Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

Research Article, Issue 4

Analytical Methods in Environmental Chemistry Journal

Journal home page: www.amecj.com/ir

AMECJ

Separation and determination of lithium and manganese ions

in healthy humans and multiple sclerosis patients based on

nanographene oxide by ultrasound assisted-dispersive -micro

solid-phase extraction

Seyed Majid Nabipour Haghighia and Negar Motakef Kazemi*,a

a Department of Medical Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic

Azad University, Tehran, Iran.

ABSTRACT

Lithium regulates the concentration of nitric oxide in the human

body and a high dose of nitric oxide causes multiple sclerosis (MS).

Also, the amount of manganese in the cerebrospinal uid alters the

metabolic reactions associated with MS. In this study, the mixture of

the ammonium pyrrolidine dithiocarbonate (APDC), the hydrophobic

ionic liquid [HMIM][PF6] and acetone coated on the surface of

graphene oxide nanoparticles (GONPs) and used for separation Li

and Mn in human samples by ultrasound assisted-dispersive-ionic

liquid-micro-solid phase extraction technique (USA-DIL-μ-SPE) at

pH 6.0. After extraction and back-extraction, the amount of lithium

and manganese in the blood, serum and urine samples was determined

by the ame and the graphite furnace atomic absorption spectroscopy

(F-AAS, GF-AAS), respectively. By optimizing parameters, the

LOD, Linear ranges (LR) and preconcentration factor (PF) for Li

and Mn ions were obtained (0.03 mg L-1, 0.25 μg L-1), (0.1-0.4 mg

L-1, 0.08-1.5 μg L-1) and 10, respectively (%RSD<5). The capacity

adsorption of APDC/IL/GONPs and GONPs was achieved (148.5 mg

g-1, 122.3 mg g-1) and (41.3 mg g-1, 33.7 mg g-1) for Li and Mn ions in

a static system, respectively. This method was successfully validated

by spiking samples and certied reference materials (CRM).

Keywords:

Lithium, Manganese,

Human samples,

Multiple sclerosis patients,

Nano graphene oxide,

Ultrasound assisted-dispersive-micro-

solid phase extraction

ARTICLE INFO:

Received 3 Sep 2021

Revised form 6 Nov 2021

Accepted 30 Nov 2021

Available online 29 Dec 2021

*Corresponding Author: Negar Motakef Kazemi

Email: motakef@iaups.ac.ir

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

1. Introduction

Multiple sclerosis (MS) is the most common

chronic inflammatory disease of the central

nervous system (CNS), affecting more than

2 million people worldwide and is currently

incurable. No drug can completely prevent

progressive neurodegeneration, which is usually

diagnosed with impaired movement function,

bladder control, and cognitive processes [1, 2].

Nitric oxide and its metabolites in the human

body may be involved in the pathogenesis of

several neurological disorders such as multiple

sclerosis. Several studies have shown that lithium

regulates NO levels in the central nervous system.

High levels of compounds derived from reactive

oxygen species (ROS) and reactive nitrogen

species (RNS) have been detected in blood

samples from RR-MS patients. In principle,

increased production of NO and its metabolites

in the peripheral blood of these patients has been

shown [3]. Lithium may control NO formation

in MS. There is a significant difference between

https://doi.org/10.24200/amecj.v4.i04.158

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

serum lithium levels in RR-MS patients and

healthy individuals. Extensive experimental and

clinical studies show that lithium has protective

effects on the pathogenesis of neurological

diseases through several mechanisms (activation

of nerve pathways, regulation of oxidative stress,

anti-inflammatory responses, regulation of

mitochondrial function, etc.). However, the use

of high doses of lithium may lead to toxic effects

[4]. Excessive accumulation of metal in the

CNS stimulates oxidative stress, mitochondrial

dysfunction, dysfunction of enzymes structural,

regulatory, and catalytic functions in a variety of

proteins, receptors, and carriers. Metals can cause

nerve damage in PD, AD, and MS by disrupting

mitochondrial function. The mechanism act

with lower adenosine triphosphate (ATP) and

produces ROS. Through these mechanisms,

metals cause cell death by apoptotic or necrotic

mechanisms [5]. Also, manganese is toxic to the

CNS in excessive amounts. High manganese

value can lead to a disease whose symptoms

are similar to those of Parkinson’s. Manganism

is a type of extrapyramidal movement disorder

of the Parkinson’s type that has impaired

motor and cognitive impairment due to neural

processes [6]. Determination of metal ions in

blood and body fluids can play an effective role

in diagnosing the disease and various treatments.

The characterization of nanomaterials in various

fields, especially in medical science, caused to

use for the determination of metals in human

body fluids. Recently, the nanoparticles such as

graphene oxide and activated carbon were used

to measure the concentration of lead, mercury,

lithium, silver and manganese ions in patients.

