Liquid Chromatographic Determination of Scopolamine in Hair with Suspended Drop Liquid Phase Microextraction Technique

doi:10.4236/ajac.2011.22028

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American Journal of Anal yt ical Chemistry, 2011, 2, 235-242

doi:10.4236/ajac.2011.22028 Published Online May 2011 (http://www.SciRP.org/journal/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

E-mail: [email protected], [email protected]

Received December 3, 2010; revised March 10, 2011; accepted March 14, 2011

Abstract

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

[5,6].

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].

M. MASROURNIA ET AL.

236

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-

struments.

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

M. MASROURNIA ET AL.237

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-

lows:

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

M. MASROURNIA ET AL.

238

Figure 3. The effect of stirring speed on enrichment factor.

Other extraction conditions: analyte concentration 5

µg·mL−1; 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·mL−1; 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·mL−1; 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.

M. MASROURNIA ET AL.

239

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

g·mL–1

0.9

ng·ml 0.9 - 25 ng·mL–1 4.8 - 7.50.98 76 -100 28

gastrointestinal

drug HPLC UV

0.001

g·mL–1

0.004

μg·mL–1 0.003 - 0.09

μg·mL–1 2.0 0.9998 99.9 29

viscera samples SPE-HPLC MASS 100

pg·mL–1

ng·L–1

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

Pharmaceuticals

sample

PVC

membrane

electrod

potentiometric 8 × 10–7

mol·dm–3 - 10-2 - 10–6

mol·dm–3 1.5 0.999 99 32

biological

samples

MIP-SPE

-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

-HPLC PAD 0.1

ng·ml–1 10 ng·ml–1 10 – 10,000

ng·ml–1 5.4 0.9978 89 - 95 This work

Dynamic Linear Range.

M. MASROURNIA ET AL.

240

Table 2. The result of scopolamine determination in the difference hair samples with relative recoveries.

Recovery%

Scopolamine Founded(ng·mL–1)Scopolamine Added(ng·mL–1)

Sample

Hair

_ 0.00 0.00

94.0

9.4

96.0

100

0.00

95.0

19.0

97.5

39.7

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

work.

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