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Applications of dispersive liquid–liquid micro-extraction in forensic toxicology
Accepted Manuscript Title: Applications of dispersive liquid-liquid microextraction in forensic toxicology Author: Rajeev Jain, Ritu Singh PII: DOI: Reference:
S0165-9936(15)00255-1 http://dx.doi.org/doi:10.1016/j.trac.2015.07.007 TRAC 14528
To appear in:
Trends in Analytical Chemistry
Please cite this article as: Rajeev Jain, Ritu Singh, Applications of dispersive liquid-liquid microextraction in forensic toxicology, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi:10.1016/j.trac.2015.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Applications of dispersive liquid-liquid microextraction in forensic toxicology
2
Rajeev Jain1*, Ritu Singh2*
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1
5
Home Affairs, Govt of India, H. No. 16, Lachit Borphukhan Path, Tetelia, Gotanagar, Guwahati
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(Assam) – 781033, India
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2
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Rajasthan, Kishangarh, Ajmer (Rajasthan) – 305817, India
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*Corresponding Authors email: +, [email protected]
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Central Forensic Science Laboratory, Directorate of Forensic Science Services, Ministry of
Department of Environmental Science, School of Earth Sciences, Central University of
Fax:+91-361-2571148
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Highlights
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Applications of DLLME for drugs of abuse and poisons
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Protocols of DLLME for various biological matrices
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Capability of DLLME for simultaneous derivatization and extraction for drugs of abuse
15
Hyphenation of DLLME with injector port silylation
16 17
Abstract
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Among several techniques available for micro-extraction, one which is becoming an increasingly
19
popular choice of forensic toxicologists is dispersive liquid-liquid microextraction (DLLME).
20
DLLME is a simple, fast, inexpensive and environmentally benign microextraction technique
21
which offers high enrichment factors and extraction efficiencies. Its coupling with broad range of
22
analytical instruments makes it a versatile microextraction method. DLLME has found wide
23
range of applications in the field of forensic toxicology such as analysis of narcotic substances,
24
drugs of abuse, hallucinogens, cannabinoids, metals, pesticides etc. Furthermore, the capability
25
of DLLME for simultaneous derivatization and extraction, and its coupling with injection port
26
silylation (IPS) makes the analysis of polar analytes by GC-MS simpler and faster. The present
27
review focuses on various applications and procedures of DLLME for various classes of drugs
28
and poisons of forensic interests since its introduction. In addition, viability of future trend for
29
DLLME in forensic toxicology has also been addressed.
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Keywords: Dispersive liquid-liquid microextraction, drugs of abuse, forensic toxicology, amphetamines, cannabinoids, opium alkaloids, benzodiazepines, cocaine
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1. Introduction
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Forensic toxicology is the scientific study of poison in relation to the law. Forensic
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toxicologists are asked for the detection and quantification of toxicant(s) in tissue, organs and
36
body fluids. Owing to highly complex nature of matrices of toxicological interests such as blood,
37
plasma, urine, tissue, saliva, vitreous humor and hair; sample preparation for the analysis of
38
various drugs/poisons still remains a challenge to the forensic toxicologist. Since, in forensic
39
analysis, every case is unique, and the nature of target analytes is unknown, therefore it is very
40
difficult to standardize the whole analytical procedure, like in other branches of analytical
41
chemistry. In such cases systematic toxicological analysis (STA) is required. Moreover, the
42
sample availability in most of the toxicological cases is limited and the analysis is usually
43
untargeted, therefore, it is necessary to have a sample preparation methodology which requires
44
least amount of sample. On the other hand, in cases where forensic toxicologists are asked for
45
targeted analysis of a specific drug/poison only, the analytical approach is quite easy and
46
different from STA.
47
As the drugs and their metabolites are generally present at trace levels in biological fluids;
48
the whole analytical procedure should be sensitive enough to detect the analytes with accuracy
49
and selectivity. Due to extreme complex nature of the samples received in forensic toxicology
50
laboratories, it is not recommended to subject the sample, directly for analysis on any analytical
51
instrument. Thus, a sample preparation methodology is necessary which could make the sample
52
suitable for instrumental analysis. For accurate measurements, these methodologies should:
53
require minimum amount of sample for the extraction of target analyte(s)
54
consume least amount of toxic organic solvents,
55
be time efficient
56
give reproducible recoveries,
57
produce a clean extract devoid of any impurities and matrix interferences,
58
be compatible with derivatizing reagents in case of extraction of polar analytes,
59 60
be compatible for coupling with different analytical instruments.
61
One method which is mostly practiced in forensic toxicology laboratories, for the extraction
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and preconcentration of analytes from toxicological matrices is liquid-liquid extraction (LLE). It
63
consists of three steps viz. (i) extraction (ii) derivatization (in case of polar analytes) and (iii) 3 Page 3 of 40
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preconcentration of extracted analytes. Despite of LLE wide usage, some limitations which puts
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set back to this method are; (a) they require large amount of toxic organic solvents for the
66
extraction of drugs and poisons from biological matrices, (b) time consuming, (c) multi-step
67
procedures (d) laborious (e) high economic expenses (f) low enrichment factors. Since
68
toxicological samples are quite dirty, therefore in LLE, before preconcentration, an additional
69
step of sample cleanup is also required. Apart from that, LLE require more than one extraction,
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which consumes lot of sample, and affects the reproducibility of the results too. Sometimes
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emulsion formation and matrix interferences also create hurdles in sample preparation. These
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drawbacks make analytes more prone to loss during sample preparation, which in turn affects the
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reproducibility of the analytical method. Additionally; use of large amount of toxic organic
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solvents may lead to environmental pollution and health problems. Solid-Phase extraction (SPE)
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is another extraction cum cleanup technique which is being used in forensic laboratories for long
76
time. In comparison to LLE, SPE requires lesser amount of organic solvent for extraction.
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Nowadays, commercial SPE cartridges with small amount of stationary phases (in milligrams)
78
are also available which requires only microliters of solvent for extracting analytes from
79
samples.
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With a view to improve traditional sample preparation methodologies, researchers over
81
the globe started focussing on developing microextraction techniques which consumes
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minimal/negligible amount of extraction solvent (zero or few μL), less time and labour,
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economic and on the top of all, environmental friendly. One of such techniques is solid-phase
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microextraction (SPME). It was invented in 1990s by Arthur and Pawliszyn who revolutionized
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the sample preparation procedures [1]. SPME is a solvent-less microextraction procedure which
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consists of a fiber coated with a polymeric stationary phase on surface of which suitable type of
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analytes get adsorbed or absorbed. The main advantage of SPME over conventional SPE and
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LLE includes; (a) environmentally benign (b) low sample requirement and (c) relatively higher
89
extraction efficiencies. SPME has been widely applied for the extraction of various analytes from
90
different matrices. The applications of SPME in biomedical and forensic toxicology have been
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well reviewed earlier [2, 3]. However, SPME is a costly extraction technique and fibers used for
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the extraction are very fragile and delicate in nature which needs special maintenance and
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protection. Also, SPME fibers need periodical replacement as their lifetime is limited. Besides
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these issues, the problem of sample carry over is a major setback of this method [4]. 4 Page 4 of 40
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Dispersive liquid-liquid microextraction (DLLME), a miniaturized LLE technique, was
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introduced by Rezaee et al. in 2006 [5]. Since then it gained lot of attention from the analysts and
97
has been used widely for the extraction of various types of analytes from different matrices such
98
as water, tissue, biological fluids and food matrices etc [6]. DLLME is based on ternary
99
component solvent system which consists of aqueous phase, disperser solvent and extraction
100
solvent. In classical DLLME, the extraction solvent is heavier than water. The mixture of
101
disperser solvent and extraction solvent is injected rapidly in aqueous phase, which quickly
102
results in the formation of a cloudy solution. This cloudy solution consists of tiny droplets of
103
extraction solvent dispersed throughout the aqueous phase achieving an enormous contact area
104
between these two, consequential in fast equilibrium. This is followed by centrifugation at high
105
speed, which settles down the droplets of heavy density extraction solvent as sedimented phase,
106
which is further used for analysis (Fig. 1). In classical DLLME, usually toxic chlorinated
107
solvents are used as extraction solvents e.g. chloroform (CF), trichloroethylene (TCE), carbon
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tetrachloride (CCl4) etc. In order to surmount this limitation, Xu et al. [7] proposed a modified
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version of DLLME based on solidification of floating organic droplet (DLLME-SFO) which
110
used low density extraction solvents having melting points below room temperature, such as 1-
111
dodecanol, 1-undecanol, hexadecanol etc. The DLLME-SFO procedure consists of rapid
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injection of mixture of disperser and extraction solvent into aqueous sample followed by
113
centrifugation. By virtue of low density of extraction solvent, a droplet of extraction solvent
114
starts floating over the surface. Now the sample vial is transferred into an ice bath for some time
115
which facilitates the solidification of floating organic droplet due to its lower melting point
116
below room temperature. The solidified droplet is then transferred into another conical vial
117
where it is allowed to melt and then subjected for analysis (Fig. 2) [7]. Some researchers
118
combined DLLME with other extraction techniques such as SPE and molecularly imprinted
119
polymer-SPE (MISPE) where the eluent collected from SPE is preconcentrated using DLLME
120
[8-9].
121
Since its introduction, DLLME has been applied for the determination of several drugs
122
and poisons of forensic interest such as opiates, hallucinogens, amphetamines, cocaine and its
123
metabolites, antidepressants, barbiturates, cannabinoids, metals and pesticides etc. This review
124
article gives a comprehensive view over applications of DLLME in the field of forensic
125
toxicology along with the detailed outline of the procedures followed for varied classes of 5 Page 5 of 40
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forensic drugs and poisons. The article also provide comparative assessment of various
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advancements made in the field of DLLME in terms of selectivity, sensitivity, enrichment factors
128
(EFs), matrix effect; cost, speed and complexity of analysis. The viability of future trend for
129
DLLME in forensic toxicology has also been addressed. Table 1 listed the analytes for which
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DLLME has been used for extraction and preconcentration from forensic samples and the types
131
of disperser and extraction solvent used along with their respective limit of detections (LODs)
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and EFs.
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2. Applications of DLLME in forensic toxicology
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2.1. Analysis of amphetamines
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Amphetamines are categorized under synthetic central nervous system stimulants which
137
include amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA)
138
and 3,4-methylenedioxymethamphetamine (Ecstasy or MDMA). According to World Drug
139
Report 2014, the global seizure of amphetamine type stimulants (ATS) from 2003-2012 is 144
140
tons. ATS (excluding ecstasy) comprises of second most commonly used illicit drugs with 13.9
141
million to 54.8 million estimated users [10].
142
A DLLME-SFO procedure was developed for the extraction of AMP and MA from urine
143
samples [11]. In this procedure, disperser solvent was replaced with a surfactant, i.e. sodium
144
dodecyl sulfate (SDS). Since surfactants are amphiphilic in nature, they are soluble in both
145
extraction solvent as well as aqueous phase. Also they possess several analytical advantages of
146
disperser solvents such as non-toxicity, cost effectiveness and compatibility with mobile phase of
147
high performance liquid chromatography (HPLC). For extraction, in 5 mL of urine sample (pH
148
6.4), 0.015 g NaCl was added followed by addition of 56.5 µL of SDS solution (concentration 70
149
µg L-1). To this solution, 31 µL of 1-undecanol (extraction solvent) was injected rapidly to form
150
cloudy solution followed by vortex mixing and centrifugation. After centrifugation the floating
151
organic droplet of 1-undecanol was solidified by keeping the sample vial in ice for 5 min. The
152
solidified floating organic droplet was allowed to melt in a separate vial and 20 µL of this was
153
injected into HPLC for analysis. The LOD for AP and MA were found to be 2 and 3 µg L-1,
154
respectively [11]. Recently, another DLLME-SFO technique for AP and MA in urine samples
155
was published using 1-undecanol as extraction solvent, whereas acetonitrile (MeCN) was used as
156
disperser solvent. Separation and identification of analytes were performed using HPLC with
157
ultraviolet detector (HPLC-UV). The LODs for AP and MA were 8 and 2 µg L-1 with a signal to
158
noise ratio of 3 [12]. In comparison to SDS [11], use of MeCN as disperser solvent in DLLME-
159
SFO, made no significant differences in terms of sensitivity and speed of analysis speed.
160
Djozan et al. [13] applied DLLME for the determination of MA and MDMA in urine
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samples by gas chromatography-flame ionization detection (GC-FID). Analytes were extracted
162
from urine samples by MI-SPE. As compared to SPE, the major advantage of MIP is its
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selectivity towards the target analyte molecules, which is attributable to its analyte specific
164
cavities formed in course of their synthesis [13]. MIP was synthesized by precipitation
165
polymerization, using MA as a template. The sample was loaded on MI-SPE cartridge and eluted
166
with methanol (MeOH). This MeOH was mixed with butylchloroformate (BCF) which served as
167
disperser solvent as well as derivatizing reagent. After centrifugation, 1 µL of sedimented phase
168
of BCF was injected into GC-FID. The LODs found for MA and MDMA were 2 and 18 ng mL-1,
169
respectively. The applicability of the developed method was demonstrated in two positive cases
170
of MA and MDMA by gas chromatography-mass spectrometry (GC-MS). DLLME has been
171
coupled to capillary electrophoresis with ultraviolet detection (CE-UV) for the extraction and
172
preconcentration of MDMA from urine samples. Dibromomethane (DBM, 0.606 mL) and MeCN
173
(1.508 mL) were used as extraction and disperser solvent for 4 mL of urine sample mixed with 1
174
mL of ultrapure water. The LOD for MDMA was found to be 1 ng mL-1 in spiked urine sample
175
with a signal to noise ratio of 3. Significant matrix effect observed in the study was compensated
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by plotting matrix matched calibration graphs [14]. A year later, in 2013, Kohler et al. [14]
177
applied DLLME for the extraction of 30 toxicological compounds from different class viz.
178
amphetamines and their derivatives, opiates, cocaine and its metabolites and pharmaceuticals
179
from urine samples followed by capillary CE-UV and time-of-flight mass spectrometer (TOF-
180
MS) analysis. 600 µL of dichloromethane (DCM) as extraction solvent along with 1400 µL of
181
isopropanol (IPA) as disperser solvent was injected rapidly in 4 mL of basified urine sample.
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After centrifugation, the sedimented phase of DCM was transferred in a polypropylene tube and
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10 µL of acidified MeOH was added to it. The solution was evaporated to dryness under gentle
184
stream of nitrogen and reconstituted in 30 µL of background electrolyte and water before
185
injection to CE. The extraction recoveries for AP and MDMA were found to be 76 and 75%,
186
respectively; whereas LODs for MAs and their derivatives were found to be in the range of 0.25–
187
1 ng mL-1. In comparison to previous method, no matrix effect for AMs was found [15]. Similar
188
method i.e. DLLME-CE-UV was applied for the chiral separation and determination of multiple
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illicit drugs i.e. MA, MDMA, ketamine and heroin in seized forensic sample such as banknote,
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kraft paper, plastic bags and silver paper. The samples were soaked in acetic acid and filtered
191
which was made alkaline by NaOH. To this filtrate, 0.5 mL of IPA (disperser solvent) and 41 µL
192
of CF (extraction solvent) were rapidly injected to form cloud solution. In this study, effect of pH
193
was different for amines and heroin. Amphetamines and ketamine showed increasing recoveries 8 Page 8 of 40
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up to pH 11, while recovery of heroin decreased above pH 9. Thus, pH 9 was selected as
195
optimum value for further DLLME procedure. The method offered good sensitivity for multiple
196
illicit drugs with the LODs ranging from 0.08–0.20 µg L-1 and EFs ranging from 545–611. The
197
method showed good speed of analysis and the entire target analytes could be separated within
198
10 min [16].