For this purpose, a micro-extraction procedure

based on solid-phase or liquid phase coupled to

ultrasound was used to measure ions by atomic

absorption spectroscopy [7]. Due to the toxicity

of Li and Mn in the human body, its concentration

must be determined in blood, serum and urine

samples. Many technologies have been used for

Li and Mn determination in different samples

including, the isotope dilution atomic absorption

spectrometry (ID-ET-AAS) for Li determination

in serum samples. ID-ET-AAS based on the

partially resolved isotope shift was done by

graphite furnace atomic absorption spectrometer

[8]. Also, flame emission(FES), flame atomic

absorption spectroscopy (F-AAS) and ion-

selective electrode (ISE) were used for lithium

determination in human patients [9, 10]. The

concentration of beryllium, copper, chromium,

cobalt, nickel, magnesium and iron in the blood

of MS patients was determined by ICP-MS [11].

Moreover, the inductively coupled plasma sector

field mass spectrometry (ICP-SFMS) and MC-

ICP-MS was used for Li determination in serum

samples [12, 13]. The Li in human samples was

determined by high-resolution atomic absorption

spectrometry (HR-AAS) and machine learning

data(MLD) [14]. In addition, Mn ions in herbal

teas were evaluated by FAAS and ICP-OES [15].

The cation exchange chromatography coupled to

field ICP-MS was used for Mn determination in

cerebrospinal fluid [16]. As in previous research,

the Li and Mn were determined in metals of

human blood samples by AAS after sample

preparation techniques. Recently, the different

treatments such as; the dispersive liquid-liquid

microextraction (DLLME) [17], the cloud point

extraction procedure (CPE) [18], and the solid-

phase extraction (SPE) [19, 20] were used for

metal analysis in various samples. Among them,

the SPE as low cost, simple and good recovery is

preferred to other methods.

In this research, a new adsorbent based on

APDCIL/GONPs was used for extraction of Li

and Mn in human biological samples by USA-

DIL-μ-SPE procedure. Li and Mn ions were

determined by F-AAS and ET-AAS, respectively

after sample treatment. The method was validated

by spiking real samples and CRM analysis in

human blood, serum and urine samples.

2. Experimental

2.1. Instruments

The different instruments include a magnetic

stirrer (Four E’S Scientific, model MI0102003)

with a variable speed made in china, a digital

scale with an accuracy of one-thousandth of a

gram made in Japan, a digital pH meter model

744 made in Switzerland, centrifuge with 3500-

4000 rpm made in Iran (IRANLABXPO),

hematocrit centrifuge with 12,000 rpm(AHN

myLab® hematocrit centrifuge, AHN

Biotechnologie GmbH, Germany), a magnetic

healer, oven (Iran Lab), Austrian microwave

device (Anton Paar, Vienna Austria), the sharp

polyethene pipes and Pyrex glass, the tube and

balloon mixer, and the automatic sampling for

different volumes between 0.01 mL to 1 mL were

used in this research. The Mn and Li concentration

was determined by flame atomic absorption

spectrometer (FAAS, GBC 906, double beam,

Aus.). The air-acetylene (C2H2), the deuterium

lampas was used for Li evaluation. The limit of

detection (LOD) and linear range of F-AAS for Li-

ions in standard solutions was obtained 0.32 mg

L-1 and 1-4 mg L-1, respectively. The light of HCL

for lithium was adjusted by maximum energy at

a wavelength of 670.8 nm, slit of 0.5 nm and 5.0

mA. The ultra-trace analyzer, the electrothermal

atomic absorption spectrophotometer (ET-AAS,

GBC, Aus.) was used for the determination of Mn

in human blood samples. The limit of detection

(LOD) and linear range of Mn ions with ET-

AAS was obtained 0.27 μg L-1 and 2.5-15 μg L-1,

respectively The Avanta software was used for

calculating absorption results by the F-AAS and

ET-AAS.

2.2. Reagents

The ultra-pure reagents were purchased from

the Merck or Sigma Companies (Germany). All

standard solutions and samples were diluted by

ultra-pure distilled water (DW, R≥18MΩ cm-1)

