1. Introduction

Copper and zinc are essential ions for the human

body. Copper has different forms such as CuS,

CuS2, CuFeS2 and CuSO4.5H2O 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-1 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 a,* and Seyed Mojtaba Mostafavi b

a Department of Pharmacology and Toxicology, Pharmaceutical Sciences Research Center, Faculty of Pharmacy,

Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran

b Department of Chemistry, Iranian-Australian Community of Science, Hobart, Tasmania, Australia

ABSTRACT

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

L−1; 15.2 µg L−1) and (100 -505 µg L−1; 41- 153 µg L−1), 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.

Keywords:

Amoxicillin drug,

Copper and zinc,

Serum and urine samples,

Dispersive ionic liquid cloud point

extraction procedure,

Flame atomic absorption spectrometry

ARTICLE INFO:

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

https://doi.org/10.24200/amecj.v3.i03.111

------------------------

Research Article, Issue 3

Analytical Methods in Environmental Chemistry Journal

Journal home page: www.amecj.com/ir

AMECJ

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-1; 111.32 μg dL-1), and the mean value

for serum zinc was at the lower edge of the normal

value (12.24 ± 1.04 μmol L-1; 80.01 μg dL-1), 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-1) were

selected. Zinc lamp with wavelength 213.9 nm, slit

0.5 nm and current 5.0 mA (0.4-1.5 mg L-1) 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-1

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

Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43

pKa3 = 8.49) for amoxicillin (CAS N:26787-78-

0) was considered. 1-Methyl-3-octylimidazolium

hexauorophosphate (CAS Number: 304680-36-

2; C12H23F6N2P; [MOIM][PF6]) was purchased

from Sigma, Germany. The pH of the samples was

adjusted up to 7 with a phosphate buffer (HPO4/

H2PO4; 0.2 M).

Fig. 1. The schema of amoxicillin

2.2. Characterizations

Amoxicillin, as organic compounds with

the penicillin core structure was used as

ligand in this study. Amoxicillin is structurally

characterized by a penam ring (C5H7NOS) bearing

two methyl groups and an amide group. Fourier

transform infrared (FT‐IR) spectra were recorded

from KBr pellets using a spectrophotometer

FTIR Shimadzu (Kyoto, Japan). For amoxicillin,

the C-O stretching vibrations show intense IR

absorptions, due to the considerable change in

the molecular dipole moment produced by this

vibration mode (Fig. 2). Powder X-ray diffraction

(XRD) was conducted on a X-ray diffractometer.

X-ray diffraction analysis of pure amoxicillin and

the optimized formulation was done by X-ray

powder diffractometer (PW 3040/ 60 Xpert PRO,

Panlytical, Netherlands). The X-ray diffraction

patterns were recorded using Cu Kα radiations

(λ=1.5405980Ả), a current of 30 ma, and a voltage

of 50 Kv. The samples were analyzed over 5–35

(2θ range) with a scan step size of 0.02s and 0.5s

per step (Fig. 3). Scanning electron microscopy

(SEM) images were obtained using a Tescan Mira-

3 Field Emission Scanning Electron Microscope

(FE-SEM). The external and internal morphology

of the amoxicillin was studied by scanning electron

microscopy (Fig. 4).

Fig. 2. FT‐IR spectra of pure amoxicillin

Fig. 3. XRD spectra of pure amoxicillin

Fig. 4. FE-SEM for pure amoxicillin

Simultaneously determination of Cu / Zn in human samples Kian Azami et al

2.3. Sample Preparation

Serum potentially contains elements and proteins

which were produced in the human body.

