1. Introduction
Copper and zinc are essential ions for the human
body. Copper has different forms such as CuS,
, CuFeS
and CuSO
O in environment.
The high concentrations of Cu and Zn more than
2 ppm in human blood are toxic and the range
between essentiality limit and toxicity form is
very small. Copper and zinc has normal ranges
between 0.8-1.6 mg L
in different ages for the
human serum and urine samples [1]. Zinc is
used as cofactor for many enzymes in the human
body. Zinc effect on cell structure of human body,
the structure of protein, the gene expression,
the immune system, and the growth in children.
Zinc deciency cause to many diseases such as,
diarrhea, a compromised immune system, night
blindness, hair loss, and the taste alterations [1-
Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43
Simultaneously determination of copper and zinc in human
serum and urine samples based on amoxicillin drug by
dispersive ionic liquid- liquid microextraction coupled to
ame atomic absorption spectrometry
Kian Azami
and Seyed Mojtaba Mostafavi
Department of Pharmacology and Toxicology, Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran
Department of Chemistry, Iranian-Australian Community of Science, Hobart, Tasmania, Australia
In this work the effect of amoxicillin on copper and zinc (Cu and Zn)
deciency was evaluated by determining of Cu and Zn concentration
in human serum and urine samples. By dispersive ionic liquid cloud
point extraction procedure (DIL-CPE), 0.03 g of pure amoxicillin
drug was added to mixture of 0.1 g of hydrophobic ionic liquid and
0.2 mL acetone which was injected to 2 mL of serum or urine samples
which was diluted with DW up to 10 mL. The cloudy solution was
shacked for 7 min and Cu and Zn ions was extracted based on sulfur
group on amoxicillin ligand at pH of 7 by DIL-CPE. Then, the solution
centrifuged and after back extraction with I mL of nitric acid (0.2 M),
the remained solution was determined by ame atomic absorption
spectrometry (F-AAS). The enrichment factor (EF), LOD and linear
range (LR) for copper and zinc was obtained (9.92; 9.81), (28.5 µg
; 15.2 µg L
) and (100 -505 µg L
; 41- 153 µg L
), respectively.
The results showed us, the concentration of the Cu and Zn ions can be
decreased by increasing amoxicillin drug dosage in human body. The
mean value for serum copper/zinc ratio was obtained 1.11 ± 0.28. The
DIL-LME method was validated by ICP-MS analysis and spike of
real samples for Zn and Cu ions in serum and urine samples.
Amoxicillin drug,
Copper and zinc,
Serum and urine samples,
Dispersive ionic liquid cloud point
extraction procedure,
Flame atomic absorption spectrometry
Received 11 Jun 2020
Revised form 5 Aug 2020
Accepted 28 Aug 2020
Available online 29 Sep 2020
*Corresponding Author: Kian Azami
Email: Kianazami@yahoo.com
Research Article, Issue 3
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
Simultaneously determination of Cu / Zn in human samples Kian Azami et al
2]. Total zinc is about 40 mg per day for adults
over 20 years. Copper helps to transportation of
iron, energy production, the pigmentation of skin,
hair, and eyes. Copper acts as an antioxidant for
defending of cell damage which was caused by free
radicals. Copper deciency cause to anemia, low
white blood cell count, loss of myelin, multiple
sclerosis (MS), the loss of pigmentation, the
impaired growth and osteoporosis [3]. The copper
intake from food/water or supplements has 10 mg
per day and over limit accumulates in the liver.
Based on previous studies, a ratio of 8-15 mg of
zinc for every 1 mg of copper reported. However,
this ratio seems to be more important for human
bodies mechanism [1-4]. Zinc is absorbed in the
small intestine by a carrier-mediated mechanism.
Under normal physiologic conditions, transport
processes of uptake are not saturated. The mean
value for serum copper was normal range (17.47 ±
3.31 μmol L
; 111.32 μg dL
), and the mean value
for serum zinc was at the lower edge of the normal
value (12.24 ± 1.04 μmol L
; 80.01 μg dL
), while
the mean value for serum copper/zinc ratio was
1.44 ± 0.31 ranging from 0.65 to 2.67 [5-7]. Copper
and zinc concentration in liquid phase can be
determined directly by inductively-coupled plasma
atomic emission spectrometry (ICP-AES) [8] or
electrothermal atomic absorption spectrometry
(ET-AAS) [9] with low detection limit (LOD). The
conventional ame atomic absorption spectrometry
(F-AAS) [10] was used in many laboratories and
had low interferences ions as compared to ICP-
AES or ETAAS. As difculty matrix in human
blood or serum patients a sample preparation is
require for preconcentration/separation/extraction
of ions from samples before determination. Many
procedures for metal determination in water and
human matrix was used with different analytical
techniques and reagents [11,12]. The Liquid–
liquid extraction by using salophen as an complex
reagents [13], the sandwich supported liquid
membrane [14], the modied carbon based solid
phase extraction [15], the solid-phase extraction
on MWCNTs - D2EHPA-TOPO [16], dispersive
liquid–liquid microextraction of copper (II) by
oxinate chelate [17] and dispersive liquid-liquid
microextraction-slotted quartz tube-ame atomic
absorption spectrometry [18] are well-known
procedures for preconcentration and separation of
trace copper or zinc from different matrix.
In this work, the pure amoxicillin antibiotic
drug was used as chelating agent for copper and
zinc (Cu/Zn) extraction in human serum and urine
samples by DIL-CPE procedure at optimized
pH. The Cu and Zn deciency was evaluated by
determining its concentration in human serum/
urine samples by F-AAS. The Cu and Zn ions can
be separated from liquid phase by hydrophobic
ionic liquid as green solvent.
2. Experimental
2.1. Apparatus and Reagents
A ame atomic absorption spectrometer model, with
an air-acetylene ame, was used for copper (II) and
zinc (II) determination in human serum and urine
samples (Shimadzu, F-AAS, model 680, Tokyo,
Japan). Copper based on wavelength 324.7 nm, slit
0.5 nm and lamp current 3.0 mA (1-5 mg L
) were
selected. Zinc lamp with wavelength 213.9 nm, slit
0.5 nm and current 5.0 mA (0.4-1.5 mg L
) were
used. A pH meter with glass electrode was adjusted
the pH of human samples (Metrohm, E-632). For
validation, the electrothermal atomic absorption
spectrophotometer (ET-AAS, GBC 932) and ICP-
MS in real samples as certied reference material
(CRM) was used for determination of copper (II)
and zinc (II) in serum and urine samples. The
calibration curve of copper (II) and zinc (II) with
injecting 20 µL of standard solution to graphite
tube were used. All the reagents with analytical
grade were used. Deionized-distilled water (DW,
Millipore, USA) was prepared for experimental
run. The copper (II) and zinc (II) solutions were
prepared by appropriate diluting a 1000 µg L
of Cu and Zn solution (Merck) with DW. The
pure amoxicillin (Fig.1), acetone and ionic liquid
purchased from Sigma Alderich (Switzerland)
and Merck Company (Germany). Buffer solutions
were prepared by standard methods. The
pKa1 = 2.68 carboxyl, (pKa2 = 7.49 amine, and