Research Article, Issue 1

Analytical Methods in Environmental Chemistry Journal

Journal home page: www.amecj.com/ir

AMECJ

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

1. Introduction Fe

into Fe

which was bonded to transferrin [2].

Copper enter to biochemical reactions and human

physiology. In biological matrix such as plasma,

copper complex to proteinsas caeruloplasmin and

albumin or amino acids [3]. The defect of central

nervous system caused to multiple sclerosis (MS)

disease in human due to demyelination of nerve

fibers of the brain and spinal cord. Based on

previous studies, the genetic, immunological and

environment factors have main source for creating

MS in humans with disability. But, the environment

factors such as heavy metals such as copper, zinc,

cadmium and lead has significant role in MS [5, 6].

The deadline of time exposure of heavy metals in

In-vitro extraction and separation of copper ions from human blood

samples based on amoxicillin/clavulanic acid by ultrasound assisted-

dispersive centrifuge liquid-liquid micro extraction

*, Jamileh Esmaeilia,b, Samira Shirooiec and Azam Bakhtiariand

a Ph.D Student in Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran , Iran

bDepartment of Pharmacology, Tehran University of Medical Sciences

C Sciences Research center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

*,d Department of Pharmacology Tehran University of medical Sciences

Copper (Cu) acts as a main co-factor in humans

and enter into cuproenzymes that catalyze electron

transfer reactions (ETR) required for cellular

respiration, iron oxidation, neurotransmitter

biosynthesis, antioxidant, peptide amylation. The

Cu intake has been associated with toxic effects

in humans include influenza-like syndrome ,

hemolysis and kidney failure due to Cu sulfate

intake [1]. Cu exists in blood plasma in Cu bound

to caeruloplasmin (65–71%) and convert the iron

*Corresponding Author: Azam Bakhtiarian

Email: bakhtiar12@yahoo.com

https://doi.org/10.24200/amecj.v3.i01.94

A R T I C L E I N F O:

Received 18 Nov 2019

Revised form 9 Jan 2020

Accepted 12 Feb 2020

Available online 30 Mar 2020

Keywords:

Copper,

Separation,

Human blood,

Amoxicillin/clavulanic acid,

Ultrasound assisted-dispersive centrifuge

liquid-liquid micro extraction

A B S T R A C T

The low concentration of copper (Cu2+) can be effected on the

central nervous system (CNS) and caused to multiple sclerosis

(MS). Although many antibiotics can treat the bacterial infections

but some of antibiotics decrease essential metal concentrations in

human body and must be controlled by determining. In this study,

in-vitro extraction of copper (Cu2+) with amoxicillin/clavulanic

acid (AMOXC) has been studied due to interacting with metals.

By procedure, Cu2+ ions were separated from blood samples by

ultrasound assisted-dispersive centrifuge liquid-liquid micro

extraction (USA-DC-LLME). The mixture of AMOXC (0.01

g), ionic liquid ([BMIM][PF6]) and acetone injected to 10 ml

of serum blood sample at human pH=7.2. After extraction, the

concentration of Cu2+ ions was determined by flame atomic

absorption spectrometry (F-AAS). The LOD, enrichment factor

(EF), linear range (LR) and working range (WR) were obtained 6

μg L-1, 9.92, 0.018-0.5 mg L-1 and 0.02-2.58 mg L-1, respectively

(RSD<1.1%). The validation of technology was confirmed by

ICP and spiking samples.