199
Recently, SPE has been combined with DLLME for the preconcentration of MDA,
200
MDMA,
201
methylenedioxypropylamphetamine (MDPA) from urine and plasma samples, followed by GC-
202
FID analysis. Ten mL aliquot of sample was passed through C18 SPE cartridge previously
203
preconditioned with 2 mL of acetone (ACE). The adsorbed analytes on SPE cartridge were then
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eluted with 1.5 mL of MeCN. This MeCN was used as disperser solvent along with 35 µL of
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carbon disulphide (CS2) as extraction solvent. After centrifugation, 2 µL of CS2 was injected into
206
GC-FID for analysis. The relative recoveries varied between 76–92.5% for all analytes. LODs
207
were found to be in the range of 0.05–7 µg L-1 [17].
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2.2. Analysis of sedative and hypnotic drugs
3,4-methylenedioxyethylamphetamine
(MDEA)
and
3,4-
209
Another important group of drugs for forensic toxicologist for which DLLME methods
210
has been developed, are sedatives and hypnotics, such as barbiturates and benzodiazepines
211
(BZD). BZD are widely used for the treatment of insomnia, convulsive attacks and other
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psychological disorders and are also linked with harmful effects such as addiction with regular
213
use either with alcohol or alone.
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Fernandez et al. [18] combined ultrasound-assisted DLLME (US-DLLME) with HPLC
215
and ultra-performance liquid chromatography (UPLC) for the comparative analysis of seven
216
BZDs i.e. alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam and
217
tetrazepam. MeOH was used as deproteinizing agent for plasma samples and subsequently as
218
disperser solvent along with chloroform as extraction solvent. To 500 µL of plasma, 2 mL of
219
MeOH was added and centrifuged. The supernatant was mixed with 250 µL of CF and rapidly
220
injected into 4.5 mL of ultrapure water (pH 9). The mixture was then subjected to ultrasonication
221
for 2 min followed by centrifugation and analysis. Under optimized conditions, method exhibited
222
linearity in the range of 0.01–5 µg mL-1. The LODs were varied for different columns in the
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range of 1.7–10.6 ng mL-1. Apart from that, the total analysis time including DLLME and UPLC 9 Page 9 of 40
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was less than 15 min [18]. Though HPLC and UPLC showed similar results, however in terms of
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sensitivity, speed and reduction in solvent and injection volume, UPLC was found to be superior.
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A year later, same research group again coupled US-DLLME method to UPLC for the
227
determination of seven BZD in urine and waste water samples. Design of experiment (DOE) i.e.
228
Doehlert design was applied for the screening of some important factors which affects the
229
extraction efficiency of DLLME, such as sample pH, extraction solvent volume and
230
ultrasonication time etc. Under optimized conditions, 1.6 mL of ACE along with 160 µL of CF
231
was injected rapidly into 0.5 mL of aqueous sample whose pH was adjusted to 9. Before
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subjecting the sample to centrifugation, ultrasonication was applied for 4.5 min to enhance the
233
extraction efficiency, which was found superior to manual shaking of 1 min in preliminary
234
experiments. All the BZD were separated within 4 min or UPLC run time. The proposed method
235
reported BZD recoveries from urine samples in the range of 96–114% with percent relative
236
standard deviation (%RSD) in the range of 4.3–9.9%. The method was found to be sensitive in
237
the range of 0.6–6.2 ng mL-1. This protocol took total 17 min for analysis of BZD in urine and
238
hospital wastewater [19]. In the same year, DLLME as a preconcentration technique was
239
combined with SPE for the analysis of three BZD i.e. diazepam, midazolam, and alprazolam in
240
urine and other samples. Analytes were adsorbed on octadecyl silica SPE column and eluted with
241
ACE. This elute (0.5 mL) was used as disperser solvent along with 40 µL of CF as extraction
242
solvent. Analysis of sedimented phase was performed using GC-FID. Combination of SPE with
243
DLLME offered very high EFs (in the range of 3895–7222) and sensitivity (LODs for tested
244
BZD were achieved in the range of 0.02–0.05 µg L-1). To cope up with the matrix interferences,
245
urine samples were diluted with ultrapure water (in ratio of 1:4) before analysis which resulted in
246
good relative recoveries in the range of 90-98% [20].
247
Flunitrazepam
is
another
widely used
BZD,
which
is
metabolized
to
7-
248
aminoflunitrazepam in human body. 7-aminoflunitrazepam is commonly used as a biomarker to
249
determine the concentration of flunitrazepam. An LC-ESI-MS method has been developed for
250
the analysis of 7-aminoflunitrazepam in urine samples using DLLME as an extraction technique.
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Before extraction urine samples were basified using ammonia and NaCl was added to increase
252
the ionic strength of the solution. IPA (disperser solvent, 500 µL) along with DCM (extraction
253
solvent, 250 µL) was rapidly injected into urine sample and subjected for centrifugation to settle
254
down the droplets of extraction solvent. The sedimented phase of DCM was transferred to 10 Page 10 of 40
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another vial and evaporated to dryness followed by reconstitution in mobile phase i.e.
256
MeCN:water (20:80 v/v). In order to achieve maximum EFs, different combinations of disperser
257
solvent and extraction solvent were tested. Among them, 500 µL of IPA and 250 µL of DCM
258
gave maximum EFs up to 20. EFs were calculated as the ratio between peak area of 7-
259
aminoflunitrazepam after and before extraction. LOD reported in this study i.e. 0.025 ng mL-1,
260
was found superior to previously reported methods in literature which were in the range of 1–5.6
261
ng mL-1. Owing to quantification, good selectivity for 7-aminoflunitrazepam could be achieved
262
in multiple reaction monitoring (MRM) mode but at the cost of analysis time which reached up
263
to 47 min [21]. In another study, DLLME coupled to HPLC was employed for the extraction of
264
three BZDs i.e. alprazolam, oxazepam and diazepam from urine samples. One mL of Ethanol
265
(EtOH) was used as disperser solvent along with 102 µL of DCM which were injected into 5 mL
266
of distilled water/urine samples followed by centrifugation and analysis. EFs in the range of
267
208–226 and relative recoveries up to 98% could be achieved by proposed DLLME method. The
268
method presented good sensitivity and LODs in the range of 0.3–0.7 µg L-1[22]. From above, it
269
is clear that for determination of BZDs, DLLME-UPLC protocol [18, 19] is more time efficient
270
(analysis time ≤ 17 min) in comparison to DLLME-HPLC method (analysis time 27 min).
271
Khodadoust et al. [23] successfully applied DLLME-HPLC method for the quantitative
272
determination of chlordiazepoxide, a BZD, in various samples such as water, urine, plasma and
273
chlordiazepoxide tablets. Central composite design (CCD) was applied to investigate the effect
274
of different parameters such as volume of extraction and disperser solvent, ionic strength and pH
275
value, on extraction efficiency of DLLME. Under optimum conditions, 210 µL of CF (extraction
276
solvent) and 1.8 mL of MeOH (disperser solvent) were injected in aqueous sample followed by 5
277
min of centrifugation at 5000 rpm. The matrix effect was lowered by diluting the urine and
278
plasma samples. For chlordiazepoxide, this method was proven to be robust, rapid requiring an
279
analysis time of less than 15 min and highly sensitive with a detection limit of 0.0005 µg mL-1.
280
Further, the chromatograms of actual urine sample showed no interfering peaks, which confirm
281
the selectivity of this method towards chlordiazepoxide.
282
Zarei et al. [24] reported a simple analytical method based on coupling of DLLME with
283
UV-Visible spectrophotometry for the determination of barbituric acid in water and biological
284
samples. Firstly, barbituric acid was reacted with p-dimethylaminobenzaldehyde resulting in a 11 Page 11 of 40
285
colored product which gave maximum absorption at 468 nm. The colored complex thus formed
286
was extracted in CF (extraction solvent, 100 µL) using MeOH as disperser solvent. The LOD
287
achieved by proposed method i.e. 0.002 µg mL-1, was superior to previously reported method
288
such as CE (0.070 µg mL-1), voltametry-MIP (1.6 µg mL-1) etc. Recoveries of barbituric acid in
289
tablets, urine and serum sample were found to be in the range of 94.2–105% with 1.64% of RSD.
290
In order to demonstrate the selectivity of the proposed method for barbituric acid (50 ng mL-1),
291
interference studies were also performed with variety of ions and compounds (200 µg mL-1)
292
which revealed that presence of different ions/compounds in water have no significant influence
293
on extraction.
294
2.3. Analysis of opium alkaloids, opiates and other alkaloids
295
Opium is the dried latex obtained from opium poppy i.e. Papaver somniferum. It contains
296
several alkaloids among which five major alkaloids are morphine, codeine, thebain, narcotine
297
and papeverine. Amongst these five, first three are well known for their analgesic properties. For
298
the determination of these opium alkaloids (morphine, papevarine, codeine, noscapine and
299
thebain) in human urine samples, a simple, rapid and sensitive method has been developed by
300
combining DLLME with HPLC-UV. A mixture of ACE (disperser solvent, 1000 µL) and CF
301
(extraction solvent, 88 µL) was injected rapidly into 10 mL of stock standard solution of opium
302
alkaloid or human urine samples which results in cloudy solution. This is followed by
303
centrifugation and analysis of sedimented phase by HPLC-UV. The effect of varied parameters
304
such as type and volume of extraction and disperser solvent, pH and salt addition on EFs of
305
DLLME procedure was studied. In this case, salt addition did not show any positive influence on
306
EF, hence was not used for real sample analysis. The LODs for opium alkaloids were found to be
307
in the range of 0.2–10 µg L-1 with EFs of 63–104.5. The DLLME-HPLC-UV method was found
308
to be rapid, as it took lesser time (<4 min) in comparison to previously reported methods (>20 –
309
40 min). Similarly, LODs of the present method (0.2–10 µg L-1) were found much higher as
310
compared to previously published methods i.e. 1.6–120 µg L-1[25].
311
In their subsequent publication, DLLME with SFO was applied for the extraction and
312
preconcentration of opium alkaloids in human plasma followed by HPLC analysis. Here, 1-
313
undecanol was used as extraction solvent which is having low density than water and lower
314
melting points than room temperature. Opium alkaloids were extracted from plasma sample after 12 Page 12 of 40
315
protein precipitation using 15% zinc sulfate-MeCN (50:40, v/v) solution. DLLME was
316
performed by rapidly injecting a mixture of ACE (500 µL) along with 1-undecanol (30 µL) into
317
5 mL of aqueous solution containing 1% NaCl (pH 9). After centrifugation, sample was placed
318
in an ice bath to solidify the floating organic droplet. This SFO was transferred to another vial,
319
where it was allowed to melt and subjected for HPLC analysis. The LODs achieved were 0.5–5
320
µg L-1 for all analytes with EFs in the range of 110.4–165 [26]. With a view to downgrade the
321
matrix effect, urine samples were subjected to dilution prior to DLLME in both methods [25,
322
26].
323
Meng et al. [16] successfully coupled CE-UV with DLLME for determining heroin in
324
forensic samples. IPA (0.5 mL) and CF (41 µL) were used as disperser and extraction solvents,
325
respectively, and injected into 5 mL of aqueous sample basified up to pH 9. After centrifugation,
326
sedimented phase of CF was subjected to analysis by CE-UV. Extraction recovery of heroin by
327
DLLME was found to be 82.1% with 3% of RSD. The method displayed wide range of linearity
328
from 0.15 to 6000 µg L-1, with an EF of 611. The LOD for heroin was 0.05 µg L-1. Heroin was
329
determined along with amphetamines and ketamine simultaneously. The method was found to be
330
rapid requiring about 15 min for sample preparation and analysis.
331
Fentanyl, alfentanil and sufentanil are three widely used synthetic opioid analgesics,
332
which are structurally different from opium alkaloids but have similar pharmacological effects.
333
Fentanyl is 50 to 100 times more powerful than morphine, whereas alfentanil is about 5 to 10
334
times more potent than fentanyl. Saraji et al. [27] compared two microextraction techniques i.e.
335
DLLME and hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) for the extraction of
336
fentanyl, alfentanil and sufentanil from biological fluids. In case of DLLME, fentanyl, alfentanil
337
and sufentanil were extracted using MeOH (2 mL) as disperser solvent along with CF (162 µL)
338
as extraction solvent from urine and plasma samples. After centrifugation, the sedimented phase
339
was evaporated and reconstituted in 120 µL of MeCN:water (50:50 v/v). For HF-LLLME,
340
analytes were extracted for 20 min at 45°C using 0.05 M sulfuric acid as acceptor phase. When
341
compared, DLLME exhibited lower range of detection limits that ranged from 0.4 to 1.9 μg L-1
342
whereas HF-LLLME showed LODs in the range of 1.1 to 2.3 μg L-1. The EFs for DLLME (275–
343
325) were also greater than HF-LLLME (190–237). Additionally, DLLME was found to be
344
faster than HF-LLLME for the extraction of analytes from urine and plasma samples. The 13 Page 13 of 40
345
separation and identification of analytes was carried out on HPLC equipped with UV-Vis diode
346
array detector. Recently, a DLLME-GC-MS method has been developed for the determination of
347
fantanyl in urine samples using chlorobenzene and 2-propanol as disperser and extraction
348
solvent, respectively. Interestingly, in this protocol the amount of urine sample used for DLLME
349
was greatly reduced to 800 µL without compromising the LOD of the method viz. 1 ng mL-1
350
[28].
351
Ranjabari et al. [29] reported a DLLME method coupled to HPLC-UV for the
352
preconcentration and analysis of methadone in four matrices i.e. human urine, saliva, plasma and
353
sweat samples. Methadone is a synthetic opioid which is used in the treatment of opiate
354
dependence. For the DLLME of methadone in 10 mL of aqueous samples, 2.5 mL of MeOH and
355
250 µL of CF were used as disperser and extraction solvents, respectively. Sedimented phase
356
obtained after centrifugation was evaporated and reconstituted in HPLC grade MeOH and
357
injected in HPLC system for analysis. Before DLLME, to keep methadone completely in its
358
molecular form, pH was adjusted at 10. LOD of methadone were studied in distilled water, urine,
359
plasma, saliva and sweat and were found to be in the range of 0.22–25.12 ng mL-1 with EFs in
360
the range of 98.26–100.34. As compared to traditional methods, such as SPE-HPLC and LLE-
361
HPLC, this method revealed greater sensitivity. Simultaneous extraction and analysis of 12
362
opiates (morphine, codeine, ethylmorphine, fentanyl, pethidine, buprenorphine, nalbuphine,
363
dextromethorphan,
364
diphenylpyrrolidine and 6-monoacetylmorphine) were performed using DLLME coupled to CE-
365
TOF-MS. Isopropanol (1.4 mL) and DCM (0.6 mL) were used as disperse and extraction solvent
366
to extract opiates from basified urine sample. The study displayed LODs in the range of 0.25–10
367
ng mL-1. Except for D-propoxyphene, dextromethorphan and methadone, no other opiates
368
demonstrated any matrix effect [15].
methadone,
D-propoxyphene,
2-ethylidene-1,5-dimethyl-3,3-
369
For the first time, DLLME has been hyphenated with injection port silylation (IPS) by
370
Jain et al. [30] and has been applied for the determination of quinine (QN) in urine samples. In
371
this procedure, QN has been extracted from urine samples using EtOH and DCM as dispersive
372
and extraction solvent, respectively. After centrifugation, 1 µL of sedimented phase was injected
373
in GC-MS followed by co-injection of 1 µL BSTFA+TMCS (99:1 v/v) i.e. derivatizing reagent.
374
The added advantage of this procedure is that it eliminates the need of lengthy reaction time, 14 Page 14 of 40
375
extra heating conditions and large volume of derivatizing reagent for the derivatization of
376
quinine. Analysis of QN by DLLME-GC-MS method took less than 18 min. Furthermore,
377
coupling of IPS with DLLME resulted in a fast, eco-friendly and economic analytical method.
378
The LOD for quinine in urine sample was found to be 5.4 ng mL-1. More recently, Fernandez et
379
al. [31] united DLLME with HPLC-PDA (photodiode array detector) for the extraction and
380
simultaneous determination of morphine, 6-acetylmorphine, methadone, cocaine and
381
benzylecgonine in plasma samples. For the deproteinization purpose, MeCN was used which was
382
further served as disperser solvent along with CF as extraction solvent. The LODs of the
383
proposed method for analytes under investigation were found to be in the range of 13.9–28.5 ng
384
mL-1. To access the selectivity of the method, several blank samples were analyzed by developed
385
method. The method showed good selectivity, as there were no interferences from matrix
386
components at the retention time of target drugs.