from Millipore (Bedford, USA). The standard

stock solution of lithium (LiCl, CASN: 7447-

41-8) and manganese (MnCl2, CAS N.:189302-

40-7) was purchased from Sigma Aldrich

(1000 mg L-1 in 1% nitric acid 500 mL). The

calibration standard solutions of lithium (0.1,

0.2, 0.3, 0.4 mg L-1) and manganese (0.1-1.5 μg

L-1) were prepared by diluting of stock solution

with DW (1000 mg L-1). Nitric acid (HNO3),

hydrochloric acid (HCl, CAS N.: 7647-01-0),

sodium hydroxide (NaOH, CAS N.; 1310-73-2),

potassium hydroxide (KOH, CAS N.: 1310-58-

3), and all other reagents were purchased from

Merck, Germany. The pH was adjusted by suitable

buffer solutions. The different buffer solutions

such as the sodium phosphate (H3PO4 / NaH2PO4,

0.15 mol L-1) for pH 1.5-3, the ammonium acetate

buffer (CH3COOH / CH3COONa) for pH 4-5.5,

the sodium borate (NaBO2/HCl) for pH 7 and

the ammonium chloride (NH3/NH4Cl) were used

for pH 8-10. Graphene oxide was prepared by

the Petroleum Industry Research Institute (RIPI)

and coated with a mixture of ionic liquid/APDC

(99%, ammonium pyrrolidine dithiocarbonate,

CAS N.: 5108-96-3, EC Number: 225-834-4)

and acetone. Hydrophobic ionic liquid (1-Hexyl-

3-methylimidazolium hexafluorophosphate,

[HMIM][PF6], CAS N.: 304680-35-1) was

prepared from Sigma, Germany.

2.3. Synthesis of APDC-IL coated on graphene

oxide

The graphite oxide was prepared by modified

Hummer technique by oxidation of natural

graphite powder in the laboratory of RIPI.

The GO was prepared by peeling off graphite

oxide. First, 5 g of graphite powder and 2.5 g

of NaNO3 were combined with 120 mL H2SO4

(98%) and shaken vigorously for 30 min in an

ice bath (0-5 °C). Simultaneously with stirring,

15 g of KMnO4 was gradually added, and the

temperature was tuned at below 15 °C. The ice

bath was then removed, and the mixture was

stirred at 35 °C until it gradually turned brown

form and then diluted gently with 250 mL of

water. The reaction temperature rose rapidly

to 98 °C with boiling and its colour changed

to dark brown. Then, 30% H2O2 solution was

added, and the colour of the mixture changed

to a bright yellow colour, indicating complete

oxidation of graphene. The graphene oxide

was washed by rinsing and centrifugation with

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

dilute hydrochloride solution and then continued

several times with deionized distilled water to

neutralize the filtered solution. The graphene

oxide suspension was centrifuged and sonicated

for 15 minutes at 3000 rpm to obtain graphene

oxide nanosheets. Finally, the prepared GONPs

was air-dried for two hours in two stages at 55

°C. Ammonium pyrrolidine dithiocarbamate

(APDC) was mixed with ionic liquid 1-hexyl-

3-methylimidazolium hexafluorophosphate

(hydrophobic) in the presence of acetone at 25

°C for 10 minutes. Then an organic mixture

(ionic liquid, acetone, ammonium pyrrolidine

dithiocarbamate) was used at 55 ° C for coating

of graphene oxide (IL /APDC-NGO) [21-23].

2.4. General Procedure

By procedure, a sample of lithium was prepared

by diluting 1 ml of blood, urine and serum sample

with DW up to 10 mL. Also, 10 mL of human

biological samples were used for manganese

separation at optimized pH. 25 mg of APDC/IL/

GONPs adsorbent was used to separate lithium

and manganese ions from the blood, serum

and urine of MS patients by the ultrasound

assisted-dispersive-ionic liquid-micro-solid

phase extraction technique (USA-DIL-μ-SPE).

The standard solution of lithium (0.1 - 0.4 mg

L-1) and manganese (0.1 – 1.5 μg L-1) as the

lower and upper limit of quantification (LLOQ,

ULOQ) were used. After adding adsorbent to

the human samples, it was placed in an ultrasonic

bath for 5 minutes and the lithium and manganese

ions were physically and chemically adsorbed by

a sulfur bond of APDC ligand and the surface of

the graphene oxide, respectively (Mn+2/Li+→NGO;

Mn+2/Li+→: S─IL-NGO). By the proposed

Fig. 1. Determination and separation lithium and manganese ions in human biological samples based

on APDC/IL/GONPs by USA-DIL-μ-SPE procedure

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

procedure, lithium (Li) and manganese (Mn) ions

are extracted by coordinate covalent bond at pH 5

and 6, respectively (more than 95%). The results

showed us, at high pH, lithium and manganese ions

were precipitated as hydroxide ions (Mn(OH)2 and

LiOH). After the separated phase by centrifuging

(3 min, 3500 rpm), the extracted lithium and

manganese ions were trapped in the bottom of the

conical tube by IL/GONPs. Then, the upper liquid

phase was set aside with an auto-sampler and the

loaded ions on the adsorbent (APDC/IL/GONPs)

were back-extracted by the nitric acid solution (500

μL, 0.3 M) by shaking and centrifuging for 1 minute.