Sample preparation helps to reduce time, errors

and interferences in analytical chemistry. The

sample collection and handling have improved

the sensitivity, selectivity, and reproducibility of

Cu/Zn analysis in serum samples. For increasing

accuracy and precision of results, the human serum

samples were prepared. First, the glass laboratories

placed in mixture of sulfuric acid and nitric acid

(ultra pure grade; 0.5 molar: 0.5 molar) for 24

hours and washed for ten times with DW. The Cu

and Zn concentrations in serum have an important

limit concentration (~0.8-1.5 mg L-1) and So, the

human serum sampling, storage and analysis must

be carefully done. In addition, 10 mL of the human

serum were prepared from personnel of multiple

Sclerosis patients in Iran (MS; 25 Men, 25 women,

25-55 age), based on the world medical association

declaration of Helsinki (WMADH). Clean and

sterilized syringes with plastic needles were

purchased for Merck, Germany for serum blood

sampling. The human biological samples were

maintained frozen in refrigerator (below -4°C).

For long-term storage of serum samples, we placed

samples at −20, −80 °C or using liquid nitrogen.

Urine samples were prepared and storage based on

standard method in human samples.

2.4. Extraction Copper and Zinc Procedure

As shown in Figure 5, the copper and zinc were

simultaneously extracted based on pure amoxicillin

by DIL-CPE procedure. Firstly, 0.1 g of hydrophobic

ionic liquid [MOIM][PF6] and 0.2 mL acetone was

mixed together and then 0.03 g of pure amoxicillin as

a antibiotic drug was added. The mixture was injected

to diluted serum or urine sample (2:10) by 2 mL of

syringe with PVC needle. The cloudy solution was

obtained and shacked for 7 min by shaker accessory.

Then, the Cu or Zn ions was extracted from serum or

urine samples based on sulfur bonding of amoxicillin

at pH of 7 by DIL-CPE procedure (Cu─:S:─Zn).

After centrifuging for 3 minute (3500 rpm), ionic

liquid separated from serum sample in end of conical

tube. The Cu or Zn ions were simply back extracted

with 0.5 mL of nitric acid (0.2 M) and diluted with

DW up to 1 mL. Finally, the remained solution was

determined by ame atomic absorption spectrometry

(F-AAS). The recovery of proposed method based on

pure amoxicillin antibiotic drug was achieved for Cu

and Zn extraction by the recovery equation (mean of

RSD% < 2.2; more than 95%). The Cp is the primary

concentrations of Cu or Zn in sample and CF is the

nal concentration of Cu or Zn by DIL-CPE/F-AAS

procedure (n=10, Eq. A).

Recovery % = (Cp-CF)/Cp×100

(EQ.A)

Fig. 5. Simultaneously extracted of Cu and Zn in serum and urine samples based on amoxicillin

by DIL-CPE procedure

36 Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43

3. Results and Discussion

The DIL-CPE procedure provides novel and

interesting approach based on amoxicillin drug for

extraction of copper and zinc from human serum

and urine samples. In order to obtain favorite

separation and quantitative extraction of Cu and

Zn ions with high sensitivity and precision, the

analytical parameters of proposed DIL-CPE

method must be optimized.

3.1. Optimization of pH

The pH of human serum or urine is main factor

for efficient extraction of Cu and Zn ions by

DIL-CPE procedure. The retention of Cu

and Zn ions by amoxicillin ligand has been

investigated at different pH from 2 to 11 with

buffer solutions containing (100 µg L−1, 500

µg L−1) and ( 50 µg L−1, 150 µg L−1) for copper

and zinc as LLOQ and ULOQ concentrations.

It showed that amoxicillin ligand (pHPZC = 5.6),

the extraction efficiencies of Cu (II) and Zn(II)

were improved with the increase of pH values

more than 6 and the quantitative extraction

were obtained at pH 6-8 and then the recoveries

were reduced at pH more than 9. Consequently,

the Cu and Zn ions quantitative extracted at

pH 7 (Fig.6). Due to serum pH, the extraction

mechanism of Cu (II) and Zn(II) ions based on

amoxicillin ligand is mainly depended on the

electrostatic attractions of deprotonated sulfur

groups of amoxicillin ligand with the positively

charged Cu2+ and Zn2+ cations. At low pH (pH<

pHPZC), the amoxicillin ligand have positively

charged (SH2

+) as a protonation system. So,

the extraction efficiency can be decreased

due to the electrostatic repulsion between the

Cu2+ and Zn2+ cations and positively charge

of ligand. In addition, by increasing pH, the

sulfur groups in amoxicillin ligand becomes

negatively charged (R-S:2-….Cu/Zn) and so

the electrostatic attraction between negatively

charged sulfur groups and positively charged

Cu2+ and Zn2+ cations increased. In more pH

(pH>9) the extraction were decreased due to the

Fig. 6. The pH effect on simultaneously copper and zinc extraction with pure amoxicillin

