Anal. Methods Environ. Chem. J. 5 (2) (2022) 51-59
Research Article, Issue 2
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Application of pipette-tip solid-phase extraction technique
for fast determination of levooxacin from wastewater
sample using cobalt metal-organic framework
Mohammad Abbaszadehbezi a, Mohammad Reza Rezaei Kahkha a,*, Alireza Khammara
and Morteza Mehdipour Rabouri b
a Faculty of Health, Zabol University of Medical Sciences, Zabol, Iran
b Occupational Health Engineering Department, Kerman University of Medical Sciences, Kerman, Iran
ABSTRACT
In this research, a miniaturized solid-phase extraction method based
on pipette tip solid-phase extraction (PT-SPE) was employed for
the determination of levooxacin. Cobalt metal-organic framework
(CoMOF) was used as an adsorbent. Levooxacin was determined
using high-performance liquid chromatography and UV detection
(HPLC-UV). Important parameters that inuence the extraction
efciency (i.e. pH, amount of adsorbent, extraction time, volumes
of sample, and eluting solvent) were tested and optimized. Results
indicated that the proposed method was validated over the range of
0.70 - 150.0 µg L-1. The relative standard deviation (RSD%) was
below 2.75% for the levooxacin. The limit of detection (LOD)
of this method is 0.041 µg L-1. The preconcentration factor (PF)
was obtained at 200 and the analysis time was around 10 min that
conrming the reliability and accuracy of this method for extraction
of levooxacin. The PT-SPE procedure based on CoMOF adsorbent
was efciently extracted for levooxacin more than 95%. In a static
system, the adsorption capacity of CoMOF adsorbent for levooxacin
was obtained at 156.7 mg g-1 (n=10). The validation of results was
successfully obtained for levooxacin values based on the spiking
real samples before determination by the HPLC technique.
Keywords:
Cobalt metal-organic framework,
Levooxacin,
Pipette tip,
Preconcentration,
Solid-phase extraction,
High-performance liquid
chromatography
ARTICLE INFO:
Received 19 Feb 2022
Revised form 26 Apr 2022
Accepted 20 May 2022
Available online 30 Jun 2022
*Corresponding Author: Mohammad Reza Rezaei Kahkha
Email: m.r.rezaei.k@gmail.com
https://doi.org/10.24200/amecj.v5.i02.185
------------------------
1. Introduction
Antibiotics are among the drugs that are widely
used in medicine and veterinary medicine and
through various routes such as agricultural
runoff, direct discharge from municipal
wastewater treatment plants, human waste,
direct disposal of medical waste, veterinary, and
industry[1, 2]. As a result, their presence has been
observed in local streams and around the world,
especially in streams that directly receive treated
wastewater[3]. Large amounts of these compound
residues remain in agricultural soils after being
applied, which leach into the groundwater and
can also be found in run-off waters[4]. Due to
the mentioned toxicological effects, sensitive
and reliable analytical methods are needed for
the determination of trace amounts of these
compounds[5]. Levooxacin is a broad-spectrum
antibiotic that prevents the growth of bacteria, so
it is classied as a bacteriostatic[5, 6]. It is widely
used in the treatment of patients with Covid-19 in
recent two years[7]. Several methods have been
applied for determining levooxacin residues
52
in aqueous samples, including, solid-phase
microextraction (SPME)[8, 9], dispersive-solid
phase extraction[10, 11], molecularly imprinted
polymer (MIP)[12, 13]and other techniques based
on microextraction principles[14, 15]. The pipette
tip (PT) is a micro-scale of SPE that is used for
the separation and extraction of environmental
pollutant samples[16]. This method used small
volume and low consumption of any solvents that
satised by green chemistry rules. It differs from
common SPE in that a small amount of sorbent
is inserted into a pipet tip, and it is relatively
inexpensive without a special auxiliary device
for extraction. In general terms, an advantage of
pipet tips for sample preparation is that extraction
can be carried out faster and more facile than
conventional SPE cartridges. Many SPE methods
based on adsorbents (silver nanoparticles
(AgNPs) coating on micro glassy balls (MGB),
graphene oxide-packed micro-column, magnetic
nanoparticles, cadmium Sulde Nanoparticles,
amine-functionalized bimodal mesoporous silica
nanoparticles) were used for extraction heavy
metals, organic materials and drugs in different
matrixes such as, water, human, vegetable,
food and drug samples [17-22]. In recent years,
many types of research have been focused on
the synthesis and application of metal-organic
framework (MOF) compounds. The MOFs are
one of major approaches for making sorbents
with large surface area. MOFs are a new class
of porous compounds consisting of organic
linkers coordinated to inorganic metal nodes[23].
Thermal and chemical stability, porosity, their
tailorable structures and properties are some
advantages of MOFs for several purposes such
as separation, sensing drug delivery and the
removal of toxic materials from air and water[24,
25]. In this work we used cobalt metal-organic
framework(Co-MOF) for the preparation of a
novel solid-phase adsorbent. Then pipette–tip
solid-phase extraction methods were used and
developed for preconcentration and extraction of
levooxacin.