Finally, the concentration of lithium and manganese

ions was determined by the F-AAS and ETAAS,

respectively after diluting eluent up to 1 mL with

0.5 ml of DW. Also, the extraction/separation of

lithium and manganese ions based on GONPs in

human biological samples and the standard solution

was investigated in various pH. For urine analysis,

10 mL of sample was used under similar conditions.

For validation of the method, the standard reference

materials (SRM) and spike samples were used for

standard and human samples. For calibration of

lithium and manganese ions, 10 mL of a standard

aqueous sample containing lithium (0.05, 0.1,

0.2, 0.3 and 0.4 mg L-1) and manganese (0.1, 0.2,

0.4. 0.5, 1.0, 1.5 μgL-1) were prepared from stock

solutions. In addition, the ICP-MS were used to

validate the real samples.

3. Results and Discussion

3.1. Characterization of APDC/IL/graphene

oxide

Fourier transform infrared (FT-IR) spectra were

recorded by a Perkin Elmer spectrophotometer.

The X-ray diffraction (XRD) was obtained based

on a Panalytical X’Pert PRO X-ray diffractometer.

The Bruker (D) FRA-106/S spectrometer was used

for Raman spectra. Scanning electron microscopy

(SEM) images were reported by a Tescan Mira-

3. The transmission electron microscopes images

(TEM, JEM 2100 plus) was achieved by JEOL

Company, Germany.

3.1.1.Electron microscope images

As Figure 2(a, b) and 3(a, b), the scanning electron

microscopy (SEM) and transmission electron

microscope (TEM) images show that the APDC/

IL/GONPs and GONPS has similar multi-layered

with a smooth surface and some with folds.

Wrinkled areas are at the APDC/IL/GONPs and

GONPS level due to the presence of oxygenated

functional groups (such as carboxyl, hydroxyl,

and carbonyl). The APDC/IL/GONPs and GONPS

both consist of randomly accumulated and

wrinkled thin sheets.

Fig. 2a.SEM image of APDC/IL/GONPs Fig. 2b.SEM image of GONPs

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

3.1.2.XRD and FT-IR analysis

In the XRD analysis of graphene oxide, a sharp

peak was observed at 2θ = 12.267 (d = 0.723)

with the usual GONPs nanoparticle diffraction

peak. The distance d increases from 0.33 to

0.72 nm after the conversion of graphite to

graphene oxide nanoparticles, which may be

due to the formation of abundant oxygenated

functional groups on the graphene oxide

surface. In addition, the peaks at 2θ = 12° and

2θ = 42.58o are related to the diffraction planes

of (002) and (100) respectively, which can be

observed in the XRD patterns of both GONPs

and APDC/IL/GONPs. It showed that the

XRD intensity of the peak at 2θ = 12° for the

APDC/IL-coated on GONPs was significantly

decreased (Fig.4a and b).

Oxygenated functional groups on the surface of

graphene oxide nanoparticles by FT-IR analysis

can be seen in the following diagram. Accordingly,

the groups C=O and COOH/OH are represented by

the peak of 1728 cm-1 and 3300 cm-1, respectively.

The C-O bonding is shown at peak 1011 cm-1 and

the peak C= C is located at 1590 cm-1 by FTIR. As

physically coated APDC/IL on GONPs any peak

wasn’t added to FTIR of GO (Fig.5).

Fig. 3a.TEM image of APDC/IL/GONPs Fig. 3b.TEM image of GONPs

Fig. 4a. XRD of the GONPS Fig. 4b. XRD of the APDC/IL/GONPS

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

3.2. Optimization of Method

To achieve optimal conditions in solid-phase

extraction for preconcentration/separation of

lithium and manganese in human biological

samples, the parameters such as pH, the amount of

sorbent, the sample volume, the amount of ionic

liquid, the concentration of APDC ligand, the

shaking and centrifuge time, were investigated.

3.2.1.The pH optimization

The pH effects on the extraction of Li and Mn ions

by APDC/IL/GONPs. So, the various pH between

2-11 was studied for Li and Mn extraction in human

biological matrixes. The results showed, the sulfur

group on the surface of GONPs adsorbent was

caused to extraction Li and Mn ions at pH of 5-7.

Due to the mechanism, the extraction was reduced

at pH < 5 and pH > 7. Therefore, we used pH=6 for

Li and Mn extraction in human biological samples

(blood, serum, urine). The mechanism of absorption

of Li and Mn ions was obtained based on the sulfur

group (SH) on the surface of APDC/IL/GONPs. By

a sulfur group of APDC, the coordinate covalent

bond with Li and Mn ions in human liquid samples

was achieved (Mn+2/Li + →: S─IL-NGO). At pH<

pHPZC, the adsorbent had a positive charge and

due to similarity charges between Li and Mn and

adsorbent, the extraction was decreased. Also, in

pH 5 for Li and pH 6 for Mn, the surface of APDC/

IL/GONPS based on negatively charged (HS─)

coordinated with a positive charge of Li and Mn. At

pH more than 7, Li and Mn ions have participated

as hydroxyl forms (Li OH, Mn(OH)2). Therefore,

we used pH=6 for further studies (Fig. 6).