by DIL-CPE procedure

Simultaneously determination of Cu / Zn in human samples Kian Azami et al

formation of hydroxyl complexes of Cu (II) and

Zn(II) ions.

3.2. Effect of amoxicillin dose

Different amounts of amoxicillin ligand

(AMOX-L) in the range of 5 to 50 mg were

tested on the recoveries of Cu (II) and Zn(II)

ions for the presented DIL-CPE procedure. The

results were shown in Figure 7. It was found that

30 mg and 25 mg of AMOX-L was sufficient

for quantitative recoveries of copper and zinc

ions in serum and urine samples, respectively.

So, 30 mg of AMOX-L was used as optimum

amount of AMOX-L for further works. Higher

amount of AMOX-L had no significant effect

on the extraction of Cu (II) and Zn(II) ions.

Due to capture of Cu (II) and Zn(II) on surface

of AMOX-L and the metal concentration in

solution, it came to equilibrium with each other.

Eventually, 30 mg of AMOX-L was used as

ligand for further work.

3.3. Effect of the IL amount

In the presented DIL-CPE method, 1-Methyl-3-

octylimidazolium hexafluorophosphate

(C12H23F6N2P; [MOIM][PF6]) as hydrophobic ionic

liquid was used as green extraction solvent in

order to separate the Cu (II) and Zn(II) ions which

was complex with AMOX-L as a coordination

complex ions in the serum and urine samples.

The effect of [MOIM][PF6] amounts on the

extraction efciencies of presented method

was studied within the range of 0.05-0.25 g for

10 mL of standard solution, serum and urine

samples containing 30 mg of AMOX-L, copper

values (100 µg L−1, 500 µg L−1) and zinc values

( 50 µg L−1, 150 µg L−1) as LLOQ and ULOQ

concentrations. The results showed that the

quantitative recoveries were obtained with 0.08 g

of [MOIM][PF6]. Therefore, in order to achieve a

suitable preconcentration, 0.1 g of IL was chosen

as optimum leading to a nal IL for serum and urine

samples (Fig.8). Moreover, the effect of IL for

Fig. 7. The effect of AMOX-L on simultaneously copper and zinc extraction

by DIL-CPE procedure

38 Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43

Cu and Zn extraction with the same experiments

were evaluated without and AMOX-L. Based on

results, the efcient extraction for Cu and Zn ions

were obtained less than 24% and 19% for urine

and serum as complexation Cu and Zn with amino

acids such as cysteine (Cys) and proteins (Cu/

Zn….Pr /Cys). Therefore the IL had low effect

on Cu and Zn extraction at optimum conditions.

In addition, by increasing the ultrasonication

time up to 60 min, the almost 27% and 31% of

Cu and Zn ions extracted by 0.1 g of IL without

any AMOX-L. These results conrm the critical

role of AMOX-L as complex agent for Cu and Zn

extraction.

3.4. Effect of eluents

The elution solutions were optimized in order to

obtain the maximum back-extraction Cu and Zn

ions from IL with the minimum concentration

and volume of the elution solution. By DIL-

CPE method, the different elution solutions were

selected with high recovery. The coordination

of Cu and Zn cations with AMOX-L was

dissociated and ions released into the aqueous

phase in acidic pH. For evaluation of the type,

the concentration and the volume of acid

solutions for back extraction ions from ligand,

1000 µL of different mineral acids solutions such

as HCl, H3PO4, HNO3 and H2SO4 (0.1-0.5 mol

L-1) were examined by DIL-CPE procedure. The

results showed that 0.2 mol L-1 HNO3 (0.5 mL)

quantitatively back-extracted Cu and Zn from

ligand/IL (Fig. 9).

