Potential application of amino acids in analytical toxicology

ABSTRACT

The ability of extraction and preconcentration of small quantities of substances from biological
samples is important in analytical sciences, particularly forensic medicine. In the present study, we
evaluated the binding potential of amino acids to produce a new solid phase microextraction fiber
based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine
in urine sample. Raw CNTs were first carboxylated and then functionalized with 3 amino acids
including glutamate, arginine, and cysteine. Functionalization was confirmed by FTIR analysis,
Raman spectroscopy and SEM imaging. The functionalized CNTs were coated on polypropylene
hollow fiber and used for preconcentration. The results of HPLC analysis in isocratic elution mode
using acetonitrile-sodium acetate (10:90, v/v; pH 4; 0.01 M) as the mobile phase showed that amino
acids are able to adsorb morphine and the prepared fiber could preconcentrate a very low con-
centration of morphine (0.25 ppb) in urine matrix. In addition, the fiber was successfully used for up
to 30 times with no significant loss in the extraction efficiency. Lowest limit of detection (LOD) and
limit of quantitation (LOQ) was 0.07 and 0.25, respectively. Also, the lowest and best recovery of the
fiber was 87.8% and 139% at LOQ, which belonged to glutamate and arginine, respectively. The
fibers based on amino acids can be used for the detection of a small amount of morphine in biological
samples, which are not detectable by conventional methods. Simple mechanism of these fibers in
preconcentrating morphine makes them a novel candidate for detection of other opiates and drugs of
abuses in crime scene investigations and postmortem examinations several days after exposure.
1. Introduction

Detection of ultratrace amount of analytes in biological sample is very important in analytical science,
especially in forensic toxicology. This is particularly important when a small amount of analytes are
available in the sample or a very limited amount of sample is accessible. To overcome these
limitations and to increase the accuracy of detection techniques, several strategies including
preconcentration methods have been developed [1]. Preconcentration helps to remove interfering
analytes from sample and enhance the efficiency of detection. So far, several strategies such as liquid-
liquid extraction (LLE), also known as solvent extraction, and solid-phase microextraction (SPME)
methods have been introduced and developed for preconcentration and micro-extraction of the
desired analytes [2,3]. SPME is based on the interaction between analytes and the sorbent, which is
typically coated on the surface of a fiber. These sorbents cannot usually detect or preconcentrate the
desired analytes specifically. For this reason, using molecules with selective or semi-selective affinity
to a specific ligand as the sorbent of SPME fiber may enhance the accuracy and precision of analyte
detection. Amino acids are particularly interesting molecules, which may be useful in detection of the
desired molecules.
Varieties of cellular processes are mediated through amino acids as individual amino acids or as a
critical element in the peptide and protein sequence [4]. Amino acids and derivatives such as
tryptamine, serotonin and catecholamines contribute in many biological processes including
regulation of the endocrine system. Also, it is known that amino acids and their derivatives can act as
cellular messengers. Enzymes that are the main catalysts in biological systems are made of amino
acids, which some of them in the active sites have critical role in enzyme activity [5]. Amino acid
residues in protein structure can also involve in mediating homeostasis and antioxidant defense [6,7].
Individual amino acids may also contribute in translational control of protein synthesis or binding a
specific molecule to the target, mediating cell signaling [8,9]. In addition, it is reported that some
amino acid residues are critical for a specific activity such as localization of a particular ligand in the
cell [10,11]. The basis of all of these activities is the interaction between amino acids and a target
molecule. Therefore, since amino acids are able to bind to particular ligands according to their
structure and net charge, it is expected that amino acids can be considered as a potential molecules in
binding to specific molecules [12]. Amino acids are composed of an asymmetric carbon called carbon
α, which is bound to four different atoms including a hydrogen atom, carboxyl (COOH) group, the
amino (NH2-) group and a specific side chain (R-), which gives different structures, features and
biological properties to each amino acid. Due to presence of the dissimilar side chain in the structure,
each amino acid can act as potential binding site of a specific molecule in the cell or on the cell
membrane or even in biological samples. This can be important in diagnostic medicine as well as
analytical science for detection or preconcentration of specific analytes or molecules in the cells or in
biological samples.
On structural point of view, every amino acid can be considered as potential targeting agent to bind
to a specific molecule. In the present study, efficiency of three amino acids from three different groups
with different chemical and biological features was investigated for potential use in separation and
analytical science. Due to specific binding features of some amino acids to various molecules, the
HF-SPME method using amino acids as the sorbent may be an alternative choice to preconcentrate
and detect a very trace amount of morphine in biological sample. Aminofunctionalized carbon
nanotubes (CNTs) were used to reinforce the fiber for extraction and preconcentration of morphine
in the urine samples. The extracted samples were then analyzed by high performance liquid
chromatography (HPLC).
2. Materials and methods