Copper Extraction in Blood by Amoxicillin Clavulanic Acid Jamileh Esmaeili, et al

Anal. Method Environ. Chem. J. 3 (1) (2020) 55-62

56 Anal. Method Environ. Chem. J. 3 (1) (2020) 55-62

human blood samples depended to kind of heavy

metals and chorionic/acute exposure (cadmium was

seen in blood up to 1 hour as acute exposure). In

chorionic exposure the concentration of heavy metals

can be followed in nail or hair. Copper used for

many products as an essential element in nature and

copper has concentration between 80-180 μg dL

-1

blood of human adults (Mean= 1.5 mg L

-1

). Copper

exposure in industry occurs primarily through

inhaled particulates and according to studies, copper

can be a cause of the pathogenesis of MS [6]. The

value of copper (Cu

) effect on the central nervous

system (CNS) and reducing of their concentration

caused to numerous CNS disorders such as multiple

sclerosis (MS), Wilson disease, Alzheimer and

Parkinson’s diseases [7-9]. Also, some of antibiotics

caused to decrease essential metals such as Zn

and

in human body. So, in present of antibiotics,

determination of Zn

and Cu

in human matrixes is

very important. Some receptors such as β-ketoenol-

bipyridine and ketoenol–pyrazole were functioned

on silica hybrid adsorbent and other sorbents [10,

11]. For determination of copper the different

analytical techniques such as, ET-AAS [12, 13],

ion selective electrode, Square wave‐adsorptive

anodic stripping voltammetric (SW‐ASV) [14]

and flame atomic absorption spectrometry (F-AAS)

[15] were used in different samples such as blood,

waters and tissues(fish)[16]. High interferences in

human blood are main problems for determination

copper by instruments. So, sample preparation

must be used before analysis by instruments. The

different procedures such as, cloud point extraction

(CPE)[9], liquid-liquid extraction (LLE) [15],solid-

phase extraction (SPE) in human biological fluid

(blood, serum and urine) [17,18-22] and dispersive

liquid-liquid microextraction (D-LLME) based on

ionic liquids (IL) [23,24,15] were used before Cu

analysis. Recently, a applied dispersive liquid-liquid

microextraction was used for separation/extraction

of ions from liquid samples. In this study, USA-

DC-LLME based on AMOXC was used for efficient

extraction of Cu ions in human biological samples

before determination by F-AAS. The hydrophobic

ionic liquid was used for simple separating of Cu-

AMOXC from blood samples.

2. Experimental

2.1. Instrumental

The spectra GBC 906 double beam atomic

absorption spectrophotometer was used for copper

determination (FAAS, GBC, Model; Plus 906,

Australia). The air-acetylene as fuel gas based on

background correction was selected for F-AAS.

The light of HCL adjusted on burner by vertical and

horizontal positions. The Avanta software of spectra

was used for collecting data. The copper hollow

cathode lamp (HCL, slit=0.7) with wavelength of

327.4 nm and current of 4.0 mA was adjusted in

vertical and horizontal position. For copper analysis,

the limit of detection, working range was obtained

0.06 mg L

-1

and 0.2-24.0 mgL

-1

, respectively by

F-AAS. All samples as minimum volume were used

by auto-sampler injector (0.5-5 mL). The pH values

of the solutions were measured by a digital pH

meter (Metrohm 744), especially in human samples.

All samples were shaken with Thermo accessory as

mixer (250 rpm, USA) and centrifuged with Thermo

fisher (1000-4500 rpm, USA).

2.2. Reagents

The ultra-trace reagents with analytical grade such as;

HNO3, HCl, NaOH, copper salt and acetone solutions

were purchased from Merck (Darmstadt, Germany).

The pure AMOXC as amoxicillin trihydrate:

potassium clavulanate (4:1) was purchased from

sigma, Germany (Product N: SMB00607, Batch

N: 128M4800V, 75% AMOX and less than 15%

clavulanic acid and less than 15% water) (Fig.1).