3.2.2.Optimization of adsorbent amount

The efcient extraction of Li and Mn ions in

human samples were studied by different amount

of APDC/IL/GONPs. For this purpose, the various

mass of APDC/IL/GONPs was evaluated at pH=6.

For Li and Mn extraction, 2-40 mg of APDC/IL/

GONPs in blood, serum, urine and standard solution

were studied and optimized by the USA-DIL-μ-

SPE procedure. Based on the results, the efcient

extractions for Li and Mn ions were obtained by 20

mg of adsorbent in human biological samples. So,

25 mg of APDC/IL/GONPs was used for further

study (Fig. 7).

Fig.5. FT-IR analysis of the APDC/IL/GONPs

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

3.2.3.Optimization of sample volume and eluent

The effect of eluent on extraction Li and Mn

ions based on APDC/IL/GONPs were studied

at optimized pH. At low pH, the covalent bond

between the metal and sulfur group was dissociated

and the Li and Mn ions released into liquid

phase. Therefore, the inorganic acid solutions

with different concentrations and volumes (HCl,

HNO3, H3PO4, H2SO4) were used to evaluate the

recovery of the back-extraction process for Li and

Mn ions in human biological samples. The eluent

concentrations between 0.2-2.0 mol L-1 was studied.

The efcient extraction was obtained by HNO3 (0.5

M, 0.25 mL) (Fig. 8). Also, the volume of human

samples and the standard solution was evaluated

from 2.0 mL to 20 mL for Li and Mn concentration

(0.1-0.4 mg L-1, 0.1-1.5 µg L-1). As result, the high

recovery occurred for 10 mL of human biological

samples at pH 5-7 (Fig.9).

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

Fig.6. The effect of pH on Li/Mn extraction Fig.7. The effect of adsorbent mass on Li /Mn extraction

2 3 4 5 6 7 8 9 10 2 5 10 15 20 25 30 40 50 60

Fig.8. The effect of eluents

on Li/Mn back-extraction

Fig.9. The effect of sample volume

on Li /Mn extraction

2 5 8 10 12 15 20

0.2 0.3 0.5 1 2

3.2.4.Optimization of APDC ligand on adsorbent

The concentration of APDC is one of the

important parameters which should be optimized

by the USA-DIL-μ-SPE procedure method.

For optimizing, 0.1 × 10−6-1.0 × 10−6 mol L−6 of

APDC ligand was used in the human biological

sample. The results showed that, by increasing

ligand concentration up to 0.5 × 10−6 mol L−1, the

recoveries are also increased. Figure 10 shows that

0.35 × 10−6 mol L−1 of APDC was necessary to

obtain the maximum extraction efciency. So, the

amount of chelating agent (APDC) between 0.1-1

μmol L-1 was investigated and 0.35 μmol L-1 was

found the best amount for Li and Mn extraction.

3.2.5.Optimization of time, reusability and

absorption capacity

The dispersion of APDC/IL/GONPs in human

blood, serum, urine or standard solution had a

critical role for extraction Li(I) and Mn(II). The

time extraction depended on chemical adsorption

between the sulfur group with metals at pH 5-7. By

the USA-DIL-μ-SPE procedure, the shaking time

was examined between 1- 10 min. It has occurred

that the shaking of 5.0 min was the favourite time for

ions extraction in samples. In this study, the time of

shaking and centrifuging process were investigated

and 5 min was found suitable for the shaking and

3 min for centrifuging process (3500 rpm). After

sonication for 5 min, the APDC/IL/GONPs was

trapped at the end of the conical tube and then the

upper liquid phase was put out by auto-sampler. The

reusability of APDC/IL/GONPs was obtained with

many cycles extraction/back-extraction process.

The results showed the adsorbent can be used for

12 cycles. The absorption capacities for cadmium

depended on the characterization of APDC/IL/

GONPs and surface area. The absorption capacities

of APDC/IL/GONPs for Li and Mn was achieved

at 148.5 mg g-1, 122.3 mg g-1, respectively.

3.2.6.Interference cations and anions

The effect of interference of ions on Li and Mn

extraction in human biological samples were

studied by the USA-DIL-μ-SPE procedure. The

concentrations of ions were added to the standard

solution and human samples with lithium (0.1

- 0.4 mg L-1) and manganese (0.1 – 1.5 μgL-1) at

optimized conditions. The results showed the

interference of ions couldn’t decrease the efcient

extraction of Li and Mn ions at pH=6. (Table 1).