2.1. Chemicals and apparatus

N, N-Dimethylformamide (DMF), ethanol, acetone, and 1-octanol were obtained from Merck
(Darmstadt, Germany). Stock standard solutions of morphine were provided by a collaborative
laboratory at Mashhad University of Medical Sciences at a concentration of 1 µg ml −1 in deionized
water. All standard solutions were stored at 4 °C and protected from light until use. Acetonitrile
(ACN) and water HPLC grade were obtained from Merck. The HFs (200 µm thick wall, 0.6 mm inner
diameter and 0.2 µm average pore size) was purchased from Membrana (Wuppertal, Germany).
Fourier-transform infrared spectroscopy (FTIR) spectra from the samples were recorded on an FT-
IR 8400S spectrophotometer (Shimadzu, Japan) in KBr tablets. Modifications of CNTs were con-
firmed by Raman spectroscopy (Teksan, Takram P50C0R10, at laser wavelength of 532 nm).
Structure of the synthesized nanocomposite as well as HFs was investigated by scanning electron
microscopy coupled with Energy Dispersive X-ray (SEM/EDX) (TESCAN MIRA3-FEG, Czech
Republic). Metrohm-744 digital glass electrode pH meter (Switzerland) was used to measure the pH
values in the solution. All experiments were carried out at room temperature (22 ± 0.5 °C), unless it
is stated.
2.2. Preparation of nanocomposite and SPME fiber

The nanotubes were first carboxylated using concentrated solution of HNO3 and H2SO4 (3:1) as
described elsewhere [1]. After carboxylation, the prepared CNTs were functionalized with 3 amino
acids including glutamate (Glu), arginine (Arg), and cysteine (Cys) with different functional groups.
For this purpose, 15 mg CNTs were dis-solved in DMF (10 ml) and were then mixed with each of the
Glu (5 mg), Arg (5 mg), and Cys (5 mg) and sonicated for 1 h at 60 °C. Subsequently, 5 mg sodium
nitrite (NaNO2) was added to the solution and sonicated for additional 12 h. The resulted materials
were then filtered through 0.45 µm PTFE membrane and washed with deionized water. To produce
the fibers, amino acid-functionalized nanocomposites were coated on the provided HFs by sonication
in a solution containing the homogenized nanocomposite - octanol for 6 h at 45 °C. Afterwards, the
two ends of fibers were sealed to not allowing the re-lease of nanocomposite during the time. Prior
to coating, HFs were washed in acetone for 5 min to remove impurities. Then, the prepared fibers
were dried for use.
2.3. Evaluation of fiber efficiency and HPLC analysis

For HPLC analysis, a serial dilution of morphine (0.1–1000 ng/ml) was prepared in urine, and the
produced fiber was immersed into urine sample containing 1 ml of the prepared morphine solution
and sonicated for 5 min. Afterwards, the fiber was placed in a microtube and washed with 200 μl
methanol 20% in deionized water and sonicated for additional 5 min. Mechanical stability in addition
to robustness of the fibers for microextraction of morphine was evaluated at different environmental
conditions. For this purpose, extraction efficiency of each fiber was evaluated in different ranges of
pH (4–12), temperature (10–50 °C), and salt concentrations (NaCl, and CaCl2) as a model of
interfering analytes. Because, the major route of morphine excretion is through kidney, the fibers
were used to trace morphine only in urine matrix.
An analytical C18 reverse phase column (0.46 × 25 cm, manufactured by Macherey-Nagel GmbH
& Co., Duren, Germany) was used for HPLC analysis. A mixture composed of acetonitrile-sodium
acetate (10:90, v/v; pH 4; 0.01 M), at the flow rate of 0.6 ml/min was iso-cratically applied to elute
sample over 10 min. After microextraction, 15 μl of the samples were injected on to HPLC system
(Knauer, Berlin, Germany) and the absorbance was monitored at 280 nm using Smartline Photodiode
Array UV detector (Knauer, Berlin, Germany). The peak area was measured and the corresponding
concentration was determined based on its standard curve using different concentrations of standard
of interest. Before HPLC analysis, the wavelength of maximum absorbance (λmax) for morphine was
determined at 285 nm using Cecil 9200 Single Beam UV/VIS Spectrophotometer.
3. Results