The Cu(II) stock standard solution was prepared as

nitrate salt of 1000 mg L

-1

(ppm) solution in 1 %

HNO

. The standard solutions were prepared daily

by dilution of the standard solution of Cu(II) by

ultra-pure water(UPW). High purity distilled water

had already prepared from Millipore (Bedford,

USA). 1-Ethyl-3-methylimidazolium acetate (CAS

N: 143314-17-4), 1-Ethyl-3-methylimidazolium

tetrafluoroborate (CAS N: 143314-16-3), 1-Ethyl-3-

methylimidazolium hexafluorophosphate (CAS N:

155371-19-0), and 1-butyl-2,3-dimethylimidazolium

Copper Extraction in Blood by Amoxicillin Clavulanic Acid Jamileh Esmaeili, et al

hexafluorophosphate ([BDMIM] [PF

]) as a

hydrophobic

ionic liquids were was selected for

separation AMOXC from blood sample. The T-X100

with analytical grade were purchased from Merck

Co., Darmstadt, Germany. The pH was adjusted

with buffer solutions (Merck, Germany). Sodium

phosphate and citric acid was used for phosphate

citrate buffer for PH=2.0–7.5.

2.3. Sample preparation

The glasses were washed with a HNO

solution

(0.5M) for at least 24 h and thoroughly rinsed 10

times with ultra-pure water. For sampling, 20 ml of

samples of blood were collected from subjects and

control peoples (N=30, men, 30-55 age). 10 mL of

blood sample mixed with heparin (pure, 20 micro

liter) and storage in low temperature. Then, 10 mL of

blood samples were stay at the room temperature for

30 minutes to coagulate and then centrifuged at 3000

rpm for 5 minutes. Next, serums were separated by

a sampler. Plasma was also prepared by procedure

by centrifuging process. The human blood/serum

samples were maintained at –20 °C up to 72 h. In

humans with low sample volume of blood, it

diluted up to 20 mL and then was used. The Ethical

Committee of Azad University

(SN.IAU.ُSRB.940522664) confirmed the human

sample method.

2.4. Procedure

By USA-DC-LLME procedure, the 10 mg of pure

AMOXC powder (15 micro molar conc.) mixed

with ionic liquid ([BMIM][PF6], IL, 0.1 g) in

present of 500 micro liter of acetone/ethanol, then,

the mixture was injected to 10 ml of serum blood

sample at human pH=7.2 (Fig.2). After 5 min

shaking time, the hydrophobic ionic liquid BMIM]

[PF6] was separated by centrifuging at 3500 rpm for

4 min in conical glass tube. The upper liquid phase

separated by auto-sampler accessory (1-100 mL)

and remained phase (IL-AMOXC/Cu2+) were back-

extracted from IL phase by decreasing pH up to 1

(HNO3, 0.5 M, 0.5 mL). Finally the concentration

of Cu2+ ions was simply determined by F-AAS after

dilution with DW up to 1 mL.

3. Results and discussion

For extraction copper from blood samples all

parameters (pH, sample volume, amount of AMOXC

and IL) must be optimized. The serum, blood, urine

samples was prepared from patients with copper

disease such as multiple sclerosis. After tuning pH

with favorite buffers, the sample determines by

ultrasound assisted-dispersive centrifuge liquid-

liquid micro extraction (USA-DC-LLME) coupled

to F-AAS.

Fig. 1. Schema of amoxicillin trihydrate: potassium

clavulanate

Fig. 2. The copper extraction from blood samples based on AMOXC by USA-DC-LLME

58 Anal. Method Environ. Chem. J. 3 (1) (2020) 55-62

3.1. PH effect

The effect of sample pH on complexation of Cu(II)

ions on AMOXC was studied for 0.2 mg L

-1

and

2.0 mg L

-1

of Cu(II) (lower limit of quantification;

LLOQ and upper limit of quantification; ULOQ)

from different pH 1-12. The complexation of

AMOXC-Cu was depended on the pH of solutions

and function group of ligand. The results showed,

the maximum extraction of Cu(II) was achieved in

human pH (7.0), and the recovery values for Cu(II)

were below 5% in acidic or basic pH. The extraction

mechanism of Cu(II) ions with AMOXC was highly

referred to pH and complex formation between

Cu(II) ions and dative sulfur bonding of AMOXC

(Fig. 3). The sulfur groups can be deprotonated (:S-

R) at wide range of pH from 6 to 9. The extraction

efficiency of Cu(II) at pH values below 6 and upper

of 8 cannot occurred due to similarity charge or

precipitation of hydroxyl copper (Cu(OH)