3.5. Effect of sample volume

As shown in Figure 10, the effect of sample

volume on the extraction of copper and zinc in

serum and urine samples were evaluated and

optimized by different volumes from 1- 25 mL

containing copper values (100 µg L−1, 500 µg

L−1) and zinc values ( 50 µg L−1, 150 µg L−1) as

LLOQ and ULOQ concentrations by DIL-CPE

procedure. As shown in Fig. 10, satisfactory

recoveries were obtained between 2-10 mL for

urine and serum samples. In addition, the high

sample volume caused to signicantly decrease

the extraction Cu and Zn in human samples.

Therefore, 10 mL of human samples was used for

further investigation.

3.6. Effect of time

Fig. 8. The effect of [MOIM][PF6] on simultaneously copper and zinc extraction

by DIL-CPE procedure

Simultaneously determination of Cu / Zn in human samples Kian Azami et al

Dispersion is main factors for DIL-CPE procedure

and allows to perfectly contacting of the Cu and Zn

cations with AMOX-L as complex agent. Due to

the favorite dispersion of the ligand into the liquid

phase, the recovery of extraction phase increased.

The effect of the ultrasonication time on the DIL-

CPE procedure based on AMOX-L was studied

within the range of 1–10 min. The results showed, by

increasing time up to 2.5 min, the relative extraction

increased and after this time remained constant. So,

the time of 2.5 min selected for Cu and Zn extraction

for further studies. On the other hand, after complex

ions with AMOX-L, the centrifugation was needed

to accelerate the separation IL from liquid phase.

Fig. 10. The effect of sample volume on extraction of copper and zinc by DIL-CPE procedure

Fig. 9. The effect of eluents on back-extraction of copper and zinc extraction

by DIL-CPE procedure

40 Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43

Therefore, different times for centrifuging were

examined between 1-5 min at 3500 rpm. The result

showed that 2.0 minutes is sufcient to perfect

separation phase.

3.7. Interference study

For evaluating of the analytical application in real

samples, the important interference of coexisting

ions effected on copper and zinc extraction in

serum and urine samples were studied by the DIL-

CPE procedure. For this proposed, the different

amounts of the interfering ions added to 10 mL of

liquid solution containing 500 µg L−1 of copper and

150 µg L−1 of zinc. The results showed, the most of

the coexisting cations and anions had no effect on

the extraction of Cu and Zn ions under optimum

conditions (SD of recovery < ±5%). In fact, the

tolerable concentration ratio of interference of

coexisting ions (M) per Cu and Zn (M/Zn2+ or M/

Cu2+) for Hg2+, Ag+ and Au3+ - was less than 50 and

30 for zinc and copper, respectively. This ratio was

almost 100-200 for Ni2+, Pb2+ and Co 2+ ions. The

results showed that the AMOX-L have favorite

ligand for Cu and Zn extraction despite the high

concentrations of the coexisting ions (Table 1).

3.8. Analytical gures of merit

The analytical characteristics for Cu and Zn

extraction in human serum and urine samples were

Interfering Ions in serum (M)

Mean ratio

(CM /C Cu(II))Recovery (%)

Cu(II) Cu(II)

V3+, Fe 3+ 800 97.2

Cd2+, Mn2+ 600 98.1

I-, Br-, F-, Cl-1200 99.3

Na+, K+1000 98.4

Ca2+, Mg2+ 750 97.1

CO3

2-, PO4

3-, NO3

-900 97.7

Ni2+, Co2+ 100 96.9

Pb2+ 700 98.2

Hg2+, Ag+, Au3+ 30 97.3

Interfering Ions in serum (M)

Mean ratio

(C M /C Zn(II))Recovery (%)

Zn(II) Zn(II)