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

Fig.10. The effect of APDC concentration on Li /Mn extraction by the USA-DIL-μ-SPE procedure

0.1 0.2 0.3 0.35 0.4 0.5 0.6 0.8 0.9 1

3.2.7.Method validation

The concentration of Li and Mn in human blood

and standard samples was determined by the USA-

DIL-μ-SPE procedure. The results were obtained

based on the average of three analyses in blood

samples. The rate of recovery showed that the

proposed method has good accuracy and precision

in the blood matrix. The recovery of spiked blood

samples and the standard solution was more than

95%. This method was satisfactory for the analysis

of analytes in the human blood samples. Lithium

and manganese concentrations were studied in

MS patients (40, subject) and healthy people (40,

control) by the USA-DIL-μ-SPE procedure. Mean

lithium and manganese concentrations in control

groups were signicantly lower than MS patients.

In addition, for the validation of the method,

standard reference materials (SRM) for Li and

Mn were examined by the adsorbent. The results

of blood samples in standard reference samples

(SRM) was satisfactorily conrmed the accurate

concentration of Li and Mn ions. The following

table shows the validation of the USA-DIL-μ-SPE

procedure based on APDC/IL/GONPs by spiking

Table 1. Interference cations and anions for Li and Mn extraction based

on APDC/IL/GONPs by the USA-DIL-μ-SPE procedure

Interfering Ions(A)

Mean ratio

(CA /C Li(I))

Mean ratio

(CA /C Mn(II))Recovery (%) Recovery (%)

Li Mn Li Mn

Ni2+, Co2+, Se2- 700 850 98.3 97.9

Cr3+, Al3+, Ag+ 600 700 97.1 97.3

I-, Br-, F-, Cl-1000 1100 97.8 98.4

Na+, K+, Ca2+, Mg2+ 1200 1300 98.8 99.1

CO3

2-, PO4

3-,NO3

-800 900 96.6 97.0

Zn2+, Cu2+ 500 750 97.5 98.5

Pb2+, Mo2+, Fe2+ 850 650 98.2 97.3

Hg2+ 100 80 96.2 98.5

Table 2. Validation of Mn determination in serum, blood and urine of MS patients by spiking

samples with the standard solution by the USA-DIL-μ-SPE procedure coupled to ET-AAS

Sample Added (μg L-1) Mn aFound (μg L-1) Mn (%) Recovery

Blood

--- 0.052 ± 1.244 ---

0.5 0.117 ± 1.729 97.0

1.0 0.135 ± 2.251 100.7

Serum

--- 0.063 ± 1.036 ---

0.5 0.072 ± 1.531 99.0

1.0 0.203 ± 1.984 94.8

Urine

--- 0.033 ± 0.784 ---

0.5 0.065 ± 1.293 101.8

1.0 0.084 ± 1.781 99.7

aMean of three determinations of samples ± condence interval (P = 0.95, n =8)

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

real samples and standard reference materials.

ICP-MS was used to prepare standard reference

samples (SRM) for validation of methodology. In

this study, the method was validated by spiking

real samples with a standard Li and Mn solution

in blood, serum and urine samples (Table 2

and 3). Also, the Li and Mn were validated by

ICP-MS. Validation of Mn and Li extraction in

human samples was obtained based on APDC/Il/

GONPs and standard reference materials (ICP-

MS analysis) by the USA-DIL-μ-SPE procedure

(Table 4 and 5). The results demonstrated the high

extraction and recovery for Li and Mn in human

blood matrixes. As intra and inter-day analysis, the

40 MS patients were compared to healthy people

by the USA-DIL-μ-SPE procedure (40 Men, 25-55

age, Iran) (Table 6). As Table 6, the concentration

of lithium and manganese ions in the human body

uids for MS patients is signicantly higher than

the concentration of healthy people.

3.2.8.Discussion

The analysis of biological uids is one of the most

appropriate forms of evaluating environmental

Table 3. Validation of Li determination in serum, blood and urine of MS patients

by spiking samples with the standard solution by the USA-DIL-μ-SPE procedure coupled to F-AAS

Sample Added (mg L-1) Li aFound (mg L-1) Li (%) Recovery

Blood

--- 0.07 ± 1.56 ---

1.0 0.12 ± 2.61 105.0

1.5 0.14 3.02± 97.3

Serum

--- 0.12 ± 2.16 ---

1.0 0.15 ± 3.11 95.0

1.5 0.18 ± 3.63 98.0

Urine

--- 0.033 ± 1.88 ---

1.0 0.065 ± 2.92 104.0

1.5 0.084 ± 3.33 96.6

aMean of three determinations of samples ± condence interval (P = 0.95, n =8)

Table 4. Validation methodology for Mn determination in human samples based on APDC/Il/

GONPs and standard reference materials (ICP-MS analysis) by the USA-DIL-μ-SPE procedure

(%) Recovery

Found a

µg L-1

Certied b

µg L-1

Added

µg L-1

Samples b

98.40.06 ± 1.210.02 ± 1.23------Serum

97.00.12 ± 2.18------1.0

102.50.04 ± 0.810.01 ± 0.79-----Blood

96.00.05 ± 1.29------0.5

97.10.06 ± 0.980.02 ± 1.01------Urine

101.00.09 ± 1.99------1.0

aMean of three determinations of samples ± condence interval (P = 0.95, n =8)

bICP-MS analyzer for blood, serum and urine as CRM (µgL-1)

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

exposure to pollutants such as toxic metals.