2. Material and methods
2.1. Reagents
All chemicals were of analytical grade.
Cobalt nitrate (Co(NO3)2.6H2O) and pyridine
2,6-dicarboxyilic acid (98%) were purchased
from Aldrich (Millipore-Sigma, USA). Ethanol
and N,N-dimethyl formamide (DMF) were
obtained from Merck KGaA (Germany). Other
reagents such as the hydrochloric acid ( HCl, CAS
N: 7647-01-0,), the sulphuric acid (H2SO4, CAS
N: 7664-93-9) with high purity were purchased
from Sigma, Germany. The pH of the samples
is tuned by the sodium acetate (CH3COONa/
CH3COOH), the sodium phosphate (HPO4
2-/
NaH2PO4 -) and the ammonium buffer for pH of
4-5, 5-8 and pH 8-10, respectively. All the plastic
and glass tubes were put on the HNO3 solution
(0.5 M, v/v) for at least 2 days and then washed
with DW many times.
2.2. Instrumental analysis
The chromatographic analysis was carried out on
Cecil HPLC system (Cecil, England), equipped
with a UV detector. A reverse-phase ACE-C18
column (250 mm × 4.6 mm i.d.) was used for
separation of the analyte. The mobile phase was a
mixture of trimethylamine phosphate buffer (1%,
Ph=4.30) and acetonitrile (12/88, v/v) at a ow
rate of 1.0 mL min-1. The column temperature was
kept at 30oC and the detection wavelength was set
at 280 nm. The injection volume was 10 µL.
2.3. Synthesize of Co-MOF
Synthesize of adsorbent was described in one of
our previously reported works[26]. First, 1.85
mmol of pyridine 2,6-dicarboxyilic acid and 5.62
mmol of cobalt nitrate were dissolved in 14 mL
of ethanol. After this, the solution was transferred
into a Teon reactor with a tight cap and kept
for 7 hours at 85oC. The products were washed
with dimethyl formamide (DMF). After mixing
and dissolving the reactants, the clear solution
radiated in the ultrasound bath for 13 min at
working conditions of 160 W, 1 kJ and 21 kHz.
Synthesized Co-MOF was stored at 4 oC.
Anal. Methods Environ. Chem. J. 5 (2) (2022) 51-59
53
2.4. Pipette- tip solid phase extraction
procedure
5 mg of Co-MOF was put into the pipette-tip.
The extraction of levooxacin was performed by
attaching this pipette-tip extractor to a variable
pipette. Then 100 µl of the aqueous standard
solution containing 1 mg mL-1 of levooxacin
in a 5 mL glass test tube was withdrawn into the
sorbent and dispensed back into the same tube.
Before the optimization of the number of draw/
eject cycles, the extraction was optimized with
10 cycles. The adsorbed levooxacin on the
surface of the Co-MOF was eluted with 1000 µl
of a mixture of methanol-acetonitrile (90:10, v/v)
into a 2-mL glass vial, also with 10 aspirating/
dispensing cycles (Fig.1). So, the levooxacin
was determined based on Co-MOF adsorbent by
pipette tip solid-phase extraction procedure (PT-
SPE).
3. Results and discussion
3.1. Optimization of extraction conditions
For achieving the highest extraction efciency,
several parameters that inuenced the extraction
procedure were investigated and optimized as
follows.
3.1.1.Type of eluting solvent
Several solvents including, methanol, acetonitrile,
deionized water, mixture of water- acetonitrile and
mixture of methanol- acetonitrile were investigated for
obtaining the best extraction efciency. With different
polarities were evaluated to desorb levooxacin from
the Co-MOF sorbent. Experiments showed that the
levooxacin was completely desorbed by a mixture
of methanol and acetonitrile (90:10, v/v)
3.1.2.Effect of pH
The effect of sample pH on the recovery of
levooxacin was investigated between 2.0 and
10.0, using a one molar NaOH or HCl. As shown in
Figure 2, a pH value of 7.0 has highest extraction
efciency. However; in stronger acidic and basic
media, extraction efciency was decreased.
3.1.3.Effect of amount of sorbent
To obtain high extraction efciency with good
recoveries of levooxacin, the amount of sorbent
for pipette-tip extraction was changed between
1-5 mg. The adsorption ability of Co-MOF was
increased by increasing amount of nanocomposite
up to 2 mg. After that, the extraction recovery
became constant and hence, amount of adsorbent
was optimized at 2 mg (Fig. 3).
Solid-Phase Extraction for determination of levooxacin Mohammad Abbaszadehbezi et al
Fig. 1. The levooxacin determination based on Co-MOF adsorbent by pipette tip
solid-phase extraction procedure (PT-SPE)
54
3.1.4.Effect of volume of eluting solvent
We tried to obtain the smallest volume of eluent
solvent which provides the highest enrichment
factor. Volumes between 5 to 50 µL of a mixture
of methanol- acetonitrile were examined. As
shown in Figure 4, between 15 and 30 µL of the
eluting solvent, the recovery of levooxacin is at
its highest value, which means a larger volume of
eluting solvent provides a better elution. Therefore,
the eluting volume of 20 µL was selected for further
experiments.