3.1. Characterization and structural analysis of the synthesized nanocomposite

The desired characteristics of the synthesized nanocomposite were verified by FTIR and Raman
spectroscopy measurements in addition to SEM imaging. FTIR and Raman spectra were analyzed
using Spectragryph version 1.2.7 software, and the results showed that the synthesized
nanocomposites were effectively functionalized with amino acids (Fig. 1a). The frequency of amine
group was shown at 3439 cm−1. Also, the peak at 1637 cm−1 can be attributed to amide bound. As
well, the frequency for C˭O stretch of amide bound was recognized at 1715 cm−1. In addition, the
peak at 1511 cm−1 may also be indicative of amide C (=O) NH bound, and the frequency at 1574 cm−1
can also be related to NH group. Raman spectroscopy of CNTs shows two main peaks at 1350 cm−1
and 1550 cm−1, which are related to the defects in the graphitic structure (D), and the longitudinal
stretching of the nanotube (G-band). According to the data of Raman spectroscopy, changes in Raman
intensities of D and G peaks confirmed the results of FTIR analysis, which indicated alteration of
CNT structure (Fig. 1b). SEM images also showed that the diameter of fibers were 30 nm (Fig. 2a–
c). In addition, content analysis of the synthesized nanocomposites using SEM/EDX showed that
functionalization was successfully completed with the synthesized nanocomposite, since all of the
expected atoms (C, H, O, S, and N) were found in nanocomposite structure. Since, sulfide and
nitrogen groups are only present in amino acids (S in Cysteine), so that the bonding between
functional groups was confirmed. Microstructure of the HFs was also demonstrated in Fig. 2d
3.2. Morphine detection

For detection of morphine, the samples were prepared by spiking different concentrations of
morphine (0.1–1000 ng/ml) in morphine-free urine. The results showed that the amino acid-
functionalized fibers could preconcentrate morphine in urine sample and the microextraction process
led to detection of a very small amount of morphine (0.25 ng/ml), while only higher concentrations
of morphine (greater than 10 ng/ml) could be detected by HPLC system before micro-extraction.

3.3. Optimization of microextraction process

Due to dissimilar side chain of amino acids, each fiber had different behavior, particularly at
different environmental conditions such as temperature, pH, salt, and extraction time. The results
showed that the fiber did not consistently adsorb morphine, and the reaction equilibrated shortly
after rapid adsorption of morphine to the fibers. In other word, long-term exposure of the fiber to
morphine can reduce the efficiency of adsorption. Robustness evaluation showed that the fibers
could preconcentrate morphine in urine sample at different environ-mental condition; however, the
extraction efficiency varied depending on the condition. Our data showed that the optimum
condition for microextraction was sonication for 5 min at 40–45 °C in neutral pH (Fig. 3a-c);
however, every 10 °C of variation from optimum temperature (increase or decrease) led to 10–30%
reduction in the efficiency of fibers. Efficiency of the fibers was also significantly reduced in the
presence of salts (Fig. 3d), which are thought to compete with morphine in binding to the fibers. In
addition, peak area measurement (for fibers containing amino acids as sorbent) showed that
morphine can more efficiently bind to Arg relative to Cys and Glu. Validity parameters of the
method was also evaluated and described as data coefficient of variation (Table 1). Our experiment
also showed that the produced fibers can be used for 30 times with high efficiency.