387
Nicotine is the principle alkaloid of tobacco and cotinine is one of its metabolite in
388
human body. A DLLME-SFO method coupled to HPLC-UV has been proposed for the
389
determination of nicotine and cotinine in urine samples. In this method, addition of disperser
390
solvent has shown negative impact on the extraction efficiency of cotinine. Therefore, author
391
added no disperser solvent in DLLME procedure; however, manual shaking was applied to form
392
the emulsion of extraction solvent i.e. mixture of undecanol and CF (1:1 v/v). Herein, due to
393
difference in the polarity of low density extraction solvents (viz. 1-undecanol, 1-dodecanol and
394
hexadecane) and cotinine, none of them was able to extract cotinine. Therefore, a mixture of
395
binary extraction solvent was used to extract both nicotine and cotinine simultaneously. The
396
LODs found for both analytes was 0.002 µg mL-1 [32].
397
2.4. Analysis of cannabinoids
398
Cannabinoids are naturally occurring terpeno-phenolic compounds found in Cannabis
399
plant. The main psychoactive and principle compounds of cannabis plants are Δ9-
400
tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN). CNB is a major
401
breakdown product of THC [33]. Moradi et al. [34] developed an analytical method combining
402
SA-DLLME coupled to HPLC-UV for the determination of cannabinoids (THC, CBD and CBN)
403
in urine samples. For DLLME, an environment friendly surfactant i.e. tetradecyl tremethyl
404
ammonium bromide was used as disperser solvent along with toluene as extraction solvent. In 15 Page 15 of 40
405
this study, for disperser solvent, cationic, anionic and non-ionic surfactant; and for extraction
406
solvent, toluene, 1-octanol, and 1-dodecanol were screened using one-variable-at-a-time (OVAT)
407
approach. For the optimization of selected factors (pH, volume of toluene, ionic strength and
408
surfactant concentration), face centered cube CCD was used. Under the optimized conditions,
409
LOD were found to be in the range of 0.1–0.5 µg L-1, with preconcentration factors ranging from
410
190–292. The proposed SA-DLLME-HPLC-UV method took about 15 min for a single analysis.
411
Both positive and negative matrix effect was observed when the proposed SA-DLLME method
412
was applied to real urine samples, as relative recoveries for target analytes were found to be in
413
the range of 83.2–117.1.
414
2.5. Analysis of antidepressant drugs
415
Antidepressants are class of psychoactive drugs which are used for the treatment of major
416
depressive disorders e.g. tricyclic antidepressants (TCA). However, the overdose of TCA may
417
results in arrhythmia, hypertension and in some cases death. Cases of suicides are also prominent
418
due to self-ingestion of TCAs [35]. Xiong et al. [36] developed a DLLME-HPLC-UV method
419
for the extraction and determination of psychoactive drugs such as amitryptiline, clomipramine
420
and thioridazine in urine samples. Factors which impacts the extraction efficiency of DLLME
421
such as type and volume of extraction and disperser solvent, pH and ionic strength were
422
optimized before performing real sample analysis. Rapid injection of MeCN (0.5 mL, disperser
423
solvent) and CCl4 (20 µL, extraction solvent) into 5 mL of aqueous solution resulted into
424
formation of a cloudy solution which was centrifuged. In case of aqueous standard, a tiny droplet
425
of CCl4 was sedimented at the bottom of centrifuge tube, but in case of urine samples, lipids got
426
co-sedimented along with CCl4. For analysis, the co-sedimented lipid was dissolved in 200 µL of
427
MeCN, filtered and then analyzed by HPLC. The LODs were reported in the range of 3–8 ng
428
mL-1 for all target analytes with absolute recoveries of 96–101% in urine samples. Although
429
under the selected conditions, there were no interferences of endogenous substances from urine
430
sample on the determination of target analytes, however, the additional step of filtration of co-
431
sedimented lipid made the procedure more time consuming and labour-intensive.
432
DLLME was hyphenated with GC-MS for the analysis of five TCAs drugs i.e.
433
imipramine, desipramine, amitriptyline, nortriptyline and clomipramine in urine samples. Since
434
the structure of desipramine and nortriptyline has a secondary amino group, hence, derivatization 16 Page 16 of 40
435
was required to convert them into less polar and more volatile derivatives which are amenable
436
for GC-MS analysis. For this purpose, 5 µL of acetic anhydride was used for derivatization of
437
desipramine and nortriptyline. Acetic anhydride was injected into aqueous sample along with
438
disperser and extraction solvent i.e . MeOH and CCl4, respectively. Combination of DLLME
439
with GC-MS was found to be rapid and sensitive in comparison to previously reported methods
440
such as LLE-LC, SPME-LC and LPME-LC which took 40–180 min for analysis of same
441
analytes. The LODs of TCAs subjected to DLLME-GC-MS analysis were found to be in the
442
range of 0.2–0.5 ng mL-1 [35]. As there is no formation of lipidic solid sediment at the bottom of
443
centrifuge tube, the present method eliminates the need of filtering the sample before injection,
444
thus, one of the added advantage of this method in comparison to previously mentioned method
445
[36] is that, the sedimented phase could be injected directly in GC-MS [35].
446
A newly developed DLLME-HPLC-UV method was utilized to determine two TCA
447
drugs i.e. imipramine and trimipramine in urine samples after optimization by response surface
448
methodology (RSM). Disperser and extraction solvent were MeCN and CF, which were selected
449
on the basis of results obtained from OVAT approach. Seven factors were optimized using RSM
450
viz. volume of extraction and disperser solvent, salt percentage, pH, centrifugation time, reaction
451
time and centrifugation speed. Under optimized experimental conditions, the LOD observed for
452
imipramine and trimipramine were 0.6 ng mL-1 which was much lower in comparison to earlier
453
publications such as SPE-GC-nitrogen phosphorous detector (SPE-GC-NPD), CE-TOF-MS,
454
HPLC-MS, SPE-HPLC-UV etc. The EFs were 161.7 and 186.7 for imipramine and
455
trimipramine, respectively. The proposed method could be able to determine the concentration of
456
target analytes in urine samples after 5 hr of 10 mg dosage that was about 21 ng mL-1. To
457
compensate the matrix effect, instead of diluting the urine samples, they were firstly hydrolyzed
458
using 10M KOH followed by ultrasonication for 5 min. Alkaline hydrolysis allowed the
459
precipitation of most of the interfering compounds such as carbamide, uric acid, calcium salt etc,
460
whereas, ultrasonication breaks down the lipid material [37].
461
Most recently, a new DLLME protocol which replaces the use of disperser solvent with a
462
sugar cube i.e. solid based DLLME (SB-DLLME), has been proposed for antidepressants
463
(fluoxetine, fluvoxamine, tranylcypromine,
464
(mexiletine). In this procedure, for simultaneous derivatization and extraction of targeted drugs,
nortryptiline) and an antiarrythmic drug
17 Page 17 of 40
465
extraction solvent (1,1,2,2-tetrachloroethane) and derivatizing reagent (BCF) were added on a
466
sugar cube which served as disperser solvent. This mixture was added in aqueous sample
467
followed by manual shaking which allowed dissolving of solid disperser i.e. sugar cube. After
468
centrifugation, sedimented phase was analyzed by GC-FID. The LODs and EFs of target
469
analytes were obtained in the range of 1–15 µg L-1 and 228–268 [38].
470
For the first time, DLLME was combined with electromembrane extraction (EME) by
471
Seidi et al. [39] to determine three TCA i.e. amitryptiline, trimipramine and doxepine in urine
472
and plasma samples. A hollow fiber packed from one end and inserted with a cathode from
473
another end was used to extract the analytes. The acceptor solution (100 mM HCl solution) was
474
filled inside the hollow fiber. This fiber was dipped in the liquid sample along with anode. After
475
turning on the electric voltage, extraction was continued for 14 min. The acceptor phase was then
476
transferred into 1 mL of alkaline solution (pH 12) which was subjected to DLLME using MeOH
477
(disperser solvent, 150 µL) and CCl4 (extraction solvent, 10 µL). Complete analysis including
478
EME, DLLME and GC-FID took less than 35 min. Duloxetine, a drug widely used in the
479
treatment of major depressive disorders was determined using HPLC with fluorescence
480
detection. Analyte was extracted by using DLLME-SFO technique employing 1-undecanol as
481
extraction solvent. In this method the sample matrix i.e. plasma was deproteinized using zinc
482
sulfate and MeCN. Therefore, there was no need to use any disperser solvent, as MeCN was
483
acting itself as a dispersant [40].
484
2.6. Analysis of hallucinogens
485
Hallucinogens are drugs that alter the perception and mood of an individual, without
486
stimulating or inhibiting brain activities [41]. Lysergic acid diethylamide (LSD) is considered as
487
one of the most potent hallucinogen. Other than LSD; MDMA and phencyclidine (PCP) are two
488
most widely used hallucinogen drugs. For the analysis of these three drugs (LSD, MDMA and
489
PCP) in human urine samples, Rodríguez et al. [42] combined DLLME method with capillary
490
zone electrophoresis (CZE) and UV detection. Diluted urine sample was basified with 30%
491
ammonia and subjected to DLLME with MeCN and DBM as disperser and extraction solvent,
492
respectively. Factors influencing DLLME such as volume of extraction solvent, volume of
493
disperser solvent and amount of NaCl was optimized by using CCD. The LODs were found to be
494
in the range of 1–4.5 ng mL-1 for all the three analytes. Since the authors found statistically 18 Page 18 of 40
495
significant differences between the slopes of external aqueous standard and DLLME treated
496
aqueous standard at confidence level of 95% for each analyte, they recommended the analysis
497
using matrix matched calibration curves.
498
2.7. Analysis of pesticides
499
Self-poisoning by pesticides is one the most common method of suicide in developing
500
countries. According to a report of World Health Organization (WHO) around 30% of total
501
suicides in low and middle income countries between 1990 and 2007 were due to self-poisoning
502
from agricultural pesticides [43]. Therefore, highly sensitive and rapid analytical methods are
503
extremely needed for the analysis of pesticides in matrices of toxicological importance e.g.
504
tissue, blood and urine. Mudiam et al. [44] have developed a low density-DLLME method
505
coupled to GC-electron capture detection (GC-ECD) for the determination of cypermethrin in
506
tissue and blood samples of cypermethrin treated rats. In this method, tissue samples such as
507
brain, liver and kidney were firstly homogenized in ACE and then centrifuged. The supernatant
508
ACE was used as disperser solvent and mixed with n-hexane (extraction solvent) and rapidly
509
injected into ultrapure water for preconcentration of cypermethrin in n-hexane. Blood samples
510
were diluted with water and subjected to DLLME with the similar procedure. The LOD for
511
cypermethrin in tissue was found to be in the range of 0.098 – 0.314 ng mg-1, whereas in blood,
512
the LOD was 8.6 ng mL-1. Since n-hexane is lighter than water, it eliminates the need of
513
centrifugation while performing low density-DLLME, consequently, in this method the upper
514
layer of n-hexane was used directly for GC-ECD analysis. The same research group developed
515
another US-DLLME method coupled to GC-MS for the analysis of endosulfan and its
516
metabolites (endosulfan ether, endosulfan hydroxyether, endosulfan lactone, endosulfan alcohol
517
and endosulfan sulphate) in urine samples. Significant factors for DLLME such as volume of
518
extraction solvent, volume of disperser solvent, ionic strength, pH, extraction time,
519
centrifugation speed and centrifugation time, were screened using Placket-Burman Design
520
(PBD). The most significant factors which were obtained by PBD were further optimized by
521
CCD. Under optimized conditions, ACE and TCE were used as disperser and extraction solvents,
522
respectively. The LODs for all the analytes in urine were found to be in the range of 0.049–0.514
523
ng mL-1 [45].
19 Page 19 of 40
524
Five triazole pesticides (myclobutanil, uniconzole, penconazole and hexaconazole) have
525
been analyzed in rat plasma samples by HPLC with diode array detection (HPLC-DAD) after
526
extraction and preconcentration using temperature controlled ionic liquid-DLLME. Ionic liquid
527
i.e. 1-hexyl-3-methylimidazolium hexafluorophoshphate was used as extraction solvent together
528
with MeOH as a disperser solvent. Heating temperature and ultrasonication was the factors that
529
significantly affected the recoveries of triazole pesticide from plasma samples. The LODs and
530
EFs for triazole pesticides were found to be in the range of 4–6 µg L-1 and 178-197, respectively.
531
Although the proposed method exhibited high LODs and EFs, the total analysis time which
532
includes ultrasonication, cooling, centrifugation and HPLC run time, was quite high (~45 min)
533
for a single analysis [46].
534
3-phenoxybenzoic acid (3-PBA) and 4-phenoxy-3-hydroxybenzoic acid (OH-PBA) are
535
major metabolite and biomarker of pyrethroid pesticides exposure. A single step derivatization
536
cum extraction method for the determination of 3-PBA in rat brain samples using methyl
537
chloroformate (MCF) as derivatizing reagent in UA-DLLME procedure is developed and
538
combined with large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC-
539
MS-MS). MeOH was used to homogenize rat brain samples and its supernatant after
540
centrifugation was further utilized as disperser solvent. To this MeOH, MCF was added as
541
derivatizing reagent together with TCE as extraction solvent. After DLLME procedure, a volume
542
of 10 µL of sedimented phase was injected in GC-MS-MS system. The LODs for 3-PBA and
543
OH-PBA were found to be 3 and 13 ng g-1, respectively. In this method, the derivatization and
544
extraction could be achieved in a single step as MCF is capable of derivatizing polar analytes
545
directly in aqueous medium at room temperature within seconds [47]. In the same year another
546
method was reported by Mudiam et al. [48] for the determination of 3-PBA in rat liver and blood
547
samples. MIP was synthesized for the selective extraction of 3-PBA from complex biological
548
matrices and the eluent obtained after MISPE was subjected for DLLME followed by IPS in hot
549
GC-MS-MS injection port. Chlorobenzene (CB) served as extraction solvent along with MISPE
550
eluent of MeOH as disperse solvent. Under optimized conditions, the LOD for 3-PBA in blood
551
and liver is 1.82 ng mL-1 and 0.0045 ng mg-1, respectively. Primary advantage of coupling
552
DLLME with MISPE is targeted extraction of 3-PBA from biological matrices which in turn
553
enhances method selectivity and specificity. Additionally, use of IPS instead of conventional in-
554
vial silylation saves time and reagent as well as also cut down the cost of derivatization. 20 Page 20 of 40
555
2.8. Analysis of metals
556
Metals are being used as poisons for centuries because of their easy availability, potency
557
and tastelessness and their symptoms are similar to many of the common poisons [49]. Hydride
558
generation atomic absorption spectrometry (HG-AAS) was coupled to DLLME for the ultra-
559
trace determination of Arsenic (As) in urine and whole blood. For this purpose, ammonium
560
pyrrolidine dithiocarbamate (at pH 4) was used to form complex with As (III). The As (III)
561
complex with ammonium pyrrolidine dithiocarbamate was extracted in ionic liquid. Various
562
factors such as concentration of complexing agent, amount of ionic liquid, centrifugation time
563
and sample volume were studied. Under the optimum conditions LOD and linear range were
564
achieved to be 5 ng L−1 and 0.02-10 μg L−1, respectively [50]. Similarly, DLLME was applied
565
for the extraction of lead (Pb) from human urine samples and determination by graphite furnace
566
AAS. In this method, Pb was chelated with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone during
567
the DLLME procedure wherein EtOH and CCl4 were used as disperser solvent extraction
568
solvent, respectively. At a signal to noise ratio of 3, the LOD of the proposed method was found
569
to be 39 ng L-1. Since interferences could be produced due to the competition of other metal ions
570
for the chelating agent and their subsequent co-extraction with Pb, the authors investigated
571
potential interferences of different metal ions in the developed method. The results revealed that
572
most of the alkaline and alkaline earth metal ions had no interference with the extraction for the
573
reason that their complexes which are formed with chelating reagent had very low stability
574
constants in comparison to Pb-chelating reagent complex [51]. Later on, DLLME method was
575
combined with laser induced-thermal lens spectrometry (LI-TLS) for the determination of Pb in
576
human blood serums. Herein, 1,5-diphenyl thiocarbazone (dithiozone) was used as chelating
577
reagent along with EtOH and CCl4 as disperser and extraction solvent, respectively. The LOD for
578
present method was 0.01 µg L-1. To reduce the matrix effect, a mixture of potassium cyanide and
579
sodium citrate were added in the sample as masking agent. With the sample preparation time of
580
less than 3 min, sample determination time of 6 s, and consumption of microlitres of toxic
581
organic solvents, this method was found to be very rapid and cost-effective [52].