V3+, Fe 3+ 900 98.7

Cd2+, Mn2+ 750 97.6

I-, Br-, F-, Cl-1100 98.8

Na+, K+900 98.2

Ca2+, Mg2+ 950 96.6

CO3

2-, PO4

3-, NO3

-1100 98.7

Ni2+, Co2+ 200 97.5

Pb2+ 800 96.7

Hg2+, Ag+, Au3+ 50 98.0

Table 1. The effect of interferences ions on extraction of Cu(II) and Zn (II) in serum samples

by DIL-CPE procedure

Simultaneously determination of Cu / Zn in human samples Kian Azami et al

studied by the purposed DIL-CPE procedure. The

intra-day analytical performance was shown in

Table 2 for the multiple sclerosis patients (50) and

healthy peoples (50). Under the optimal conditions,

the linearity for the Cu (II) and Zn(II) concentration

ranges between 100 -505 µg L−1 and 41- 153 µg

L−1, respectively as a lower limit of quantication

(LLOQ) and upper limit of quantication (ULOQ)

with mean correlation coefcient of R2=0.9997

for Intra-day analysis. The LOD and LOQ are as

an analytical signal three times higher than the

background noise and three times higher than

LOD, respectively. The precision of the AMOX-L/

DIL-CPE procedure showed by the relative

standard deviation (RSD %) for ten replicate

determination containing 100 µg L−1 of Cu and

Zn concentration which was obtained lower than

2.2%. The enrichment factor was calculated based

on calibration curve and curve tting rule (tga=m1/

m2). According to proposed method, a favorite

linear ranges and satisfactory EF were achieved for

determination of Cu(II) and Zn (II) ions in human

samples. The mean value for serum copper/zinc

ratio in MS patients was obtained 1.11 ± 0.28 which

was lower than normal range as 1.44 ± 0.31.

3.9. Analysis of real and certied samples

The DIL-CPE procedure was used for

determination of copper and zinc in serum and

urine samples by AMOX-L ligand. The validation

of method was obtained based on spiking samples

by known concentration of Cu (II), and Zn(II).

The efcient recovery was achieved by spiking

samples, which conrms the accuracy of the

Table 2. Determination of copper and zinc in serum by DIL-CPE procedure (Mean intra-day

and inter –day analysis for 50 MS and 50 healthy peoples (HP); µgL-1)

Serum Sample

a Mean of MS (n=50) a Mean of HP (n=50) Data Subject

Intra-day Inter day Intra-day Inter day r P value

Copper 965.4 ± 51.6 972.1 ± 58.7 1154.5 ± 62.6 1168.7 ± 71.4 0.107 <0.001

Zinc 658.4 ± 29.5 666.2 ± 33.8 875.3 ± 44.6 861.9 ± 48.8 0.123 <0.001

Mean of three determinations of samples ± condence interval (P = 0.95, n =10)

Correlations are based on Pearson coefcients (r). Statistical signicance will be observed if P < 0.001

Table 3. Validation of developed the DIL-CPE procedure based on AMOX-L for Cu

and Zn determination by spiking real samples (µgL-1)

Sample Added Cu Added Zn Found Cu* Found Zn* Recovery(%)Cu Recovery(%)Zn

Serum ------ ------ 235.5 ± 11.2 189.6 ± 9.3 ------ ------

200 200 429.6 ± 20.8 380.4 ± 18.7 97.1 95.4

Urine ------ ------ 197.3 ± 10.1 201.2 ± 9.4 ------ ------

200 200 394.8 ± 17.6 404.7 ± 18.9 98.7 101.7

Serum ------ ------ 252.7 ± 11.2 168.8 ± 8.3 ------ ------

250 150 499.8 ± 23.6 315.5 ± 14.7 98.8 97.8

Urine ------ ------ 171.4 ± 8.1 142.3 ± 6.6 ------ ------

150 150 324.2 ± 13.5 288.7 ± 14.2 101.8 97.6

* Mean of three determinations of samples ± condence interval (P = 0.95, n =10)

All samples (2 mL) diluted with DW up to 10 mL( Dilution factor: DF=5)

42 Anal. Method Environ. Chem. J. 3 (3) (2020) 32-43

DIL-CPE procedure (Table 3). The recoveries of

spiked samples for Cu and Zn were ranged from

96 to 105%, which demonstrated that the DIL-

CPE procedure was satisfactory for determination

copper and zinc in urine and serum samples. On

the other hand, the certied standard reference

materials (CRM) were prepared in serum and urine

sample by ICP-MS and used for the validation

methodology (Table 4).