Metals are important constituents widely used in

different industrial processes and can be present in

biological uids, namely urine, as a consequence

of occupational exposure. Although atomic

absorption spectrometric techniques, (ame or

graphite furnace mode; FAAS and GFAAS) are

a powerful analytical tool for the determination

of trace metals. Determination of metals in urine,

serum and blood samples is very difcult due to

various factors, particularly low metal content and

high salt content of the sample matrix. The use

of a separation and preconcentration technique in

the analytical process can solve these problems

and lead to easy determination of trace metals in

urine, serum and blood samples. There are many

methods for preconcentration/separation of trace

heavy metals from human biological samples

[24]. Komatsu et al reported determination of Li

in whole blood by using colorimetric determination

of lithium ions with a low limit of detection about

0.054 mM, and the coefcient of variance below

6.1%. A portion of whole blood has been placed on

the end of the separation unit, plasma in the sample

is automatically transported to the detection unit,

which displays a diagnostic color [25]. This method

has higher LOD and RSD% as compared to the USA-

DIL-μ-SPE procedure. Also, a wide linear range

Table 5. Validation methodology for Li determination in human samples based on APDC/Il/

GONPs and standard reference materials (ICP-MS analysis) by the USA-DIL-μ-SPE procedure

(%) Recovery

Founda

mg L-1

Certied

mg L-1

Added

mg L-1

Samples b

97.20.15 ± 3.140.05 ± 3.23------ Serum

97.50.24 ± 5.09------2.0

99.30.13 ± 2.860.02 ± 2.88-----Blood

100.80.24 ± 5.38------2.5

102.10.11 ± 2.020.01 ± 1.98------Urine

98.50.09 ± 3.99------2.0

aMean of three determinations of samples ± condence interval (P = 0.95, n =8)

bICP analyzer for blood, serum and urine as CRM (mgL-1)

Table 6. The comparing of Li and Mn concentration in MS patients with healthy people by the

USA-DIL-μ-SPE procedure

**Sample

*Patients MS * Control Patients MS

(mgL-1)

(µgL-1)

Li (mgL-1) Mn

(µgL-1)

r Li P value r Mn P value

Blood 6.19 ±

0.29

14.24 ±

0.65**

3.42 ± 0.15 3.12 ±

2.11**

0.202 <0.001 0.115 <0.001

Urine 4.35 ±

0.22

6.83 ±

0.31**

1.75 ± 0.07 2.42 ±

0.90

0.196 <0.001 0.076 <0.001

Serum 7.47 ±

0.33

1.34 ±

0.06

2.58 ± 0.12 0.63 ±

3.92

0.221 <0.001 0.109 <0.001

*Mean of three determinations of samples ± condence interval (P = 0.95, n =40)

** Sample dilution (1:10) over a linear range of Mn

Nano graphene for determination Li and Mn in MS patients Seyed Majid Nabipour Haghighi et al

between 0.1-0.4 mg L-1 was used based on APDC/

IL/GONPs. Suherman et al reported electrochemical

detection and quantication of lithium ions in

authentic human saliva using LiMn2O4-modied

electrodes. The sensing strategy is based on an

initial galvanostatic delithiation of LMO followed

by linear stripping voltammetry (LSV) to detect

the re-insertion Li+ in the analyte. The process was

investigated using powder X-ray diffraction (PXRD)

and voltammetry. LSV measurements reveal a

measurable lower limit of 50.0 µM in both LiClO4

aqueous solutions and synthetic saliva samples [26].

Filippini et al showed that the determinants of serum

manganese levels in an Italian population. They

employed an inductively coupled plasmasector eld

mass spectrometry (ICP-SFMS) instrument for Mn

determination in medium resolution mode. The LOD

for Mn-peaks in SEC-ICP-DRC-MS was calculated

as 3 σ-criterion and found between 28-35 ng L-1 [27].