582
Hair is a unique matrix for the assessment of chronic exposure of drugs and metallic
583
poisons in criminal cases. A DLLME-SFO method has been combined with flame atomic
584
absorption spectrometry (FAAS) for the determination of copper in human hair samples. In this 21 Page 21 of 40
585
method, 8-hydroxy quinoline was added in aqueous solution along with EtOH as disperser
586
solvent and 1-undecanol as extraction solvent. Hair samples were acid digested with
587
concentrated nitric acid for overnight. Perchloric acid was added to this mixture and the mixture
588
was heated at 200 °C for 1 hr. After neutralization of the solution with NaOH, the sample was
589
subjected to DLLME-SFO procedure. The LOD for copper achieved by this study was
590
comparable with other previously reported methods such as SPE and DLLME coupled to UV-
591
Vis spectrophotometry [53].
592
2.9. Miscellaneous applications of DLLME in forensic toxicology
593
Beside most routinely encountered narcotics substances and drugs of abuse, DLLME has
594
also been applied for the determination of several drugs of clinical and forensic importance.
595
Pregabalin (PRG) is an anticonvulsant drug which is used treatment of neuropathic pain and
596
epilepsy. However, in recent years, abuse and addiction potential of PRG has been proved. An
597
international team of researchers described PRG as an ideal psychotropic drug for recreational
598
intentions [54]. Recently, a study has been conducted to assess the serum levels of PRG in
599
drivers suspected of driving under the influence of drugs. PRG was detected in 206 samples in
600
which 50% of the samples have shown serum level of PRG above therapeutic range [55]. In
601
2012, Mudiam et al. [56] have reported a DLLME method coupled to GC-MS which allow
602
simultaneous derivatization and extraction of PRG in a single step using ethyl chloroformate as
603
derivatizing reagent. In the first step, pyridine was added in urine samples as a catalyst, followed
604
by rapid injection of a mixture of EtOH, TCE and ethyl chloroformate (disperser solvent,
605
extraction solvent and derivatizing reagent, respectively). In the same work, the authors also
606
developed a SPME method for extraction of PRG from urine samples. However, DLLME
607
method was proven to be superior over SPME in terms of time consumption and cost
608
effectiveness. The LOD for PRG using DLLME method under optimized conditions were found
609
to be 0.022 µg mL-1.
610
Amantadine, a drug used in the treatment of Parkinson disease, has found to cause
611
unexpected intoxication and death in trauma patients [57]. Farajzadeh et al. [58] demonstrated
612
simultaneous derivatization and extraction of amantadine in urine and plasma samples using
613
isobutyl chloroformate as derivatizing reagent along with MeOH as disperser solvent and 1,2-
614
DBM as extraction solvent. DLLME was coupled to GC-FID for the analysis of amantadine. 22 Page 22 of 40
615
Under optimized conditions, the EFs for amantadine was obtained to be in the range of 408–420.
616
The LODs in urine and plasma samples were found to be 2.7 and 4.2 ng mL-1. As done in
617
previous studies, herein, also the method of sample dilution (5 and 10 fold) was followed to
618
reduce the matrix effect. The proposed procedure was sensitive enough to detect amantadine
619
content in urine and plasma samples even after 12 to 24 hours. DLLME was coupled with CE
620
for the selective determination of psychiatric drugs (olanzapine, prochlorperazine dimaleate,
621
trifluoroperazine 2 x HCl, perphenazine, clomipramine HCl and chlorprothixene HCl) in urine
622
samples. Carbon tetrachloride and MeCN have served as extraction and disperse solvent,
623
respectively. The LODs were 0.030–0.75 ng mL-1 for all tested analytes. Excellent EFs in the
624
range of 8080 – 13410 have been achieved by combining DLLME with field-amplified sample
625
injection-CE-UV [59]. A combination of CCl4 and MeCN was used as disperser and extraction
626
solvent to extract cyproheptadine, an antihistamine drug, from urine samples. The sedimented
627
phase obtained after centrifugation was evaporated and reconstituted in mobile phase used for
628
HPLC analysis of cyproheptadine. By the DLLME-HPLC-DAD method, cyproheptadine
629
concentration as low as 13.1 ng mL-1 could be detected with relative recoveries in the range of
630
91.6–101 %. When compared to formerly reported SPE-HPLC-UV method, the proposed
631
method exhibited higher sensitivity, and the use of extraction solvent reduced drastically from
632
6000 µL to 30 µL. Additionally simplicity of operation and low analysis cost are other added
633
benefits of the present method over SPE-HPLC-UV [60].
634
Digoxin, a cardiac glycoside obtained from the foxglove plant, Digitalis lanata, is
635
categorized as toxic principle of plant origin which is occasionally ingested for the purpose of
636
suicide [61, 62]. In 2013, Cheng et al. [63] developed a method combining DLLME with
637
surface-assisted laser desorption/ionization mass spectrometry (SALDI/MS) for the analysis of
638
digoxin in urine samples using ACE and CF as disperser and extraction solvent, respectively.
639
The proposed method was found to be rapid as total analysis time of digoxin in aqueous samples
640
takes less than 10 min. The authors analyzed blank urine samples under optimized conditions to
641
check the selectivity of the method and did not found any endogenous compound in the sample
642
which could interfere with digoxin and internal standard. The LOD and EF for digoxin were 2
643
nM and 252, respectively.
23 Page 23 of 40
644
Anti-epileptic drugs are not generally abused, however, cases of accidental and
645
intentional poisoning as well driving under the influence, are frequently encountered [64].
646
Carbamazepine and zonisamide are two most widely used anti-epileptic drugs. A SA-DLLME
647
method is proposed to extract carbamazepine and zonisamide from urine and plasma samples
648
using cethyltrimethyl ammonium bromide as surfactant along with 1-octanol as extraction
649
solvent. After DLLME samples were analyzed by HPLC-UV. The LODs were found to be in the
650
range of 1.5–2.3 µg L-1 [65].
651
24 Page 24 of 40
652
3. Conclusion and future trends
653
The nature of samples which are analyzed in forensic toxicology laboratories is highly
654
complex. Therefore, a sample preparation technique is desirable which is able to extract and
655
preconcentrate the analytes from dirty and complex matrices. In forensic laboratories, LLE and
656
SPE are the two most dominant sample preparation methodologies. However, since the
657
introduction of DLLME, attention has been paid by analysts and researchers to develop protocols
658
using DLLME, for the analysis of drugs of abuse and other forensically important analytes. By
659
virtue of its simplicity, rapidity, cost effectiveness, eco-friendly nature, high EFs and recoveries
660
offered by this methodology, it has become a popular choice of forensic analysts among other
661
sample preparation methodologies available. This review summarized the applications of
662
DLLME in forensic toxicological analysis.
663
DLLME has been extensively used in the analysis of drugs of abuse, narcotics, pesticides,
664
metals and their metabolites. The ability of DLLME to couple with various analytical
665
instruments such as GC-MS, HPLC, UV visible spectrophotometer, CE etc. makes it one of the
666
most versatile microextraction techniques. DLLME has shown successful application for the
667
extraction of target analytes from routinely encountered samples in forensic toxicology
668
laboratories such as urine, blood, saliva, plasma, tissue and serum. One of the major advantages
669
which DLLME offers over other microextraction technique is its ability of simultaneous
670
derivatization and preconcentration using alkyl chloroformate as derivatizing reagents directly in
671
aqueous medium at room temperature within seconds. Coupling of IPS with DLLME also seems
672
to be promising approach for the analysis of polar analytes by GC-MS, since it eliminates the
673
need of lengthy reaction time, extra heating environment and large amount of costly silylating
674
reagents.
675
The future trends of DLLME in the field of forensic toxicology could be summarized in
676
the following aspects: DLLME has been coupled with GC-MS and HPLC for the determination
677
of pesticides from environmental water samples. Nevertheless, there are limited applications of
678
DLLME for the extraction of pesticides from biological matrices of forensic importance.
679
Similarly, DLLME has been widely applied for the determination of metals in environmental
680
samples, though, analysis of toxic metals from complex matrices such as blood, saliva, plasma,
681
urine, tissue, hair and nail is expected in coming years. Replacement of toxic chlorinated 25 Page 25 of 40
682
extraction solvents in DLLME with solvents having low toxicity is a key point of concern. For
683
this purpose, DLLME-SFO can be promoted and ionic liquids could be of immense use. New
684
perspectives are to be opened in automation of DLLME for substances of toxicological interest
685
to make this method more vibrant, versatile and universal.
686
Acknowledgements
687
Authors are grateful to Dr. S. K. Jain, Dy. Director cum Coordinator, Central Forensic
688
Science Laboratory, Guwahati, and Dr. Mohana Krishna Reddy Mudiam, Senior Scientist, CSIR-
689
Indian Institute of Toxicology Research, Lucknow, for their constant support and guidance
690
throughout the manuscript preparation and onwards.
691
Conflict of Interest
692
There are no financial or other relations that could lead to a conflict of interest.
693
26 Page 26 of 40
694 695 696 697 698 699 700 701 702 703
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778
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779
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781
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795
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811
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818
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822
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823
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824
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826
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827
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839
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841
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844
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861
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862
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863
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864 865
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867
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870
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871
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56. M.K.R. Mudiam, A. Chauhan, R. Jain, R. Ch, G. Fatima, E. Malhotra, R.C. Murthy,
877
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878
and sensitive determination of pregabalin in urine and pharmaceutical formulations after
879
ethyl chloroformate derivatization followed by gas chromatography-mass spectrometric
880
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881 882
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891
60. M. Maham, V. Kiarostami, S.W. Husain, P.A. Azar, M.S. Tehrani, M.K. Sharifabadi, H.
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899
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900
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901
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902
assisted laser desorption/ionization mass spectrometry with dispersive liquid-liquid
903
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904
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905
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906
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907
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33 Page 33 of 40
908
65. M. Behbahani, F. Najafi, S. Bagheri, M.S. Bojdi, A. Bagheri, Application of surfactant
909
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910
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911
(2013) 25-31.
912
34 Page 34 of 40
913
Figure captions
914
Fig. 1.Dispersive Liquid-Liquid Microextraction (DLLME)
915
Fig. 2. DLLME-SFO
916
35 Page 35 of 40
917
36 Page 36 of 40
918
Table 1. Applications of DLLME in forensic toxicology Extraction solvent 1-undecanol 1-undecanol isobutyl chloroformate
Analyte
Matrix
Disperser solvent
AP, MA AP, MA
urine urine
SDS MeCN
MA, MDMA
urine
MeOH
MDMA, LSD, PCP
urine
MeCN
DBM
APs and their derivatives, opiates, cocaine and its metabolites, pharmaceuticals
urine
IPA
DCM
0.1–10 ng mL-1
IPA
CF
0.08–0.20 µg L-1
MeCN
CS2
0.05–7 µg L-1
MeOH
CF
ACE
CF
ACE
CF
0.02–0.05 µg L-1
urine
IPA
DCM
urine
EtOH
DCM
water, urine, plasma, chlordiazepox
MeOH
CF
MDA, MDMA, MDEA, MDPA
seized forensic samples urine and plasma
BZD
plasma
MA, MDMA, ketamine, heroin
BZD BZD 7aminoflunitrazepam alprazolam, oxazepam, diazepam chlordiazepoxide
urine and waste water urine and other matrices
LOD
EF
Analytical technique
Ref.
2 and 3 µg L-1 8 and 2 µg L-1 2 and 18 ng mL-1 1, 4.41 and 4.52 ng mL-1
56, 48 58.5, 62.4
HPLC HPLC
[11] [12]
427, 285
GC-FID
[13]
CE-UV
[14]
CE-UV and CE-TOF-MS
[15]
CE-UV
[16]
GC-FID
[17]
HPLC & UPLC
[18]
UPLC
[19]
3895–7222
GC-FID
[20]
0.025 ng mL-1
20
LC-ESI-MS
[21]
0.3–0.7 µg L-1
208–226
HPLC
[22]
HPLC
[23]
545–611
1.7–10.6 ng mL-1 0.6–6.2 ng mL1
0.0005 µg mL1
37 Page 37 of 40
barbituric acid opium alkaloids opium alkaloids fentanyl, alfentanil and sufentanil fantanyl methadone QN morphine, 6-acetyl morphine, methadone, cocaine, benzylecgonine
ide tablets urine, serum, tablet urine plasma urine and plasma urine urine, plasma, saliva, sweat urine plasma
MeOH
CF
0.002 µg mL-1
30
ACE ACE
CF 1-undecanol
63–104.5 110.4–165
MeOH
CF
275 –325
HPLC
[27]
2-propanol
chlorobenzene
GC-MS
[28]
MeOH
CF
HPLC-UV
[29]
EtOH
DCM
0.2–10 µg L-1 0.5–5 µg L-1 0.4 to 1.9 μg L-1 1 ng mL-1 0.22–25.12 ng mL-1 5.4 ng mL-1
UV-Visible Spectrophotometer HPLC-UV HPLC-UV
GC-MS
[30]
MeCN
CF
13.9–28.5 ng mL-1
HPLC-PDA
[31]
undecanol and CF (1:1 v/v)
0.002 µg mL-1
90.5–95.2
HPLC
[32]
190–292
HPLC-UV
[34]
98.26– 100.34
[24] [25] [26]
nicotine and cotinine
urine
cannabinoids
urine
tetradecyl tremethyl ammonium bromide
toluene
0.1–0.5 µg L-1
urine
MeOH
MeOH
0.2–0.5 ng mL-1
GC-MS
[35]
urine
MeCN
CCl4
3–8 ng mL-1
HPLC-UV
[36]
urine
MeCN
CF
HPLC-UV
[37]
plasma and
sugar cube
1,1,2,2-
GC-FID
[38]
imipramine, desipramine, amitriptyline, nortriptyline, clomipramine amitryptiline, clomipramine, thioridazine imipramine and trimipramine antidepressants and
0.6 ng mL-1 each 1–15 µg L-1
161.7 and 186.7 228–268
38 Page 38 of 40
antiarrythmic amitryptiline, trimipramine and doxepine duloxetine
urine
tetrachloroethane
urine and plasma
MeOH
CCl4
0.25–15 µg-1
383–1065
GC-FID
[39]
Plasma
MeCN
1-undecanol
98
HPLC-FLD
[40]
cypermethrin
tissue and blood
ACE
n-hexane
477-689
GC-ECD
[41]
endosulfan and its metabolites
urine
ACE
TCE
2.5 ng mL-1 0.098 – 0.314 ng mg-1; 8.6 ng mL-1 0.049 – 0.514 ng mL-1
GC-MS
[42]
4–6 µg L-1
178-197
HPLC-DAD
[43]
5 ng L−1
HG-AAS
[50]
39 ng L−1
Graphite Furnace AAS
[51]
EtOH EtOH
CCl4
0.01 µg L-1
LI-TLS
[52]
PRG
urine human blood serum urine
1-hexyl-3methylimidazoli um hexafluorophosh phate (1-butyl3methylimidazol ium hexafluorophosp hate CCl4
EtOH
TCE
GC-MS
[56]
psychiatric drugs
urine
MeCN
CCl4
CE-UV
[59]
cyproheptadine digoxin
urine urine
CCl4 CF
HPLC-DAD SALDI/MS
[60] [63]
carbamazepine and zonisamide
urine and plasma
MeCN ACE cethyltrimethyl ammonium bromide
0.022 µg mL-1 0.030–0.75 ng mL-1 13.1 ng mL-1 2 nM
1-octanol
1.5–2.3 µg L-1
HPLC-UV
[65]
triazole pesticides
rat plasma
As
urine and whole blood
Pb Pb
MeOH
8080 – 13410 252
919 39 Page 39 of 40
920 921
66.