3.4. Functionality of the produced fiber on real sample

To confirm the functionality of produced fibers in real samples, morphine-positive urine samples
were collected from patients admitted to the toxicology department of Imam Reza Hospital, Mashhad,
Iran. The results demonstrated that the produced SPME fibers could preconcentrate the morphine in
urine samples as well (Fig. 4). According to our findings, concentrating power of the fibers to the
morphine was linear at two different concentration range (Table 1). In addition, limit of detection
(LOD) and limit of quantitation (LOQ) were determined for each fiber according to signal to noise
ratio. Comparison of these parameters with other similar reports showed that the sensitivity of
produced fibers for preconcentration of morphine in this study was considerably higher than other
reported methods (Table 2) [13–18].

4. Discussion

SPME has been known as efficient method for preconcentration of analytes in gaseous and aqueous
samples. Various SPME methods have been previously introduced for the microextraction of several
chemical and biological compounds such as benzene derivatives, organophosphates, amphetamines,
cocaine, and other drugs of abuse [3,19–21]. SPME is also an appropriate method for microextraction
and monitoring therapeutic drugs such as benzodiazepines, and metal ions in addition to estimate the
degree of intoxication [22,23]. The function of SPME is based on the interaction between analyte and
sorbent. For this purpose, many chemical agents have been used as the sorbent, most of which adsorb
the analytes through mass transferring with no speci-ficity and bind to a variety of analytes.
Theory of the present study was based on binding potential of in-dividual amino acids to different
molecules. Theoretically, it was ex-pected that each amino acid behave differently in binding to
morphine. Hence, in the present study we evaluated binding potential of Arg, Cys, and Glu amino
acids to morphine. Our results demonstrated that Arg, Cys, and Glu can bind to morphine with
different degree of binding affinity. According to our findings, the morphine showed greater binding
affinity to Arg than other two amino acids (Cys and Glu), indicating that the binding of morphine to
amine groups is more desirable than that of carboxyl and sulfhydryl groups. On structural view, higher
binding affinity of Arg to morphine may be attributed to more binding site (NH- group) of Arg, which
gives greater binding avidity to the analyte. Due to presence of many interfering analytes such as urea
and creatinine at very high concentration in the urine sample, micro-extraction of a very trace amount
of morphine may indicate semiselective properties of the produced fiber to morphine. Also, according
to the slope of calibration curve it was shown that the sensitivity of fibers to morphine was higher at
lower concentrations (0.25–10 ng/ml). This is particularly important in forensic medicine and
analytical toxicology in which choosing the treatment protocols for intoxicated cases or finding the
cause of poisoning are based on appropriated diagnosis of drugs of abuse. As provided in Table 1,
validity profile indexes showed that the produced microextraction fibers using amino acids as the ex-
tractor phase may be considered as reliable tools for preconcentration of morphine in small quantities.
Findings of this study showed that biological molecules such as amino acids have great potential
for use in analytical area and particularly for detection of drugs of abuses. Also, our findings suggest
that using biological molecules as sorbent may have several advantages over chemical agents such as
abundant sources of production, lower contamination, and therefore cost-effectiveness. By
considering the lower LOD and LOQ of this fiber compared to other similar methods (Table 2), it is
suggested that the introduced SPME method based on amino acids may be helpful for detection of a
very trace amount of analytes. Therefore, it is recommended that the efficiency of this fiber for pre-
concentration of morphine may be greater in other biological samples such as oral fluid, which
contains fewer interfering analytes. Based on the findings of this study and by considering potentials
of amino acids in analytical sciences, it is suggested that designing a specific sequence of proteins
and peptides may be useful and more selective for pre-concentration of the desired molecule.
5. Conclusion
The results of this study showed that SPME fibers functionalized with amino acids are appropriate
tools for preconcentration of morphine. Altogether, the introduced method based on binding affinity
of amino acids to the target molecules can be considered as a standard method of sample preparation,
especially when a very limited volume of biological sample is available for the analysis or when a
few concentrations of the analytes are in the samples. This could be of interest for postmortem and
ante-mortem examination such as crime scene investigation in which a very trace amount of
substances can be found for detection.
Acknowledgment

The results described in this paper were part of the corresponding author's PhD. thesis. The
research project was supported by the Vice Chancellorship for Research, Mashhad University of
Medical Sciences, Mashhad, Iran; grant no: 930829.
Conflict of interest

There is no conflict of interest between the authors.