4. Conclusions

A simple, rapid, reliable and sensitive method

was developed for separation and extraction of Cu

(II) and Zn(II) in serum and urine samples based

on AMOX-L by the DIL-CPE procedure. The

[MOIM][PF6] ionic liquid as a trapping phase was

used for separating of Cu/Zn-loaded AMOX-L

from liquid phase. IL helps to reducing the sample

preparation and separation time for extraction

process. Using a small amount of AMOX-L

with high extraction recovery, good precision,

minimal acid elution (500 µL) and green solvent

caused to make the efcient extraction based on

environmentally friendly for determining of Cu

and Zn in urine and serum samples. Also, the low

LOD and RSD% values as well as the quantitative

recoveries (more than 95%) were obtained in

optimized conditions. Therefore, the developed

method based on AMOX-L can be used as favorite

sample preparation in human biological samples

in short time. As obtained results, the amoxicillin

[1] Zinc, Institute of Medicine, Dietary Reference

Intakes: The essential guide to nutrient

requirements, The National Academies Press,

Washington D.C., pp. 402-413, 2006.

[2] R. K Gupta, S.S. Gangoliya, N.K. Singh,

Reduction of phytic acid and enhancement of

bioavailable micronutrients in food grains, J.

Food Sci. Technol., 52 (2015) 676-684.

[3] Copper, Institute of Medicine. Dietary Reference

Intakes: The Essential Guide to Nutrient

Requirements, The National Academies Press,

Washington D.C., pp. 304-311, 2006.

[4] J. Osredkar, N. Sustar. Copper and zinc,

biological role and signicant of copper/

zinc imbalance, J. Clinic. Toxicol., S3

(2011) 001. http://doi: 10.4172/2161-0495.

S3-001

Table 4. Validation of methodology based on ICP-MS analysis in real samples

and compared to the DIL-CPE procedure (µgL-1)

Sample Added(µgL-1) *Found(ICP-MS) *Found(DIL-CPE) Recovery

Serum(Cu) ------ 302.2 ± 7.2 297.5 ± 14.2 98.4

200 ------ 492.8 ± 22.4 97.6

Serum (Zn) ------ 182.3± 4.2 178.9 ± 8.2 98.1

150 ------ 334.2 ± 15.4 103.5

Urine (Cu) ------ 261.5 ± 6.8 257.2 ± 11.7 98.3

250 ------ 497.9 ± 24.6 96.3

Urine (Zn) ------ 192.3 ± 4.5 200.6 ± 9.5 104.3

200 ------ 399.8 ± 17.4 99.6

* Mean of three determinations of samples ± condence interval (P = 0.95, n =10)

All samples (2 mL) diluted with DW up to 10 mL( DF=5)

can be affected on copper and zinc deciency in

human body at human pH when the patients used it

by over dosage for many times.

5. Acknowledgment

The author thank to Tehran University of Medical

Sciences (TUMS). The Ethical Committee of

Iranian Petroleum Industry Health Research

Institute approved the human sample analysis by

Lab of IPIHRI (R.IPIHRI.PN.1398. 001)

6. References

Simultaneously determination of Cu / Zn in human samples Kian Azami et al

[5] E. Kouremenou-Dona, A. Dona, J.

Papoutsis, C. Spiliopoulou, Copper and zinc

concentrations in the serum of healthy Greek

adults, Sci. Total Environ., 359 (2006)76–

81.