40 Page 40 of 40
S0165-9936(15)00255-1 http://dx.doi.org/doi:10.1016/j.trac.2015.07.007 TRAC 14528
To appear in:
Trends in Analytical Chemistry
Please cite this article as: Rajeev Jain, Ritu Singh, Applications of dispersive liquid-liquid microextraction in forensic toxicology, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi:10.1016/j.trac.2015.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Applications of dispersive liquid-liquid microextraction in forensic toxicology
2
Rajeev Jain1*, Ritu Singh2*
3 4
1
5
Home Affairs, Govt of India, H. No. 16, Lachit Borphukhan Path, Tetelia, Gotanagar, Guwahati
6
(Assam) – 781033, India
7
2
8
Rajasthan, Kishangarh, Ajmer (Rajasthan) – 305817, India
9
*Corresponding Authors email: +, [email protected]
10
Central Forensic Science Laboratory, Directorate of Forensic Science Services, Ministry of
Department of Environmental Science, School of Earth Sciences, Central University of
Fax:+91-361-2571148
1 Page 1 of 40
11
Highlights
12
Applications of DLLME for drugs of abuse and poisons
13
Protocols of DLLME for various biological matrices
14
Capability of DLLME for simultaneous derivatization and extraction for drugs of abuse
15
Hyphenation of DLLME with injector port silylation
16 17
Abstract
18
Among several techniques available for micro-extraction, one which is becoming an increasingly
19
popular choice of forensic toxicologists is dispersive liquid-liquid microextraction (DLLME).
20
DLLME is a simple, fast, inexpensive and environmentally benign microextraction technique
21
which offers high enrichment factors and extraction efficiencies. Its coupling with broad range of
22
analytical instruments makes it a versatile microextraction method. DLLME has found wide
23
range of applications in the field of forensic toxicology such as analysis of narcotic substances,
24
drugs of abuse, hallucinogens, cannabinoids, metals, pesticides etc. Furthermore, the capability
25
of DLLME for simultaneous derivatization and extraction, and its coupling with injection port
26
silylation (IPS) makes the analysis of polar analytes by GC-MS simpler and faster. The present
27
review focuses on various applications and procedures of DLLME for various classes of drugs
28
and poisons of forensic interests since its introduction. In addition, viability of future trend for
29
DLLME in forensic toxicology has also been addressed.
30 31
Keywords: Dispersive liquid-liquid microextraction, drugs of abuse, forensic toxicology, amphetamines, cannabinoids, opium alkaloids, benzodiazepines, cocaine
32
2 Page 2 of 40
33
1. Introduction
34
Forensic toxicology is the scientific study of poison in relation to the law. Forensic
35
toxicologists are asked for the detection and quantification of toxicant(s) in tissue, organs and
36
body fluids. Owing to highly complex nature of matrices of toxicological interests such as blood,
37
plasma, urine, tissue, saliva, vitreous humor and hair; sample preparation for the analysis of
38
various drugs/poisons still remains a challenge to the forensic toxicologist. Since, in forensic
39
analysis, every case is unique, and the nature of target analytes is unknown, therefore it is very
40
difficult to standardize the whole analytical procedure, like in other branches of analytical
41
chemistry. In such cases systematic toxicological analysis (STA) is required. Moreover, the
42
sample availability in most of the toxicological cases is limited and the analysis is usually
43
untargeted, therefore, it is necessary to have a sample preparation methodology which requires
44
least amount of sample. On the other hand, in cases where forensic toxicologists are asked for
45
targeted analysis of a specific drug/poison only, the analytical approach is quite easy and
46
different from STA.
47
As the drugs and their metabolites are generally present at trace levels in biological fluids;
48
the whole analytical procedure should be sensitive enough to detect the analytes with accuracy
49
and selectivity. Due to extreme complex nature of the samples received in forensic toxicology
50
laboratories, it is not recommended to subject the sample, directly for analysis on any analytical
51
instrument. Thus, a sample preparation methodology is necessary which could make the sample
52
suitable for instrumental analysis. For accurate measurements, these methodologies should:
53
require minimum amount of sample for the extraction of target analyte(s)
54
consume least amount of toxic organic solvents,
55
be time efficient
56
give reproducible recoveries,
57
produce a clean extract devoid of any impurities and matrix interferences,
58
be compatible with derivatizing reagents in case of extraction of polar analytes,
59 60
be compatible for coupling with different analytical instruments.
61
One method which is mostly practiced in forensic toxicology laboratories, for the extraction
62
and preconcentration of analytes from toxicological matrices is liquid-liquid extraction (LLE). It
63
consists of three steps viz. (i) extraction (ii) derivatization (in case of polar analytes) and (iii) 3 Page 3 of 40
64
preconcentration of extracted analytes. Despite of LLE wide usage, some limitations which puts
65
set back to this method are; (a) they require large amount of toxic organic solvents for the
66
extraction of drugs and poisons from biological matrices, (b) time consuming, (c) multi-step
67
procedures (d) laborious (e) high economic expenses (f) low enrichment factors. Since
68
toxicological samples are quite dirty, therefore in LLE, before preconcentration, an additional
69
step of sample cleanup is also required. Apart from that, LLE require more than one extraction,
70
which consumes lot of sample, and affects the reproducibility of the results too. Sometimes
71
emulsion formation and matrix interferences also create hurdles in sample preparation. These
72
drawbacks make analytes more prone to loss during sample preparation, which in turn affects the
73
reproducibility of the analytical method. Additionally; use of large amount of toxic organic
74
solvents may lead to environmental pollution and health problems. Solid-Phase extraction (SPE)
75
is another extraction cum cleanup technique which is being used in forensic laboratories for long
76
time. In comparison to LLE, SPE requires lesser amount of organic solvent for extraction.
77
Nowadays, commercial SPE cartridges with small amount of stationary phases (in milligrams)
78
are also available which requires only microliters of solvent for extracting analytes from
79
samples.
80
With a view to improve traditional sample preparation methodologies, researchers over
81
the globe started focussing on developing microextraction techniques which consumes
82
minimal/negligible amount of extraction solvent (zero or few μL), less time and labour,
83
economic and on the top of all, environmental friendly. One of such techniques is solid-phase
84
microextraction (SPME). It was invented in 1990s by Arthur and Pawliszyn who revolutionized
85
the sample preparation procedures [1]. SPME is a solvent-less microextraction procedure which
86
consists of a fiber coated with a polymeric stationary phase on surface of which suitable type of
87
analytes get adsorbed or absorbed. The main advantage of SPME over conventional SPE and
88
LLE includes; (a) environmentally benign (b) low sample requirement and (c) relatively higher
89
extraction efficiencies. SPME has been widely applied for the extraction of various analytes from
90
different matrices. The applications of SPME in biomedical and forensic toxicology have been
91
well reviewed earlier [2, 3]. However, SPME is a costly extraction technique and fibers used for
92
the extraction are very fragile and delicate in nature which needs special maintenance and
93
protection. Also, SPME fibers need periodical replacement as their lifetime is limited. Besides
94
these issues, the problem of sample carry over is a major setback of this method [4]. 4 Page 4 of 40
95
Dispersive liquid-liquid microextraction (DLLME), a miniaturized LLE technique, was
96
introduced by Rezaee et al. in 2006 [5]. Since then it gained lot of attention from the analysts and
97
has been used widely for the extraction of various types of analytes from different matrices such
98
as water, tissue, biological fluids and food matrices etc [6]. DLLME is based on ternary
99
component solvent system which consists of aqueous phase, disperser solvent and extraction
100
solvent. In classical DLLME, the extraction solvent is heavier than water. The mixture of
101
disperser solvent and extraction solvent is injected rapidly in aqueous phase, which quickly
102
results in the formation of a cloudy solution. This cloudy solution consists of tiny droplets of
103
extraction solvent dispersed throughout the aqueous phase achieving an enormous contact area
104
between these two, consequential in fast equilibrium. This is followed by centrifugation at high
105
speed, which settles down the droplets of heavy density extraction solvent as sedimented phase,
106
which is further used for analysis (Fig. 1). In classical DLLME, usually toxic chlorinated
107
solvents are used as extraction solvents e.g. chloroform (CF), trichloroethylene (TCE), carbon
108
tetrachloride (CCl4) etc. In order to surmount this limitation, Xu et al. [7] proposed a modified
109
version of DLLME based on solidification of floating organic droplet (DLLME-SFO) which
110
used low density extraction solvents having melting points below room temperature, such as 1-
111
dodecanol, 1-undecanol, hexadecanol etc. The DLLME-SFO procedure consists of rapid
112
injection of mixture of disperser and extraction solvent into aqueous sample followed by
113
centrifugation. By virtue of low density of extraction solvent, a droplet of extraction solvent
114
starts floating over the surface. Now the sample vial is transferred into an ice bath for some time
115
which facilitates the solidification of floating organic droplet due to its lower melting point
116
below room temperature. The solidified droplet is then transferred into another conical vial
117
where it is allowed to melt and then subjected for analysis (Fig. 2) [7]. Some researchers
118
combined DLLME with other extraction techniques such as SPE and molecularly imprinted
119
polymer-SPE (MISPE) where the eluent collected from SPE is preconcentrated using DLLME
120
[8-9].
121
Since its introduction, DLLME has been applied for the determination of several drugs
122
and poisons of forensic interest such as opiates, hallucinogens, amphetamines, cocaine and its
123
metabolites, antidepressants, barbiturates, cannabinoids, metals and pesticides etc. This review
124
article gives a comprehensive view over applications of DLLME in the field of forensic
125
toxicology along with the detailed outline of the procedures followed for varied classes of 5 Page 5 of 40
126
forensic drugs and poisons. The article also provide comparative assessment of various
127
advancements made in the field of DLLME in terms of selectivity, sensitivity, enrichment factors
128
(EFs), matrix effect; cost, speed and complexity of analysis. The viability of future trend for
129
DLLME in forensic toxicology has also been addressed. Table 1 listed the analytes for which
130
DLLME has been used for extraction and preconcentration from forensic samples and the types
131
of disperser and extraction solvent used along with their respective limit of detections (LODs)
132
and EFs.
133
6 Page 6 of 40
134
2. Applications of DLLME in forensic toxicology
135
2.1. Analysis of amphetamines
136
Amphetamines are categorized under synthetic central nervous system stimulants which
137
include amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA)
138
and 3,4-methylenedioxymethamphetamine (Ecstasy or MDMA). According to World Drug
139
Report 2014, the global seizure of amphetamine type stimulants (ATS) from 2003-2012 is 144
140
tons. ATS (excluding ecstasy) comprises of second most commonly used illicit drugs with 13.9
141
million to 54.8 million estimated users [10].
142
A DLLME-SFO procedure was developed for the extraction of AMP and MA from urine
143
samples [11]. In this procedure, disperser solvent was replaced with a surfactant, i.e. sodium
144
dodecyl sulfate (SDS). Since surfactants are amphiphilic in nature, they are soluble in both
145
extraction solvent as well as aqueous phase. Also they possess several analytical advantages of
146
disperser solvents such as non-toxicity, cost effectiveness and compatibility with mobile phase of
147
high performance liquid chromatography (HPLC). For extraction, in 5 mL of urine sample (pH
148
6.4), 0.015 g NaCl was added followed by addition of 56.5 µL of SDS solution (concentration 70
149
µg L-1). To this solution, 31 µL of 1-undecanol (extraction solvent) was injected rapidly to form
150
cloudy solution followed by vortex mixing and centrifugation. After centrifugation the floating
151
organic droplet of 1-undecanol was solidified by keeping the sample vial in ice for 5 min. The
152
solidified floating organic droplet was allowed to melt in a separate vial and 20 µL of this was
153
injected into HPLC for analysis. The LOD for AP and MA were found to be 2 and 3 µg L-1,
154
respectively [11]. Recently, another DLLME-SFO technique for AP and MA in urine samples
155
was published using 1-undecanol as extraction solvent, whereas acetonitrile (MeCN) was used as
156
disperser solvent. Separation and identification of analytes were performed using HPLC with
157
ultraviolet detector (HPLC-UV). The LODs for AP and MA were 8 and 2 µg L-1 with a signal to
158
noise ratio of 3 [12]. In comparison to SDS [11], use of MeCN as disperser solvent in DLLME-
159
SFO, made no significant differences in terms of sensitivity and speed of analysis speed.
160
Djozan et al. [13] applied DLLME for the determination of MA and MDMA in urine
161
samples by gas chromatography-flame ionization detection (GC-FID). Analytes were extracted
162
from urine samples by MI-SPE. As compared to SPE, the major advantage of MIP is its
7 Page 7 of 40
163
selectivity towards the target analyte molecules, which is attributable to its analyte specific
164
cavities formed in course of their synthesis [13]. MIP was synthesized by precipitation
165
polymerization, using MA as a template. The sample was loaded on MI-SPE cartridge and eluted
166
with methanol (MeOH). This MeOH was mixed with butylchloroformate (BCF) which served as
167
disperser solvent as well as derivatizing reagent. After centrifugation, 1 µL of sedimented phase
168
of BCF was injected into GC-FID. The LODs found for MA and MDMA were 2 and 18 ng mL-1,
169
respectively. The applicability of the developed method was demonstrated in two positive cases
170
of MA and MDMA by gas chromatography-mass spectrometry (GC-MS). DLLME has been
171
coupled to capillary electrophoresis with ultraviolet detection (CE-UV) for the extraction and
172
preconcentration of MDMA from urine samples. Dibromomethane (DBM, 0.606 mL) and MeCN
173
(1.508 mL) were used as extraction and disperser solvent for 4 mL of urine sample mixed with 1
174
mL of ultrapure water. The LOD for MDMA was found to be 1 ng mL-1 in spiked urine sample
175
with a signal to noise ratio of 3. Significant matrix effect observed in the study was compensated
176
by plotting matrix matched calibration graphs [14]. A year later, in 2013, Kohler et al. [14]
177
applied DLLME for the extraction of 30 toxicological compounds from different class viz.
178
amphetamines and their derivatives, opiates, cocaine and its metabolites and pharmaceuticals
179
from urine samples followed by capillary CE-UV and time-of-flight mass spectrometer (TOF-
180
MS) analysis. 600 µL of dichloromethane (DCM) as extraction solvent along with 1400 µL of
181
isopropanol (IPA) as disperser solvent was injected rapidly in 4 mL of basified urine sample.
182
After centrifugation, the sedimented phase of DCM was transferred in a polypropylene tube and
183
10 µL of acidified MeOH was added to it. The solution was evaporated to dryness under gentle
184
stream of nitrogen and reconstituted in 30 µL of background electrolyte and water before
185
injection to CE. The extraction recoveries for AP and MDMA were found to be 76 and 75%,
186
respectively; whereas LODs for MAs and their derivatives were found to be in the range of 0.25–
187
1 ng mL-1. In comparison to previous method, no matrix effect for AMs was found [15]. Similar
188
method i.e. DLLME-CE-UV was applied for the chiral separation and determination of multiple
189
illicit drugs i.e. MA, MDMA, ketamine and heroin in seized forensic sample such as banknote,
190
kraft paper, plastic bags and silver paper. The samples were soaked in acetic acid and filtered
191
which was made alkaline by NaOH. To this filtrate, 0.5 mL of IPA (disperser solvent) and 41 µL
192
of CF (extraction solvent) were rapidly injected to form cloud solution. In this study, effect of pH
193
was different for amines and heroin. Amphetamines and ketamine showed increasing recoveries 8 Page 8 of 40
194
up to pH 11, while recovery of heroin decreased above pH 9. Thus, pH 9 was selected as
195
optimum value for further DLLME procedure. The method offered good sensitivity for multiple
196
illicit drugs with the LODs ranging from 0.08–0.20 µg L-1 and EFs ranging from 545–611. The
197
method showed good speed of analysis and the entire target analytes could be separated within
198
10 min [16].