3.2. Sample volume effect

The sample volume in one of the important factors

which must be optimized for Cu extraction from

blood samples by AMOXC. As evaluation extraction

efficiency, different volumes between 1.0-30 mL

of blood and serum samples was examined and

optimizes in pH=7. The results showed us that the

maximum recovery for copper extraction based

on AMOXC was achieved for less than 12 mL

for human blood samples and less than 17 mL of

standard solutions. Therefore, 10 mL and 15 mL

of sample volume was selected as optimum point

for copper extraction for blood and water samples,

respectively by USA-DC-LLME procedure (Fig.4).

3.3. AMOXC effect

The amount of ligand is very important for copper

extraction in blood samples, so, the concentration of

AMOXC in human samples must be studied for high

extraction. For this purpose, the vary concentration

between 1-50 micro molar concentration of AMOXC

was prepared and used for evaluation of copper

extraction in blood/standard samples by USA-DC-

LLME procedure. The experimental results showed,

the high extraction of copper was obtained with

more than 15 micro molar of AMOXC in 10 mL of

samples (~ 10 mg pure powder) by USA-DC-LLME

procedure (Fig. 5). Also, the extraction efficiency

more than 97% was made by 12 micro molar of

AMOXC concentration in 10 mL of water samples.

First, 10 mg of pure AMOXC powder mixed with

ethanol/acetone (0.5 mL) and then added to 0.1 g

of IL in 2 mL syringe. After shacking, the mixture

injected to 10 mL of blood samples. The extraction

efficacy was decreased about 74.6% when the

mixture added step by step without shacking.

3.4. Ionic liquid effect

Separation process for AMOXC-Cu from liquid

samples (blood, standard solution) is very difficult.

Fig. 3. The effect of pH on copper extraction based on AMOXC by USA-DC-LLME procedure

Copper Extraction in Blood by Amoxicillin Clavulanic Acid Jamileh Esmaeili, et al

The different techniques with filter, centrifuging

and organic solvents was used for separation ligands

from liquid phase. Recently, the benign solvents as

green solvents (IL) was introduced in many papers.

In this study we used different hydrophobic ionic

liquids for separation processes in blood samples.

The different volumes or gram (0.05-0.3 g) of

1-Ethyl-3-methylimidazolium acetate, 1-Ethyl-3-

methylimidazolium tetrafluoroborate, 1-Ethyl-3-

methylimidazolium hexafluorophosphate, and 1-butyl-

2,3-dimethylimidazolium hexafluorophosphate

[BDMIM][PF6] as a hydrophobic ionic liquids were

was selected for separation AMOXC from blood

sample. Based on results, 0.1 g of [BDMIM][PF6] has

more extraction efficiency as compared to others (Fig.

6). The 1-Ethyl-3-methylimidazolium acetate and

1-Ethyl-3-methylimidazolium tetrafluoroborate had

lower recovery because of solubility in liquid phase

and missed of IL. The1-Ethyl-3-methylimidazolium

hexafluorophosphate as hydrophobic IL can be

separated copper-AMOXC from blood samples but

has lower recovery (85%) as compared to [BDMIM]

[PF6] with (97.8%).

3.5. Interference ions study

Many ions such as Zn

, Mn

, Na

, K

, HCO

, SO

, Co

, exist in water or blood samples and can be

effected on complexations of copper with AMOXC

in presence of interference ions. The complexation

copper depended on power of ligand and competition

Fig. 4. The effect of sample volume on copper extraction based on AMOXC by USA-DC-LLME procedure

Fig. 5. The effect of AMOXC concentration by USA-DC-LLME procedure

60 Anal. Method Environ. Chem. J. 3 (1) (2020) 55-62

of ions in different pH. So, in optimized pH many

ions had no effect on extraction process, but some of

them may be done. Therefore the effect of important

ions which was bonding with amino acids or proteins

was studied. The recovery of copper extraction based

on AMOXC in presence of different concentration

of 1-4 mg L

-1

of cations and anions was evaluated

by USA-DC-LLME procedure. As selectivity of

determination of F-AAS, the concentration of other

ions can’t reported. So, the recovery was checked

as effect of interference ions. The results showed,

the interference ions had no effect on complexation

processes of copper – AMOXV by USA-DC-LLME

procedure (Table 1).