[6] V. rvanitidou, I. Voskaki, G. Tripsianis, S.

Flippidis, K. Schulpis, I. Androulakis, Serum

copper and zinc concentrations in healthy

children aged 3–14 years in Greece, Biol.

Trace Elem. Res., 115 (2007) 1–12

[7] Z. L.-L. Zhang, L. Lu, Y.-J. Pan, Baseline

Blood Levels of Manganese, Lead, Cadmium,

Copper, and Zinc in Residents of Beijing

Suburb, Environ. Res., 2015 140 (2015) 10–

17.

[8] Y. M. Park, J. Y. Choi, E. Y. Nho, Ch. M. Lee,

I. M. Hwang, N. Khan, Determination of

macro and trace elements in canned marine

products by inductively coupled plasma—

optical emission spectrometry (ICP-OES)

and ICP—mass spectrometry (ICP-MS), J.

Anal. Lett., 52 (2019)1018-1030.

[9] Y. Wang, J. Xie, Y. Wu, X. Hu, A magnetic

metal-organic framework as a new sorbent

for solid-phase extraction of copper(II), and

its determination by electrothermal AAS,

Microchim. Acta, 2014, 181, 949–956.

[10] S. Hamida, L. Ouabdesslam, A. F. Ladjel,

M. Escudero, J. Anzano, Determination of

Cadmium, Copper, Lead, and Zinc in Pilchard

Sardines from the Bay of Boumerdés by

Atomic Absorption Spectrometry, J. Anal.

Lett., 51 (2018) 2501-2508.

[11] T. Sahraeian, H. Sereshti, A. Rohanifar, A.

Simultaneous determination of bismuth, lead,

and iron in water samples by optimization of

USAEME and ICP–OES via experimental

design. J. Anal. Test., 2 (2018) 98–105.

[12] H. Sereshti, A.R. Far, S. Samadi, Optimized

ultrasound-assisted emulsication-

microextraction followed by ICP-OES for

simultaneous determination of lanthanum

and cerium in urine and water samples, Anal.

Lett., 45 (2012) 1426–1439.

[13] G.Bagherian, M. A. Chamjangali, H. S.

Evari, M. Ashra, Determination of copper

(II) by ame atomic absorption spectrometry

after its perconcentration by a highly

selective and environmentally friendly

dispersive liquid–liquid microextraction

technique, J. Anal. Sci. Technol., 10

(2019) 3.

[14] H. Duan, Z. Wang, X. Yuan, S. Wang, H.

Guo, X. Yang, A novel sandwich supported

liquid membrane system for simultaneous

separation of copper, nickel and cobalt in

ammoniacal solution, Sep. Purif. Technol.,

173 (2017) 323–329.

[15] B. Ebrahimi, S. Mohammadiazar, S. Ardalan,

New modied carbon based solid phase

extraction sorbent prepared from wild

cherry stone as natural raw material for the

pre-concentration and determination of

trace amounts of copper in food samples,

Microchem. J. 147 (2019) 666–673.

[16] S. Vellaichamy, K. Palanivelu,

Preconcentration and separation of copper,

nickel and zinc in aqueous samples by ame

atomic absorption spectrometry after column

solid-phase extraction onto MWCNTs

impregnated with D2EHPA-TOPO mixture,.

J. Hazard. Mater., 185 (2011) 1131–1139

[17] M.A. Farajzadeh, M. Bahram, B.G. Mehr,

J.A. Jönsson, Optimization of dispersive

liquid–liquid microextraction of copper (II)

by atomic absorption spectrometry as its

oxinate chelate: application to determination

of copper in different water samples, Talanta,

75 (2008) 832–40.

[18] G. Özzeybek, S. Erarpat, D.S. Chormey,

M. Fırat, Büyükpınar Ç, Turak F, Bakırdere

S. Sensitive determination of copper in

water samples using dispersive liquid-liquid

microextraction-slotted quartz tube-ame

atomic absorption spectrometry. Microchem

J., 132 (2017) 406–10.