199
Recently, SPE has been combined with DLLME for the preconcentration of MDA,
200
MDMA,
201
methylenedioxypropylamphetamine (MDPA) from urine and plasma samples, followed by GC-
202
FID analysis. Ten mL aliquot of sample was passed through C18 SPE cartridge previously
203
preconditioned with 2 mL of acetone (ACE). The adsorbed analytes on SPE cartridge were then
204
eluted with 1.5 mL of MeCN. This MeCN was used as disperser solvent along with 35 µL of
205
carbon disulphide (CS2) as extraction solvent. After centrifugation, 2 µL of CS2 was injected into
206
GC-FID for analysis. The relative recoveries varied between 76–92.5% for all analytes. LODs
207
were found to be in the range of 0.05–7 µg L-1 [17].
208
2.2. Analysis of sedative and hypnotic drugs
3,4-methylenedioxyethylamphetamine
(MDEA)
and
3,4-
209
Another important group of drugs for forensic toxicologist for which DLLME methods
210
has been developed, are sedatives and hypnotics, such as barbiturates and benzodiazepines
211
(BZD). BZD are widely used for the treatment of insomnia, convulsive attacks and other
212
psychological disorders and are also linked with harmful effects such as addiction with regular
213
use either with alcohol or alone.
214
Fernandez et al. [18] combined ultrasound-assisted DLLME (US-DLLME) with HPLC
215
and ultra-performance liquid chromatography (UPLC) for the comparative analysis of seven
216
BZDs i.e. alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam and
217
tetrazepam. MeOH was used as deproteinizing agent for plasma samples and subsequently as
218
disperser solvent along with chloroform as extraction solvent. To 500 µL of plasma, 2 mL of
219
MeOH was added and centrifuged. The supernatant was mixed with 250 µL of CF and rapidly
220
injected into 4.5 mL of ultrapure water (pH 9). The mixture was then subjected to ultrasonication
221
for 2 min followed by centrifugation and analysis. Under optimized conditions, method exhibited
222
linearity in the range of 0.01–5 µg mL-1. The LODs were varied for different columns in the
223
range of 1.7–10.6 ng mL-1. Apart from that, the total analysis time including DLLME and UPLC 9 Page 9 of 40
224
was less than 15 min [18]. Though HPLC and UPLC showed similar results, however in terms of
225
sensitivity, speed and reduction in solvent and injection volume, UPLC was found to be superior.
226
A year later, same research group again coupled US-DLLME method to UPLC for the
227
determination of seven BZD in urine and waste water samples. Design of experiment (DOE) i.e.
228
Doehlert design was applied for the screening of some important factors which affects the
229
extraction efficiency of DLLME, such as sample pH, extraction solvent volume and
230
ultrasonication time etc. Under optimized conditions, 1.6 mL of ACE along with 160 µL of CF
231
was injected rapidly into 0.5 mL of aqueous sample whose pH was adjusted to 9. Before
232
subjecting the sample to centrifugation, ultrasonication was applied for 4.5 min to enhance the
233
extraction efficiency, which was found superior to manual shaking of 1 min in preliminary
234
experiments. All the BZD were separated within 4 min or UPLC run time. The proposed method
235
reported BZD recoveries from urine samples in the range of 96–114% with percent relative
236
standard deviation (%RSD) in the range of 4.3–9.9%. The method was found to be sensitive in
237
the range of 0.6–6.2 ng mL-1. This protocol took total 17 min for analysis of BZD in urine and
238
hospital wastewater [19]. In the same year, DLLME as a preconcentration technique was
239
combined with SPE for the analysis of three BZD i.e. diazepam, midazolam, and alprazolam in
240
urine and other samples. Analytes were adsorbed on octadecyl silica SPE column and eluted with
241
ACE. This elute (0.5 mL) was used as disperser solvent along with 40 µL of CF as extraction
242
solvent. Analysis of sedimented phase was performed using GC-FID. Combination of SPE with
243
DLLME offered very high EFs (in the range of 3895–7222) and sensitivity (LODs for tested
244
BZD were achieved in the range of 0.02–0.05 µg L-1). To cope up with the matrix interferences,
245
urine samples were diluted with ultrapure water (in ratio of 1:4) before analysis which resulted in
246
good relative recoveries in the range of 90-98% [20].
247
Flunitrazepam
is
another
widely used
BZD,
which
is
metabolized
to
7-
248
aminoflunitrazepam in human body. 7-aminoflunitrazepam is commonly used as a biomarker to
249
determine the concentration of flunitrazepam. An LC-ESI-MS method has been developed for
250
the analysis of 7-aminoflunitrazepam in urine samples using DLLME as an extraction technique.
251
Before extraction urine samples were basified using ammonia and NaCl was added to increase
252
the ionic strength of the solution. IPA (disperser solvent, 500 µL) along with DCM (extraction
253
solvent, 250 µL) was rapidly injected into urine sample and subjected for centrifugation to settle
254
down the droplets of extraction solvent. The sedimented phase of DCM was transferred to 10 Page 10 of 40
255
another vial and evaporated to dryness followed by reconstitution in mobile phase i.e.
256
MeCN:water (20:80 v/v). In order to achieve maximum EFs, different combinations of disperser
257
solvent and extraction solvent were tested. Among them, 500 µL of IPA and 250 µL of DCM
258
gave maximum EFs up to 20. EFs were calculated as the ratio between peak area of 7-
259
aminoflunitrazepam after and before extraction. LOD reported in this study i.e. 0.025 ng mL-1,
260
was found superior to previously reported methods in literature which were in the range of 1–5.6
261
ng mL-1. Owing to quantification, good selectivity for 7-aminoflunitrazepam could be achieved
262
in multiple reaction monitoring (MRM) mode but at the cost of analysis time which reached up
263
to 47 min [21]. In another study, DLLME coupled to HPLC was employed for the extraction of
264
three BZDs i.e. alprazolam, oxazepam and diazepam from urine samples. One mL of Ethanol
265
(EtOH) was used as disperser solvent along with 102 µL of DCM which were injected into 5 mL
266
of distilled water/urine samples followed by centrifugation and analysis. EFs in the range of
267
208–226 and relative recoveries up to 98% could be achieved by proposed DLLME method. The
268
method presented good sensitivity and LODs in the range of 0.3–0.7 µg L-1[22]. From above, it
269
is clear that for determination of BZDs, DLLME-UPLC protocol [18, 19] is more time efficient
270
(analysis time ≤ 17 min) in comparison to DLLME-HPLC method (analysis time 27 min).
271
Khodadoust et al. [23] successfully applied DLLME-HPLC method for the quantitative
272
determination of chlordiazepoxide, a BZD, in various samples such as water, urine, plasma and
273
chlordiazepoxide tablets. Central composite design (CCD) was applied to investigate the effect
274
of different parameters such as volume of extraction and disperser solvent, ionic strength and pH
275
value, on extraction efficiency of DLLME. Under optimum conditions, 210 µL of CF (extraction
276
solvent) and 1.8 mL of MeOH (disperser solvent) were injected in aqueous sample followed by 5
277
min of centrifugation at 5000 rpm. The matrix effect was lowered by diluting the urine and
278
plasma samples. For chlordiazepoxide, this method was proven to be robust, rapid requiring an
279
analysis time of less than 15 min and highly sensitive with a detection limit of 0.0005 µg mL-1.
280
Further, the chromatograms of actual urine sample showed no interfering peaks, which confirm
281
the selectivity of this method towards chlordiazepoxide.
282
Zarei et al. [24] reported a simple analytical method based on coupling of DLLME with
283
UV-Visible spectrophotometry for the determination of barbituric acid in water and biological
284
samples. Firstly, barbituric acid was reacted with p-dimethylaminobenzaldehyde resulting in a 11 Page 11 of 40
285
colored product which gave maximum absorption at 468 nm. The colored complex thus formed
286
was extracted in CF (extraction solvent, 100 µL) using MeOH as disperser solvent. The LOD
287
achieved by proposed method i.e. 0.002 µg mL-1, was superior to previously reported method
288
such as CE (0.070 µg mL-1), voltametry-MIP (1.6 µg mL-1) etc. Recoveries of barbituric acid in
289
tablets, urine and serum sample were found to be in the range of 94.2–105% with 1.64% of RSD.
290
In order to demonstrate the selectivity of the proposed method for barbituric acid (50 ng mL-1),
291
interference studies were also performed with variety of ions and compounds (200 µg mL-1)
292
which revealed that presence of different ions/compounds in water have no significant influence
293
on extraction.
294
2.3. Analysis of opium alkaloids, opiates and other alkaloids
295
Opium is the dried latex obtained from opium poppy i.e. Papaver somniferum. It contains
296
several alkaloids among which five major alkaloids are morphine, codeine, thebain, narcotine
297
and papeverine. Amongst these five, first three are well known for their analgesic properties. For
298
the determination of these opium alkaloids (morphine, papevarine, codeine, noscapine and
299
thebain) in human urine samples, a simple, rapid and sensitive method has been developed by
300
combining DLLME with HPLC-UV. A mixture of ACE (disperser solvent, 1000 µL) and CF
301
(extraction solvent, 88 µL) was injected rapidly into 10 mL of stock standard solution of opium
302
alkaloid or human urine samples which results in cloudy solution. This is followed by
303
centrifugation and analysis of sedimented phase by HPLC-UV. The effect of varied parameters
304
such as type and volume of extraction and disperser solvent, pH and salt addition on EFs of
305
DLLME procedure was studied. In this case, salt addition did not show any positive influence on
306
EF, hence was not used for real sample analysis. The LODs for opium alkaloids were found to be
307
in the range of 0.2–10 µg L-1 with EFs of 63–104.5. The DLLME-HPLC-UV method was found
308
to be rapid, as it took lesser time (<4 min) in comparison to previously reported methods (>20 –
309
40 min). Similarly, LODs of the present method (0.2–10 µg L-1) were found much higher as
310
compared to previously published methods i.e. 1.6–120 µg L-1[25].
311
In their subsequent publication, DLLME with SFO was applied for the extraction and
312
preconcentration of opium alkaloids in human plasma followed by HPLC analysis. Here, 1-
313
undecanol was used as extraction solvent which is having low density than water and lower
314
melting points than room temperature. Opium alkaloids were extracted from plasma sample after 12 Page 12 of 40
315
protein precipitation using 15% zinc sulfate-MeCN (50:40, v/v) solution. DLLME was
316
performed by rapidly injecting a mixture of ACE (500 µL) along with 1-undecanol (30 µL) into
317
5 mL of aqueous solution containing 1% NaCl (pH 9). After centrifugation, sample was placed
318
in an ice bath to solidify the floating organic droplet. This SFO was transferred to another vial,
319
where it was allowed to melt and subjected for HPLC analysis. The LODs achieved were 0.5–5
320
µg L-1 for all analytes with EFs in the range of 110.4–165 [26]. With a view to downgrade the
321
matrix effect, urine samples were subjected to dilution prior to DLLME in both methods [25,
322
26].
323
Meng et al. [16] successfully coupled CE-UV with DLLME for determining heroin in
324
forensic samples. IPA (0.5 mL) and CF (41 µL) were used as disperser and extraction solvents,
325
respectively, and injected into 5 mL of aqueous sample basified up to pH 9. After centrifugation,
326
sedimented phase of CF was subjected to analysis by CE-UV. Extraction recovery of heroin by
327
DLLME was found to be 82.1% with 3% of RSD. The method displayed wide range of linearity
328
from 0.15 to 6000 µg L-1, with an EF of 611. The LOD for heroin was 0.05 µg L-1. Heroin was
329
determined along with amphetamines and ketamine simultaneously. The method was found to be
330
rapid requiring about 15 min for sample preparation and analysis.
331
Fentanyl, alfentanil and sufentanil are three widely used synthetic opioid analgesics,
332
which are structurally different from opium alkaloids but have similar pharmacological effects.
333
Fentanyl is 50 to 100 times more powerful than morphine, whereas alfentanil is about 5 to 10
334
times more potent than fentanyl. Saraji et al. [27] compared two microextraction techniques i.e.
335
DLLME and hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) for the extraction of
336
fentanyl, alfentanil and sufentanil from biological fluids. In case of DLLME, fentanyl, alfentanil
337
and sufentanil were extracted using MeOH (2 mL) as disperser solvent along with CF (162 µL)
338
as extraction solvent from urine and plasma samples. After centrifugation, the sedimented phase
339
was evaporated and reconstituted in 120 µL of MeCN:water (50:50 v/v). For HF-LLLME,
340
analytes were extracted for 20 min at 45°C using 0.05 M sulfuric acid as acceptor phase. When
341
compared, DLLME exhibited lower range of detection limits that ranged from 0.4 to 1.9 μg L-1
342
whereas HF-LLLME showed LODs in the range of 1.1 to 2.3 μg L-1. The EFs for DLLME (275–
343
325) were also greater than HF-LLLME (190–237). Additionally, DLLME was found to be
344
faster than HF-LLLME for the extraction of analytes from urine and plasma samples. The 13 Page 13 of 40
345
separation and identification of analytes was carried out on HPLC equipped with UV-Vis diode
346
array detector. Recently, a DLLME-GC-MS method has been developed for the determination of
347
fantanyl in urine samples using chlorobenzene and 2-propanol as disperser and extraction
348
solvent, respectively. Interestingly, in this protocol the amount of urine sample used for DLLME
349
was greatly reduced to 800 µL without compromising the LOD of the method viz. 1 ng mL-1
350
[28].
351
Ranjabari et al. [29] reported a DLLME method coupled to HPLC-UV for the
352
preconcentration and analysis of methadone in four matrices i.e. human urine, saliva, plasma and
353
sweat samples. Methadone is a synthetic opioid which is used in the treatment of opiate
354
dependence. For the DLLME of methadone in 10 mL of aqueous samples, 2.5 mL of MeOH and
355
250 µL of CF were used as disperser and extraction solvents, respectively. Sedimented phase
356
obtained after centrifugation was evaporated and reconstituted in HPLC grade MeOH and
357
injected in HPLC system for analysis. Before DLLME, to keep methadone completely in its
358
molecular form, pH was adjusted at 10. LOD of methadone were studied in distilled water, urine,
359
plasma, saliva and sweat and were found to be in the range of 0.22–25.12 ng mL-1 with EFs in
360
the range of 98.26–100.34. As compared to traditional methods, such as SPE-HPLC and LLE-
361
HPLC, this method revealed greater sensitivity. Simultaneous extraction and analysis of 12
362
opiates (morphine, codeine, ethylmorphine, fentanyl, pethidine, buprenorphine, nalbuphine,
363
dextromethorphan,
364
diphenylpyrrolidine and 6-monoacetylmorphine) were performed using DLLME coupled to CE-
365
TOF-MS. Isopropanol (1.4 mL) and DCM (0.6 mL) were used as disperse and extraction solvent
366
to extract opiates from basified urine sample. The study displayed LODs in the range of 0.25–10
367
ng mL-1. Except for D-propoxyphene, dextromethorphan and methadone, no other opiates
368
demonstrated any matrix effect [15].
methadone,
D-propoxyphene,
2-ethylidene-1,5-dimethyl-3,3-
369
For the first time, DLLME has been hyphenated with injection port silylation (IPS) by
370
Jain et al. [30] and has been applied for the determination of quinine (QN) in urine samples. In
371
this procedure, QN has been extracted from urine samples using EtOH and DCM as dispersive
372
and extraction solvent, respectively. After centrifugation, 1 µL of sedimented phase was injected
373
in GC-MS followed by co-injection of 1 µL BSTFA+TMCS (99:1 v/v) i.e. derivatizing reagent.
374
The added advantage of this procedure is that it eliminates the need of lengthy reaction time, 14 Page 14 of 40
375
extra heating conditions and large volume of derivatizing reagent for the derivatization of
376
quinine. Analysis of QN by DLLME-GC-MS method took less than 18 min. Furthermore,
377
coupling of IPS with DLLME resulted in a fast, eco-friendly and economic analytical method.