This method is expensive and sample preparation

needs before analysis. On the other hand, as normal

level Mn in serum or blood (µgL-1), the USA-DIL-

μ-SPE procedure has a sufcient rate. Zabłocka et al

reported an applied method for evaluation Zn, Cu and

Mn in serum /whole blood samples and their relation

to redox status in lung cancer patients. In their study

for whole blood preparation, 0.5 ml was performed

twice by microwave −technique wet mineralization

in a closed system using MLS 1200 Mega, with

mixture 1:5 of H2O2(30%) and HNO3(69–70%)

and graphite furnace atomization was used for the

determination of Mn. They reported analytical

values of Zn, Mn and Cu were: 8.97 mg L-1, 47.3

μg L-1 and 2.47 mg L-1, respectively. Mean accuracy

(n = 6) was as follows: 98,3% (Zn), 105.9% (Mn)

and 91.6% (Cu) which was comparable to the USA-

DIL-μ-SPE procedure for Mn determination [28].

In this study, we used the USA-D-μ-SPE procedure

based on APDC/IL/GONPs for micro-extraction

and determination of lithium and manganese

concentration in human biological samples. The

surface charge of GONPs is negative so, the

electrostatic attraction between negatively charged

of adsorbent and positive charge of Li+ and Mn2+

ions have occurred at optimized pH=6. In addition,

quantitative extraction of more than 95% was

observed in the optimized sample volume. It was

also noticed that higher sample volumes, partially

solubilized the ionic liquid phase, leading to non-

reproducible results and increased the amount of

GONPs. It was also observed that the extraction

efciency was remarkably affected by GONPs and

IL amount, so they were examined within the range

of 2 to 60 mg and 10 to 100 mg for GONPs and IL,

respectively. Quantitative extraction was observed at

25 mg of GONPs for Li and Mn which was lower or

similar amount as compared to other methods. The

USA-DIL-μ-SPE method was applied to determine

Li+ and Mn2+ for 1 mL and 10 ml in human biological

samples, respectively. The spiked serum and blood

were prepared to demonstrate the reliability of the

method for extraction and determination of Mn and

Li. At optimized condition, the LOD of the method

was found 0.03 mg L-1 for Li and 0.026 μgL-1 for

Mn2+ and working ranges was found 0.3-1.0 mg L-1

for Li and 0.08-5 μgL-1 for Mn. The mean of Mn2+

and Li+ concentration in blood, urine and serum of

MS patients and healthy people were determined

by the USA-DIL-μ-SPE procedure which was near

to recently reported. The results showed that the

concentration of Li+ and Mn2+ ions in the blood,

urine and serum of MS patients were higher than

healthy people, (6.19±0.29 μgL-1 vs 3.42±0.15 μgL-

1, P<0.05 for Li in the blood and 6.83±0.31 mgL-1

vs 2.42±0.90 mgL-1, P<0.05 for Mn in urine). The

adsorption capacity of APDC/IL/GONPs for Li+ and

Mn2+ ions were found at 148.5 mg g-1, 122.3 mg g-1,

respectively. This study showed that the application

of APDC/IL/GONPs as the SPE procedure is a fast

and low-cost separation route without nonabsorbent

loss. Therefore, APDC/IL/GONPs is considered

to be excellent and potential adsorbent for the

extraction of Li+ and Mn2+ ions in human biological

uids.

4. Conclusions

A novel APDC/IL/GONPs was used for separation/

extraction and determination of Li and Mn ions

in human blood, serum and urine samples by the

USA-DIL-μ-SPE procedure. The IL ([HMIM]

Anal. Methods Environ. Chem. J. 4 (4) (2021) 20-35

[PF6]) property was helped to collect the GONPs

phase from the liquid phase in the bottom of the

conical tube. By the proposed procedure, a fast and

simple, efcient extraction and perfect separation

were achieved at pH 5-7 (RSD ≤ 5%). The APDC

ligand coated on nanoparticles of GONPs enhanced

the Li and Mn extraction in blood samples. By the

USA-DIL-μ-SPE procedure many advantages such

as good reusability, perfect separation, low cost, and

high recovery (more than 95%) were achieved

as compared to ICP-MS and other techniques.

Therefore, the separation and determination of

Li and Mn ions in human samples were achieved

in optimized conditions. The results showed the

detection limits (LOD), pre-concentration factor

and the linear range of the method for Li and Mn

were obtained (0.03 mg L-1 and 0.025 μg L-1), (9.72,

10.2) and (0.1-0.4 mg L-1 and 0.08-1.5 mg L-1),

respectively. The adsorption capacity of graphene

oxide for lithium and manganese was achieved

at 148.5 mg g-1 and 122.3 mg g-1, respectively.

Validation of the method was performed by spiking

samples and standard reference materials (SRM) in

the human blood, serum and urine.

5. Acknowledgement

The Ethical Committee of Azad University

(E.C.: R.IAU.PS.REC.1398.272) was obtained

for blood sample project by the world medical

association declaration of Helsinki (WMADH)

based on guiding physicians in human body

research.

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