phase compared to
the acceptor phase, in order to achieve a high degree of
recovery of analyte in the acceptor phase. The appropri-
ate organic solvents in this work should have lower den-
sity than the water to float on the top of the aqueous
sample solution. It should be immiscible with water to
avoid dissolution in two aqueous phases, because it serves
as a barrier between them. The organic solvent should
have high viscosity to hold the microdroplet at its top-
center position (Figure 2) without using a microsyringe
as supporting device.
During this experiment, several organic solvents were
tested to investigate their effect on the extraction effi-
ciency. Five organic solvents including 1-octanol, n-
Hexane, dichloromethane, toluene and benzylalcohol,
have been examined. Among 1-octanol, Toluen, n-Hexane,
Benzylalcol, Dichlorometan, 1-octanol was selected, be-
cause of its higher viscosity, immiscible with aqueous
solution and high extraction efficiency.
3.2.2. Stirring Speed
Agitation of the sample is routinely applied to accelerate
the extraction kinetics. Increasing the stirring speed of
the donor phase enhances extraction as the diffusion of
analyte through the organic phase is facilitated and im-
proves the repeatability of the extraction method [18,19].
Therefore, the stirring speed was also optimized for a
better extraction. Different stirring rates (100 to 500 rpm)
were checked (Figure 3) Sample agitation by using ei-
ther vibration or magnetic stirring dramatically increased
extraction, but the liquid drop (acceptor phase) at the end
of the needle, to be lost under great agitation. Therefore
the stirring speed was selected as 500 rpm.
3.2.3. Extraction Time
In the first step, analyte extracted from the aqueous sam-
ple into the organic solvent that is a slow equilibrium
Copyright © 2011 SciRes. AJAC
Figure 3. The effect of stirring speed on enrichment factor.
Other extraction conditions: analyte concentration 5
µg·mL1; 1-octanol as the organic solvent; sample pH 10.0;
acceptor phase pH 3.0; T1 = 2 min, back-extraction time (T2)
= 30 min; 5 mL donor sample volume; micro-droplet vol-
ume 10 µL.
process, and mass transfer is depended on time .With the
passage of time solute molecules have sufficient time for
transfer from donor phase to interface between the donor
and organic phases and collection in organic phase.
Therefore, extraction time is a significant factor in the
extraction efficiency. The mixture of water sample and
organic solution was agitated at 500rpm. Due to the high
degree of mixing between the donor and organic phases
the mass transfer is rapid. It has been reported that longer
equilibration times do not have any significant effect on
the extraction parameters [20,21] and in this work we
observed that an equilibration time of 30.0 min (T1) is
sufficient to obtain a good extraction. SDLME is not an
exhaustive extraction technique. Although maximum sen-
sitivity is attained at the equilibrium, complete equilib-
rium needs not to be attained for accurate and precise
analysis. Increasing this time causes increased extraction
and leads to progressed enrichment factor. However,
longer extraction time will result in the dissolution of
extracted analyte in the organic phase and instability of
the droplet especially under stirring. We have tested dif-
ferent back-extraction times the results are showed in
Figure 4. On this basis, 30.0 min was selected as optimal
back-extraction time for the experiment and after 30.0
min microdroplet is dissolved. The recovery percentage
depends on the time that the analyte is in contact with the
organic phase and the acceptor solution.
3.2.4. pH of the Donor and Acceptor Phases
In three-phase microextraction process, the pH of the
donor phase is adjusted to produce molecular form of the
analyte, and the acceptor phase is adjusted to ionize it.
The difference in pH between the donor and acceptor
phases can promote the extracted analyte from donor to
acceptor phase [1,16,18,22-24].
Figure 4. The effect of extraction time (T2) on enrichment
factor. Other extraction conditions: analyte concentration 5
µg·mL1; 1-octanol as the organic solvent; sample pH 10.0;
acceptor phase pH 3.0; stirring speed 500 rpm; 5 mL donor
sample volume; micro-droplet volume 10 µL.
Scopolamine is a moderately basic drug (pKb = 7.75)
and the pH of the aqueous sample should be higher than
the pKa of the analyte, so that the analyte is neutral and
extractable into the organic phase and contrary for ac-
ceptor phase. The effect of sample pH on the method
efficiency in the range of 6 - 11, and for acceptor solu-
tion in the range of 2.5 - 7 was evaluated. Although the
peak area decreased with increasing acceptor pH, the
extraction efficiency was not affected significantly by pH
in this range.
The maximum of enrichment factor was observed in
donor phase pH = 10. The pH of acceptor phase de-
creased until the higher enrichment factor was obtained
in pH = 3 (see Figure 5).
3.2.5. Phase Volume
Generally in the three-phase liquid microextraction sys-
Figure 5. The effect of donor and acceptor phases pH on the
extraction procedure. Extraction conditions: analyte con-
centration 5 µg·mL1; 350 µL 1-octanol as organic solvent;
T1 = 2 min, back-extraction time (T2) = 30 min; stirring
speed 500 rpm; 5 mL donor sample volume.