378
The LOD for quinine in urine sample was found to be 5.4 ng mL-1. More recently, Fernandez et
379
al. [31] united DLLME with HPLC-PDA (photodiode array detector) for the extraction and
380
simultaneous determination of morphine, 6-acetylmorphine, methadone, cocaine and
381
benzylecgonine in plasma samples. For the deproteinization purpose, MeCN was used which was
382
further served as disperser solvent along with CF as extraction solvent. The LODs of the
383
proposed method for analytes under investigation were found to be in the range of 13.9–28.5 ng
384
mL-1. To access the selectivity of the method, several blank samples were analyzed by developed
385
method. The method showed good selectivity, as there were no interferences from matrix
386
components at the retention time of target drugs.
387
Nicotine is the principle alkaloid of tobacco and cotinine is one of its metabolite in
388
human body. A DLLME-SFO method coupled to HPLC-UV has been proposed for the
389
determination of nicotine and cotinine in urine samples. In this method, addition of disperser
390
solvent has shown negative impact on the extraction efficiency of cotinine. Therefore, author
391
added no disperser solvent in DLLME procedure; however, manual shaking was applied to form
392
the emulsion of extraction solvent i.e. mixture of undecanol and CF (1:1 v/v). Herein, due to
393
difference in the polarity of low density extraction solvents (viz. 1-undecanol, 1-dodecanol and
394
hexadecane) and cotinine, none of them was able to extract cotinine. Therefore, a mixture of
395
binary extraction solvent was used to extract both nicotine and cotinine simultaneously. The
396
LODs found for both analytes was 0.002 µg mL-1 [32].
397
2.4. Analysis of cannabinoids
398
Cannabinoids are naturally occurring terpeno-phenolic compounds found in Cannabis
399
plant. The main psychoactive and principle compounds of cannabis plants are Δ9-
400
tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN). CNB is a major
401
breakdown product of THC [33]. Moradi et al. [34] developed an analytical method combining
402
SA-DLLME coupled to HPLC-UV for the determination of cannabinoids (THC, CBD and CBN)
403
in urine samples. For DLLME, an environment friendly surfactant i.e. tetradecyl tremethyl
404
ammonium bromide was used as disperser solvent along with toluene as extraction solvent. In 15 Page 15 of 40
405
this study, for disperser solvent, cationic, anionic and non-ionic surfactant; and for extraction
406
solvent, toluene, 1-octanol, and 1-dodecanol were screened using one-variable-at-a-time (OVAT)
407
approach. For the optimization of selected factors (pH, volume of toluene, ionic strength and
408
surfactant concentration), face centered cube CCD was used. Under the optimized conditions,
409
LOD were found to be in the range of 0.1–0.5 µg L-1, with preconcentration factors ranging from
410
190–292. The proposed SA-DLLME-HPLC-UV method took about 15 min for a single analysis.
411
Both positive and negative matrix effect was observed when the proposed SA-DLLME method
412
was applied to real urine samples, as relative recoveries for target analytes were found to be in
413
the range of 83.2–117.1.
414
2.5. Analysis of antidepressant drugs
415
Antidepressants are class of psychoactive drugs which are used for the treatment of major
416
depressive disorders e.g. tricyclic antidepressants (TCA). However, the overdose of TCA may
417
results in arrhythmia, hypertension and in some cases death. Cases of suicides are also prominent
418
due to self-ingestion of TCAs [35]. Xiong et al. [36] developed a DLLME-HPLC-UV method
419
for the extraction and determination of psychoactive drugs such as amitryptiline, clomipramine
420
and thioridazine in urine samples. Factors which impacts the extraction efficiency of DLLME
421
such as type and volume of extraction and disperser solvent, pH and ionic strength were
422
optimized before performing real sample analysis. Rapid injection of MeCN (0.5 mL, disperser
423
solvent) and CCl4 (20 µL, extraction solvent) into 5 mL of aqueous solution resulted into
424
formation of a cloudy solution which was centrifuged. In case of aqueous standard, a tiny droplet
425
of CCl4 was sedimented at the bottom of centrifuge tube, but in case of urine samples, lipids got
426
co-sedimented along with CCl4. For analysis, the co-sedimented lipid was dissolved in 200 µL of
427
MeCN, filtered and then analyzed by HPLC. The LODs were reported in the range of 3–8 ng
428
mL-1 for all target analytes with absolute recoveries of 96–101% in urine samples. Although
429
under the selected conditions, there were no interferences of endogenous substances from urine
430
sample on the determination of target analytes, however, the additional step of filtration of co-
431
sedimented lipid made the procedure more time consuming and labour-intensive.
432
DLLME was hyphenated with GC-MS for the analysis of five TCAs drugs i.e.
433
imipramine, desipramine, amitriptyline, nortriptyline and clomipramine in urine samples. Since
434
the structure of desipramine and nortriptyline has a secondary amino group, hence, derivatization 16 Page 16 of 40
435
was required to convert them into less polar and more volatile derivatives which are amenable
436
for GC-MS analysis. For this purpose, 5 µL of acetic anhydride was used for derivatization of
437
desipramine and nortriptyline. Acetic anhydride was injected into aqueous sample along with
438
disperser and extraction solvent i.e . MeOH and CCl4, respectively. Combination of DLLME
439
with GC-MS was found to be rapid and sensitive in comparison to previously reported methods
440
such as LLE-LC, SPME-LC and LPME-LC which took 40–180 min for analysis of same
441
analytes. The LODs of TCAs subjected to DLLME-GC-MS analysis were found to be in the
442
range of 0.2–0.5 ng mL-1 [35]. As there is no formation of lipidic solid sediment at the bottom of
443
centrifuge tube, the present method eliminates the need of filtering the sample before injection,
444
thus, one of the added advantage of this method in comparison to previously mentioned method
445
[36] is that, the sedimented phase could be injected directly in GC-MS [35].
446
A newly developed DLLME-HPLC-UV method was utilized to determine two TCA
447
drugs i.e. imipramine and trimipramine in urine samples after optimization by response surface
448
methodology (RSM). Disperser and extraction solvent were MeCN and CF, which were selected
449
on the basis of results obtained from OVAT approach. Seven factors were optimized using RSM
450
viz. volume of extraction and disperser solvent, salt percentage, pH, centrifugation time, reaction
451
time and centrifugation speed. Under optimized experimental conditions, the LOD observed for
452
imipramine and trimipramine were 0.6 ng mL-1 which was much lower in comparison to earlier
453
publications such as SPE-GC-nitrogen phosphorous detector (SPE-GC-NPD), CE-TOF-MS,
454
HPLC-MS, SPE-HPLC-UV etc. The EFs were 161.7 and 186.7 for imipramine and
455
trimipramine, respectively. The proposed method could be able to determine the concentration of
456
target analytes in urine samples after 5 hr of 10 mg dosage that was about 21 ng mL-1. To
457
compensate the matrix effect, instead of diluting the urine samples, they were firstly hydrolyzed
458
using 10M KOH followed by ultrasonication for 5 min. Alkaline hydrolysis allowed the
459
precipitation of most of the interfering compounds such as carbamide, uric acid, calcium salt etc,
460
whereas, ultrasonication breaks down the lipid material [37].
461
Most recently, a new DLLME protocol which replaces the use of disperser solvent with a
462
sugar cube i.e. solid based DLLME (SB-DLLME), has been proposed for antidepressants
463
(fluoxetine, fluvoxamine, tranylcypromine,
464
(mexiletine). In this procedure, for simultaneous derivatization and extraction of targeted drugs,
nortryptiline) and an antiarrythmic drug
17 Page 17 of 40
465
extraction solvent (1,1,2,2-tetrachloroethane) and derivatizing reagent (BCF) were added on a
466
sugar cube which served as disperser solvent. This mixture was added in aqueous sample
467
followed by manual shaking which allowed dissolving of solid disperser i.e. sugar cube. After
468
centrifugation, sedimented phase was analyzed by GC-FID. The LODs and EFs of target
469
analytes were obtained in the range of 1–15 µg L-1 and 228–268 [38].
470
For the first time, DLLME was combined with electromembrane extraction (EME) by
471
Seidi et al. [39] to determine three TCA i.e. amitryptiline, trimipramine and doxepine in urine
472
and plasma samples. A hollow fiber packed from one end and inserted with a cathode from
473
another end was used to extract the analytes. The acceptor solution (100 mM HCl solution) was
474
filled inside the hollow fiber. This fiber was dipped in the liquid sample along with anode. After
475
turning on the electric voltage, extraction was continued for 14 min. The acceptor phase was then
476
transferred into 1 mL of alkaline solution (pH 12) which was subjected to DLLME using MeOH
477
(disperser solvent, 150 µL) and CCl4 (extraction solvent, 10 µL). Complete analysis including
478
EME, DLLME and GC-FID took less than 35 min. Duloxetine, a drug widely used in the
479
treatment of major depressive disorders was determined using HPLC with fluorescence
480
detection. Analyte was extracted by using DLLME-SFO technique employing 1-undecanol as
481
extraction solvent. In this method the sample matrix i.e. plasma was deproteinized using zinc
482
sulfate and MeCN. Therefore, there was no need to use any disperser solvent, as MeCN was
483
acting itself as a dispersant [40].
484
2.6. Analysis of hallucinogens
485
Hallucinogens are drugs that alter the perception and mood of an individual, without
486
stimulating or inhibiting brain activities [41]. Lysergic acid diethylamide (LSD) is considered as
487
one of the most potent hallucinogen. Other than LSD; MDMA and phencyclidine (PCP) are two
488
most widely used hallucinogen drugs. For the analysis of these three drugs (LSD, MDMA and
489
PCP) in human urine samples, Rodríguez et al. [42] combined DLLME method with capillary
490
zone electrophoresis (CZE) and UV detection. Diluted urine sample was basified with 30%
491
ammonia and subjected to DLLME with MeCN and DBM as disperser and extraction solvent,
492
respectively. Factors influencing DLLME such as volume of extraction solvent, volume of
493
disperser solvent and amount of NaCl was optimized by using CCD. The LODs were found to be
494
in the range of 1–4.5 ng mL-1 for all the three analytes. Since the authors found statistically 18 Page 18 of 40
495
significant differences between the slopes of external aqueous standard and DLLME treated
496
aqueous standard at confidence level of 95% for each analyte, they recommended the analysis
497
using matrix matched calibration curves.
498
2.7. Analysis of pesticides
499
Self-poisoning by pesticides is one the most common method of suicide in developing
500
countries. According to a report of World Health Organization (WHO) around 30% of total
501
suicides in low and middle income countries between 1990 and 2007 were due to self-poisoning
502
from agricultural pesticides [43]. Therefore, highly sensitive and rapid analytical methods are
503
extremely needed for the analysis of pesticides in matrices of toxicological importance e.g.
504
tissue, blood and urine. Mudiam et al. [44] have developed a low density-DLLME method
505
coupled to GC-electron capture detection (GC-ECD) for the determination of cypermethrin in
506
tissue and blood samples of cypermethrin treated rats. In this method, tissue samples such as
507
brain, liver and kidney were firstly homogenized in ACE and then centrifuged. The supernatant
508
ACE was used as disperser solvent and mixed with n-hexane (extraction solvent) and rapidly
509
injected into ultrapure water for preconcentration of cypermethrin in n-hexane. Blood samples
510
were diluted with water and subjected to DLLME with the similar procedure. The LOD for
511
cypermethrin in tissue was found to be in the range of 0.098 – 0.314 ng mg-1, whereas in blood,
512
the LOD was 8.6 ng mL-1. Since n-hexane is lighter than water, it eliminates the need of
513
centrifugation while performing low density-DLLME, consequently, in this method the upper
514
layer of n-hexane was used directly for GC-ECD analysis. The same research group developed
515
another US-DLLME method coupled to GC-MS for the analysis of endosulfan and its
516
metabolites (endosulfan ether, endosulfan hydroxyether, endosulfan lactone, endosulfan alcohol
517
and endosulfan sulphate) in urine samples. Significant factors for DLLME such as volume of
518
extraction solvent, volume of disperser solvent, ionic strength, pH, extraction time,
519
centrifugation speed and centrifugation time, were screened using Placket-Burman Design
520
(PBD). The most significant factors which were obtained by PBD were further optimized by
521
CCD. Under optimized conditions, ACE and TCE were used as disperser and extraction solvents,
522
respectively. The LODs for all the analytes in urine were found to be in the range of 0.049–0.514
523
ng mL-1 [45].
19 Page 19 of 40
524
Five triazole pesticides (myclobutanil, uniconzole, penconazole and hexaconazole) have
525
been analyzed in rat plasma samples by HPLC with diode array detection (HPLC-DAD) after
526
extraction and preconcentration using temperature controlled ionic liquid-DLLME. Ionic liquid
527
i.e. 1-hexyl-3-methylimidazolium hexafluorophoshphate was used as extraction solvent together
528
with MeOH as a disperser solvent. Heating temperature and ultrasonication was the factors that
529
significantly affected the recoveries of triazole pesticide from plasma samples. The LODs and
530
EFs for triazole pesticides were found to be in the range of 4–6 µg L-1 and 178-197, respectively.
531
Although the proposed method exhibited high LODs and EFs, the total analysis time which
532
includes ultrasonication, cooling, centrifugation and HPLC run time, was quite high (~45 min)
533
for a single analysis [46].
534
3-phenoxybenzoic acid (3-PBA) and 4-phenoxy-3-hydroxybenzoic acid (OH-PBA) are
535
major metabolite and biomarker of pyrethroid pesticides exposure. A single step derivatization
536
cum extraction method for the determination of 3-PBA in rat brain samples using methyl
537
chloroformate (MCF) as derivatizing reagent in UA-DLLME procedure is developed and
538
combined with large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC-
539
MS-MS). MeOH was used to homogenize rat brain samples and its supernatant after
540
centrifugation was further utilized as disperser solvent. To this MeOH, MCF was added as
541
derivatizing reagent together with TCE as extraction solvent. After DLLME procedure, a volume
542
of 10 µL of sedimented phase was injected in GC-MS-MS system. The LODs for 3-PBA and
543
OH-PBA were found to be 3 and 13 ng g-1, respectively. In this method, the derivatization and
544
extraction could be achieved in a single step as MCF is capable of derivatizing polar analytes
545
directly in aqueous medium at room temperature within seconds [47]. In the same year another
546
method was reported by Mudiam et al. [48] for the determination of 3-PBA in rat liver and blood
547
samples. MIP was synthesized for the selective extraction of 3-PBA from complex biological
548
matrices and the eluent obtained after MISPE was subjected for DLLME followed by IPS in hot
549
GC-MS-MS injection port. Chlorobenzene (CB) served as extraction solvent along with MISPE
550
eluent of MeOH as disperse solvent. Under optimized conditions, the LOD for 3-PBA in blood
551
and liver is 1.82 ng mL-1 and 0.0045 ng mg-1, respectively. Primary advantage of coupling
552
DLLME with MISPE is targeted extraction of 3-PBA from biological matrices which in turn
553
enhances method selectivity and specificity. Additionally, use of IPS instead of conventional in-
554
vial silylation saves time and reagent as well as also cut down the cost of derivatization. 20 Page 20 of 40
555
2.8. Analysis of metals
556
Metals are being used as poisons for centuries because of their easy availability, potency
557
and tastelessness and their symptoms are similar to many of the common poisons [49]. Hydride
558
generation atomic absorption spectrometry (HG-AAS) was coupled to DLLME for the ultra-
559
trace determination of Arsenic (As) in urine and whole blood. For this purpose, ammonium
560
pyrrolidine dithiocarbamate (at pH 4) was used to form complex with As (III). The As (III)
561
complex with ammonium pyrrolidine dithiocarbamate was extracted in ionic liquid. Various
562
factors such as concentration of complexing agent, amount of ionic liquid, centrifugation time
563
and sample volume were studied. Under the optimum conditions LOD and linear range were
564
achieved to be 5 ng L−1 and 0.02-10 μg L−1, respectively [50]. Similarly, DLLME was applied
565
for the extraction of lead (Pb) from human urine samples and determination by graphite furnace
566
AAS. In this method, Pb was chelated with 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone during
567
the DLLME procedure wherein EtOH and CCl4 were used as disperser solvent extraction
568
solvent, respectively. At a signal to noise ratio of 3, the LOD of the proposed method was found
569
to be 39 ng L-1. Since interferences could be produced due to the competition of other metal ions
570
for the chelating agent and their subsequent co-extraction with Pb, the authors investigated
571
potential interferences of different metal ions in the developed method. The results revealed that
572
most of the alkaline and alkaline earth metal ions had no interference with the extraction for the
573
reason that their complexes which are formed with chelating reagent had very low stability
574
constants in comparison to Pb-chelating reagent complex [51]. Later on, DLLME method was
575
combined with laser induced-thermal lens spectrometry (LI-TLS) for the determination of Pb in
576
human blood serums. Herein, 1,5-diphenyl thiocarbazone (dithiozone) was used as chelating
577
reagent along with EtOH and CCl4 as disperser and extraction solvent, respectively. The LOD for
578
present method was 0.01 µg L-1. To reduce the matrix effect, a mixture of potassium cyanide and
579
sodium citrate were added in the sample as masking agent. With the sample preparation time of
580
less than 3 min, sample determination time of 6 s, and consumption of microlitres of toxic
581
organic solvents, this method was found to be very rapid and cost-effective [52].