3.6. Validation procedure

The methodology of copper extraction must be

validated by different technologies. By spiking of

blood, serum, urine, plasma samples, the favorite

recovery more than 95% was achieved for LLOQ and

ULOQ range. So, the USA-DC-LLME procedure

was validated for copper complexation based on

AMOXC (Table 2). In addition, ET-AAS coupled

with microwave digestion was used for evaluation

of purposed procedure after dilution samples up

to 100 mL(1:10) (Table 3). As intra-day and inter-

day studies, the mean copper concentration based

on AMOXC in subjects and control groups (N=30,

men, 30-55 age) was checked by USA-DC-LLME

procedure. The results showed no significance

difference between subjects and control groups with

favorite p-value (Table 4). The mean concentration

of copper in control groups were a little higher

than Cu blood subjects. The regression analysis

and t-test were achieved between Cu in subject and

control groups. There were a correlation (0.45< r

<0.5) between blood of subject and control groups.

(p-value < 0.001).

Fig. 6. The effect of different ILs on copper separation based on AMOXC by USA-DC-LLME procedure

Table 1. Effect of interfering ions on the extraction recovery of Cu (II) ions by USA-DC-LLME procedure

Foreign Ions

Concentration ratio (Cinterferent ion/CCu2+) Recovery (%)

Standard Blood Standard Blood

K+, Na+, Li+, Cl-, F-. Mg2+,Ca2+ 1200 1000 97.5 96.8

Co2+, Ni2+ 900 700 98.2 97.3

Pb2+, Ag+600 500 97.6 97.9

Zn2+, Mn2+ 700 600 95.5 98.4

Fe2+, V3+, As3+, Mo3+ 850 800 97.0 96.8

Cd2+, Al3+ 800 550 98.2 97.3

Hg2+ 1000 900 96.5 95.7

CO3

2-, SO3

2- 950 600 99.1 98.5

Copper Extraction in Blood by Amoxicillin Clavulanic Acid Jamileh Esmaeili, et al

4. Conclusions

A simple Pharmacology and biology method based

on USA-DC-LLME was used for evaluating of

AMOXC for copper extraction in human blood

samples. The results demonstrated that AMOXC

can decreased copper concentration in blood

samples in efficient time. On the other hands

amino acids and proteins was also complexed with

copper and caused to decrease copper extraction by

AMOXC. So, the AMOXC competed with amino

acids and proteins for extraction copper and other

metals at pH 7.2. In addition, the antibiotics such

as AMOXC can treat the bacterial infections but

they can decrease essential metals (Cu) in biological

matrixes and cause disease in humans. By purposed

procedure, the copper extracted with AMOXC at

pH=7 and determined by F-AAS. The 1-butyl-

2,3-dimethylimidazolium hexafluorophosphate

([BDMIM][PF

] as hydrophobic IL was used for

separation of Cu from liquid phase. In optimized

conditions, the low LOD and RSD% values as well

as good working ranges (0.02-2.58 mg L

-1

) and high

recoveries caused to consider as a recent innovative

procedure.

5. Acknowledgements

The authors thank Azad University of Medical Sciences

for supporting this work. The Ethical Committee of Azad

University (SN.IAU.ُSRB.940522664) confirmed the

human sample method.

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Sample Added(mg L-1) *Found(mg L-1) Recovery (%)

Blood -------- 0.884 ±0.043 --------

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