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
tems, sensitivity of the method can be increased by de-
creasing the volume ratio of the acceptor to the donor
phase [22,23]. However, the volume of the acceptor so-
lution used for extraction may also be adjusted depend-
ing on the analytical technique coupled to liquid phase
microextraction. For example, in contrast to GC, sample
volumes in the range from 10 to 25 µL are easily injected
into a HPLC instrument, so the whole acceptor phase
may be analyzed, potentially providing lower detection
limits [24]. In this manner, the use of a large drop results
in an increase of the analytical response. However, larger
drops are difficult to manipulate [25]. Additionally, lar-
ger injection volumes result in band broadening [26]. Thus,
in this investigation we used 5.0 mL donor phase (P1), 350
µL organic phase (P2) and 10µL acceptor phase (P3).
3.3. Method Validation
After optimization of all affective parameters, optimal
conditions have been set to evaluate the performance of
microextraction. In this research, experimental limits of
detection were calculated as the minimum concentration
providing chromatographic signals 3 times higher than
background noise. In addition, the limit of quantification
was calculated experimentally as the minimum concen-
tration of scopolamine that provides chromatographic
signals 10 times higher than background noise. We ob-
served that LOD was 0.10 ng·mL–1. The linearity of this
method for analyte has been investigated over the ranges
10 - 10,000 ng·mL–1 of scopolamine in the blank hair
matrix. The obtained calibration equation was y =
0.0098x + 11.769 (r2 = 0.9978), where y is the absorb-
ance (in mAU) and C the concentration of scopolamine
in ng·mL–1 in the initial solution. The method showed
good repeatability (RSD% 5.4, n = 5).
For calculation of enrichment factor, response of analy-
sis after extraction by the investigated method should be
divided to response of it before extraction. After that, en-
richment factor was calculated by division of peak area
results from injection of this solution, to peak area from
initial sample. For reporting of this factor, we calculated
enrichments of three standards solution (in the initial, mid
and final reign of linear range) and the enrichment factor
of the method was reported as the average of these calcu-
lates. We obtained enrichment factor 77.0. The review of
some methods [27-33], which were used for determination
of scopolamine in the environmental and biological sam-
ples is demonstrated in Table 1. As compare the other
method low detection limits and high enrichment factors
are readily achieved in present work.
3.4. Real Sample Analysis
To show the applicability of the method, we have ana-
lyzed the two sample hair. Figure 6 shows human hair
prior (left) and after spiking with scopolamine (right)
after SDLME under optimal conditions. The concentra-
tions of scopolamine in two Hair samples are shown in
Table 2.These samples were spiked with scopolamine
standards to assess matrix effects.
These results demonstrated that the hair sample matri-
ces had little effect on the SDLME of scopolamine. It is
clear from the above discussion that SDLME was com-
bined with HPLC for the determination of scopolamine
in Hair samples may have a good potential for extraction
Table 1. Comparison between current methods for determination of scopolamine with this work.
Matrix Method Detection LOD LOQ DLR1 RSD%r 2 Recovery% Reference
Blood and urine LC-MS/APCI MASS 0.7
ng·ml 0.9 - 25 ng·mL–1 4.8 - 7.50.98 76 -100 28
drug HPLC UV
μg·mL–1 0.003 - 0.09
μg·mL–1 2.0 0.9998 99.9 29
viscera samples SPE-HPLC MASS 100
100 - 0.000
g·mL–1 - 0.9982 - 30
Plant root SPE- HPLC UV 0.8 ng - 8 - 200 g·mL–1 4.9 - 85.4 31
potentiometric 8 × 10–7
mol·dm–3 - 10-2 - 10–6
mol·dm–3 1.5 0.999 99 32
-HPLC UV 20 ng - 1 - 100
μg·mL–1 - 0.9956 79 33
plant HPLC UV 0.5
mg·L–1 1 mg·L–1 - - 0.9960 - 34
Hair samples SDLME
ng·ml–1 10 ng·ml–1 10 – 10,000
ng·ml–1 5.4 0.9978 89 - 95 This work
Dynamic Linear Range.
Table 2. The result of scopolamine determination in the difference hair samples with relative recoveries.
Scopolamine Founded(ng·mL1)Scopolamine Added(ng·mL1)
_ 0.00 0.00
Figure 6. HPLC analysis of human hair prior (left) and
after spiking with scopolamine (right) after SDLME under
optimal conditions.
and determination from biological samples.
4. Conclusions
The aim of the present study was to develop and validate
a rapid, sensitive, robust and reliable method for the
quantitative determination of the drug abuse in human
hair by
HPLC and the results obtained with the method de-
scribed above indicate that SDLME methodology is a
good alternative extraction technique for scopolamine in
hair samples.
In this study we showed that SDLME coupled with
HPLC was successfully applied for scopolamine analysis.
Compared to the most conventional extraction proce-
dures, this extraction technique requires a very little
aqueous sample solution and very little expensive and
toxic organic extractant. In our method, we introduced a
reliable qualitative and quantitative technique for deter-
mination of scopolamine at low level of concentration in
hair. In the mean time hair sample has some advantages
over the other biological samples like urine and blood,
such as long time of drug residence in the sample and
low risk of side effect in transferring to examiner. On the
other hand, this method is very fast, easy and simple. As
be showed in other researches [34], low detection limits
and high enrichment factors also are readily achieved in
present work. The results of this study are applicable in
biological material, drugs and forensic toxicology.
5. Acknowledgements
The authors would like to acknowledge the Islamic Azad
University of Mashhad, Iran for financial support of this
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