582
Hair is a unique matrix for the assessment of chronic exposure of drugs and metallic
583
poisons in criminal cases. A DLLME-SFO method has been combined with flame atomic
584
absorption spectrometry (FAAS) for the determination of copper in human hair samples. In this 21 Page 21 of 40
585
method, 8-hydroxy quinoline was added in aqueous solution along with EtOH as disperser
586
solvent and 1-undecanol as extraction solvent. Hair samples were acid digested with
587
concentrated nitric acid for overnight. Perchloric acid was added to this mixture and the mixture
588
was heated at 200 °C for 1 hr. After neutralization of the solution with NaOH, the sample was
589
subjected to DLLME-SFO procedure. The LOD for copper achieved by this study was
590
comparable with other previously reported methods such as SPE and DLLME coupled to UV-
591
Vis spectrophotometry [53].
592
2.9. Miscellaneous applications of DLLME in forensic toxicology
593
Beside most routinely encountered narcotics substances and drugs of abuse, DLLME has
594
also been applied for the determination of several drugs of clinical and forensic importance.
595
Pregabalin (PRG) is an anticonvulsant drug which is used treatment of neuropathic pain and
596
epilepsy. However, in recent years, abuse and addiction potential of PRG has been proved. An
597
international team of researchers described PRG as an ideal psychotropic drug for recreational
598
intentions [54]. Recently, a study has been conducted to assess the serum levels of PRG in
599
drivers suspected of driving under the influence of drugs. PRG was detected in 206 samples in
600
which 50% of the samples have shown serum level of PRG above therapeutic range [55]. In
601
2012, Mudiam et al. [56] have reported a DLLME method coupled to GC-MS which allow
602
simultaneous derivatization and extraction of PRG in a single step using ethyl chloroformate as
603
derivatizing reagent. In the first step, pyridine was added in urine samples as a catalyst, followed
604
by rapid injection of a mixture of EtOH, TCE and ethyl chloroformate (disperser solvent,
605
extraction solvent and derivatizing reagent, respectively). In the same work, the authors also
606
developed a SPME method for extraction of PRG from urine samples. However, DLLME
607
method was proven to be superior over SPME in terms of time consumption and cost
608
effectiveness. The LOD for PRG using DLLME method under optimized conditions were found
609
to be 0.022 µg mL-1.
610
Amantadine, a drug used in the treatment of Parkinson disease, has found to cause
611
unexpected intoxication and death in trauma patients [57]. Farajzadeh et al. [58] demonstrated
612
simultaneous derivatization and extraction of amantadine in urine and plasma samples using
613
isobutyl chloroformate as derivatizing reagent along with MeOH as disperser solvent and 1,2-
614
DBM as extraction solvent. DLLME was coupled to GC-FID for the analysis of amantadine. 22 Page 22 of 40
615
Under optimized conditions, the EFs for amantadine was obtained to be in the range of 408–420.
616
The LODs in urine and plasma samples were found to be 2.7 and 4.2 ng mL-1. As done in
617
previous studies, herein, also the method of sample dilution (5 and 10 fold) was followed to
618
reduce the matrix effect. The proposed procedure was sensitive enough to detect amantadine
619
content in urine and plasma samples even after 12 to 24 hours. DLLME was coupled with CE
620
for the selective determination of psychiatric drugs (olanzapine, prochlorperazine dimaleate,
621
trifluoroperazine 2 x HCl, perphenazine, clomipramine HCl and chlorprothixene HCl) in urine
622
samples. Carbon tetrachloride and MeCN have served as extraction and disperse solvent,
623
respectively. The LODs were 0.030–0.75 ng mL-1 for all tested analytes. Excellent EFs in the
624
range of 8080 – 13410 have been achieved by combining DLLME with field-amplified sample
625
injection-CE-UV [59]. A combination of CCl4 and MeCN was used as disperser and extraction
626
solvent to extract cyproheptadine, an antihistamine drug, from urine samples. The sedimented
627
phase obtained after centrifugation was evaporated and reconstituted in mobile phase used for
628
HPLC analysis of cyproheptadine. By the DLLME-HPLC-DAD method, cyproheptadine
629
concentration as low as 13.1 ng mL-1 could be detected with relative recoveries in the range of
630
91.6–101 %. When compared to formerly reported SPE-HPLC-UV method, the proposed
631
method exhibited higher sensitivity, and the use of extraction solvent reduced drastically from
632
6000 µL to 30 µL. Additionally simplicity of operation and low analysis cost are other added
633
benefits of the present method over SPE-HPLC-UV [60].
634
Digoxin, a cardiac glycoside obtained from the foxglove plant, Digitalis lanata, is
635
categorized as toxic principle of plant origin which is occasionally ingested for the purpose of
636
suicide [61, 62]. In 2013, Cheng et al. [63] developed a method combining DLLME with
637
surface-assisted laser desorption/ionization mass spectrometry (SALDI/MS) for the analysis of
638
digoxin in urine samples using ACE and CF as disperser and extraction solvent, respectively.
639
The proposed method was found to be rapid as total analysis time of digoxin in aqueous samples
640
takes less than 10 min. The authors analyzed blank urine samples under optimized conditions to
641
check the selectivity of the method and did not found any endogenous compound in the sample
642
which could interfere with digoxin and internal standard. The LOD and EF for digoxin were 2
643
nM and 252, respectively.
23 Page 23 of 40
644
Anti-epileptic drugs are not generally abused, however, cases of accidental and
645
intentional poisoning as well driving under the influence, are frequently encountered [64].
646
Carbamazepine and zonisamide are two most widely used anti-epileptic drugs. A SA-DLLME
647
method is proposed to extract carbamazepine and zonisamide from urine and plasma samples
648
using cethyltrimethyl ammonium bromide as surfactant along with 1-octanol as extraction
649
solvent. After DLLME samples were analyzed by HPLC-UV. The LODs were found to be in the
650
range of 1.5–2.3 µg L-1 [65].
651
24 Page 24 of 40
652
3. Conclusion and future trends
653
The nature of samples which are analyzed in forensic toxicology laboratories is highly
654
complex. Therefore, a sample preparation technique is desirable which is able to extract and
655
preconcentrate the analytes from dirty and complex matrices. In forensic laboratories, LLE and
656
SPE are the two most dominant sample preparation methodologies. However, since the
657
introduction of DLLME, attention has been paid by analysts and researchers to develop protocols
658
using DLLME, for the analysis of drugs of abuse and other forensically important analytes. By
659
virtue of its simplicity, rapidity, cost effectiveness, eco-friendly nature, high EFs and recoveries
660
offered by this methodology, it has become a popular choice of forensic analysts among other
661
sample preparation methodologies available. This review summarized the applications of
662
DLLME in forensic toxicological analysis.
663
DLLME has been extensively used in the analysis of drugs of abuse, narcotics, pesticides,
664
metals and their metabolites. The ability of DLLME to couple with various analytical
665
instruments such as GC-MS, HPLC, UV visible spectrophotometer, CE etc. makes it one of the
666
most versatile microextraction techniques. DLLME has shown successful application for the
667
extraction of target analytes from routinely encountered samples in forensic toxicology
668
laboratories such as urine, blood, saliva, plasma, tissue and serum. One of the major advantages
669
which DLLME offers over other microextraction technique is its ability of simultaneous
670
derivatization and preconcentration using alkyl chloroformate as derivatizing reagents directly in
671
aqueous medium at room temperature within seconds. Coupling of IPS with DLLME also seems
672
to be promising approach for the analysis of polar analytes by GC-MS, since it eliminates the
673
need of lengthy reaction time, extra heating environment and large amount of costly silylating
674
reagents.
675
The future trends of DLLME in the field of forensic toxicology could be summarized in
676
the following aspects: DLLME has been coupled with GC-MS and HPLC for the determination
677
of pesticides from environmental water samples. Nevertheless, there are limited applications of
678
DLLME for the extraction of pesticides from biological matrices of forensic importance.
679
Similarly, DLLME has been widely applied for the determination of metals in environmental
680
samples, though, analysis of toxic metals from complex matrices such as blood, saliva, plasma,
681
urine, tissue, hair and nail is expected in coming years. Replacement of toxic chlorinated 25 Page 25 of 40
682
extraction solvents in DLLME with solvents having low toxicity is a key point of concern. For
683
this purpose, DLLME-SFO can be promoted and ionic liquids could be of immense use. New
684
perspectives are to be opened in automation of DLLME for substances of toxicological interest
685
to make this method more vibrant, versatile and universal.
686
Acknowledgements
687
Authors are grateful to Dr. S. K. Jain, Dy. Director cum Coordinator, Central Forensic
688
Science Laboratory, Guwahati, and Dr. Mohana Krishna Reddy Mudiam, Senior Scientist, CSIR-
689
Indian Institute of Toxicology Research, Lucknow, for their constant support and guidance
690
throughout the manuscript preparation and onwards.
691
Conflict of Interest
692
There are no financial or other relations that could lead to a conflict of interest.
693
26 Page 26 of 40
694 695 696 697 698 699 700 701 702 703
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778
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779
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781
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795
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826
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841
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863
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864 865
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871
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56. M.K.R. Mudiam, A. Chauhan, R. Jain, R. Ch, G. Fatima, E. Malhotra, R.C. Murthy,
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879
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899
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900
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901
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902
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903
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904
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905
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906
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907
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33 Page 33 of 40
908
65. M. Behbahani, F. Najafi, S. Bagheri, M.S. Bojdi, A. Bagheri, Application of surfactant
909
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910
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911
(2013) 25-31.
912
34 Page 34 of 40
913
Figure captions
914
Fig. 1.Dispersive Liquid-Liquid Microextraction (DLLME)
915
Fig. 2. DLLME-SFO
916
35 Page 35 of 40
917
36 Page 36 of 40
918
Table 1. Applications of DLLME in forensic toxicology Extraction solvent 1-undecanol 1-undecanol isobutyl chloroformate
Analyte
Matrix
Disperser solvent
AP, MA AP, MA
urine urine
SDS MeCN
MA, MDMA
urine
MeOH
MDMA, LSD, PCP
urine
MeCN
DBM
APs and their derivatives, opiates, cocaine and its metabolites, pharmaceuticals
urine
IPA
DCM
0.1–10 ng mL-1
IPA
CF
0.08–0.20 µg L-1
MeCN
CS2
0.05–7 µg L-1
MeOH
CF
ACE
CF
ACE
CF
0.02–0.05 µg L-1
urine
IPA
DCM
urine
EtOH
DCM
water, urine, plasma, chlordiazepox
MeOH
CF
MDA, MDMA, MDEA, MDPA
seized forensic samples urine and plasma
BZD
plasma
MA, MDMA, ketamine, heroin
BZD BZD 7aminoflunitrazepam alprazolam, oxazepam, diazepam chlordiazepoxide
urine and waste water urine and other matrices
LOD
EF
Analytical technique
Ref.
2 and 3 µg L-1 8 and 2 µg L-1 2 and 18 ng mL-1 1, 4.41 and 4.52 ng mL-1
56, 48 58.5, 62.4
HPLC HPLC
[11] [12]
427, 285
GC-FID
[13]
CE-UV
[14]
CE-UV and CE-TOF-MS
[15]
CE-UV
[16]
GC-FID
[17]
HPLC & UPLC
[18]
UPLC
[19]
3895–7222
GC-FID
[20]
0.025 ng mL-1
20
LC-ESI-MS
[21]
0.3–0.7 µg L-1
208–226
HPLC
[22]
HPLC
[23]
545–611
1.7–10.6 ng mL-1 0.6–6.2 ng mL1
0.0005 µg mL1
37 Page 37 of 40
barbituric acid opium alkaloids opium alkaloids fentanyl, alfentanil and sufentanil fantanyl methadone QN morphine, 6-acetyl morphine, methadone, cocaine, benzylecgonine
ide tablets urine, serum, tablet urine plasma urine and plasma urine urine, plasma, saliva, sweat urine plasma
MeOH
CF
0.002 µg mL-1
30
ACE ACE
CF 1-undecanol
63–104.5 110.4–165
MeOH
CF
275 –325
HPLC
[27]
2-propanol
chlorobenzene
GC-MS
[28]
MeOH
CF
HPLC-UV
[29]
EtOH
DCM
0.2–10 µg L-1 0.5–5 µg L-1 0.4 to 1.9 μg L-1 1 ng mL-1 0.22–25.12 ng mL-1 5.4 ng mL-1
UV-Visible Spectrophotometer HPLC-UV HPLC-UV
GC-MS
[30]
MeCN
CF
13.9–28.5 ng mL-1
HPLC-PDA
[31]
undecanol and CF (1:1 v/v)
0.002 µg mL-1
90.5–95.2
HPLC
[32]
190–292
HPLC-UV
[34]
98.26– 100.34
[24] [25] [26]
nicotine and cotinine
urine
cannabinoids
urine
tetradecyl tremethyl ammonium bromide
toluene
0.1–0.5 µg L-1
urine
MeOH
MeOH
0.2–0.5 ng mL-1
GC-MS
[35]
urine
MeCN
CCl4
3–8 ng mL-1
HPLC-UV
[36]
urine
MeCN
CF
HPLC-UV
[37]
plasma and
sugar cube
1,1,2,2-
GC-FID
[38]
imipramine, desipramine, amitriptyline, nortriptyline, clomipramine amitryptiline, clomipramine, thioridazine imipramine and trimipramine antidepressants and
0.6 ng mL-1 each 1–15 µg L-1
161.7 and 186.7 228–268
38 Page 38 of 40
antiarrythmic amitryptiline, trimipramine and doxepine duloxetine
urine
tetrachloroethane
urine and plasma
MeOH
CCl4
0.25–15 µg-1
383–1065
GC-FID
[39]
Plasma
MeCN
1-undecanol
98
HPLC-FLD
[40]
cypermethrin
tissue and blood
ACE
n-hexane
477-689
GC-ECD
[41]
endosulfan and its metabolites
urine
ACE
TCE
2.5 ng mL-1 0.098 – 0.314 ng mg-1; 8.6 ng mL-1 0.049 – 0.514 ng mL-1
GC-MS
[42]
4–6 µg L-1
178-197
HPLC-DAD
[43]
5 ng L−1
HG-AAS
[50]
39 ng L−1
Graphite Furnace AAS
[51]
EtOH EtOH
CCl4
0.01 µg L-1
LI-TLS
[52]
PRG
urine human blood serum urine
1-hexyl-3methylimidazoli um hexafluorophosh phate (1-butyl3methylimidazol ium hexafluorophosp hate CCl4
EtOH
TCE
GC-MS
[56]
psychiatric drugs
urine
MeCN
CCl4
CE-UV
[59]
cyproheptadine digoxin
urine urine
CCl4 CF
HPLC-DAD SALDI/MS
[60] [63]
carbamazepine and zonisamide
urine and plasma
MeCN ACE cethyltrimethyl ammonium bromide
0.022 µg mL-1 0.030–0.75 ng mL-1 13.1 ng mL-1 2 nM
1-octanol
1.5–2.3 µg L-1
HPLC-UV
[65]
triazole pesticides
rat plasma
As
urine and whole blood
Pb Pb
MeOH
8080 – 13410 252
919 39 Page 39 of 40
920 921
66.
40 Page 40 of 40