Anal. Methods Environ. Chem. J. 5 (2) (2022) 51-59
Fig.3. Effect of amount of adsorbent on recovery of levooxacin based on Co-MOF adsorbent
by pipette tip solid-phase extraction procedure (PT-SPE)
Fig. 2. Effect of pH on recovery of levooxacin
by pipette-tip solid-phase extraction technique
55
3.1.5. Effect of volume of sample solution
The volume of sample solution is an important
factor in pipette- tip solid phase extraction.
Different volumes of sample solution were
evaluated in the range of 300 to 5000 µL.
Figure 5 shows that with increasing the sample
solution, extraction recovery of levooxacin is
also increased up to 3800 µL. Hence, this point
of sample solution was selected as optimized
volume. So, considering 20 μL of eluent, and
extraction efciency of 100 %, a preconcentration
factor of 200 was achieved
Solid-Phase Extraction for determination of levooxacin Mohammad Abbaszadehbezi et al
Fig. 4. Effect of eluent volume on recovery of levooxacin based on Co-MOF adsorbent
by pipette tip solid-phase extraction procedure (PT-SPE)
Fig. 5. Effect of sample volume on recovery of levooxacin based on Co-MOF adsorbent
by pipette tip solid-phase extraction procedure (PT-SPE)
56
3.1.6.Effect of number of aspirating/dispensing
of sample
The number of aspirating/dispensing cycles is
another important in the pipette-tip extraction.
The time of extraction depends on the number
of cycles and the volume of solution that passed
through the extractor. The results show that the
highest extraction efciency for levooxacin was
10 cycles, while using a 4000 µL of the sample.
At a higher number of cycles, the back extraction
of analytes from adsorbent into the sample solution
might occur, causing a decrease in the recovery. The
optimal number of aspirating/dispensing cycles
used for desorption of levooxacin was found to be
11 cycles at 9.5 min.
3.2. Analytical performance
The analytical performance of the suggested
pipette-tip extraction coupled with HPLC–UV
was investigated, and the results are summarized
in Table 1. Limit of detection (LOD) was obtained
based on a signal-to-noise ratio of 3. The linearity
range was studied by varying the concentration of
the standard solution from 0.1 to 200 µg L-1. The
repeatability of the method, expressed as relative
standard deviation (RSD), was calculated for
seven replicates of the standard at an intermediate
concentration (100 μg L-1) of the calibration curve.
3.3. Application of proposed method in real
samples
The suggested pipette-tip procedure was applied for
three hospital wastewater real samples. All samples
were ltered through 0.45 µm nylon membranes
before analysis and were sonicated for 10 min and
the slurry was centrifuged at 3500rpm for 5 min.
The extraction procedure was repeated three times
on each wastewater sample. For evaluation of the
analytical performance of the proposed method
in reaa samples, the samples were spiked with 3
different concentrations to investigate the matrix
effect on its determination. The results are shown in
Table 2. As can be seen, recoveries of are adequate;
therefore, we can justly this assumption that matrix
effect is negligible for the analysis of the target
analyte.
4. Conclusion
In this study, pipette-tip solid phase extraction with
a novel sorbent based on Co-MOF followed by
high performance liquid chromatography (HPLC)
has been developed for the determination of
levooxacin in wastewater samples. Due to very
high surface areas and short diffusion rate, high
adsorption capacities can obtain in a very short
time at pH=7. The optimized method is found to
be fast, economical, sensitive, accurate and simple.
The LOD, linear range (LR) and mean absorption
capacity of Co-MOF was obtained 0.041 µgL-1,
0.1 to 200 µgL-1 and 156.7 mg g-1, respectively
for 10 analyses. The range adsorption capacity of
Co-MOF adsorbent for levooxacin was obtained
at 114.9-176.8 mg g-1. Therefore, the efcient
extraction and separation of levooxacin in water
and wastewater samples were obtained by the
pipette tip solid-phase extraction procedure (PT-
SPE) before being determined with HPLC.
5. Acknowledgements
Authors hereby thanks from health laboratory
of Zabol University for cooperation to perform
experiments.
Anal. Methods Environ. Chem. J. 5 (2) (2022) 51-59
Table 1. Analytical performance of proposed PT-SPE method
Parameter Analytical feature
Dynamic range (μg L-1) 0.70-150
Repeatability 0.99
Repeatabilityb (RSD %) 2.75
Limit of detection (μg L-1) 0.041
Enrichment factor (fold) 200
Total extraction time (min) ≤ 10
57
6. Conict of Interest
The authors have declared no conict of interest.
7. References
[1] M.R.R. Kahkha, G. Ebrahimzadeh, A.
Salarifar, Removal of Metronidazole residues
from aqueous solutions based on magnetic
multiwalled carbon nanotubes by response
surface methodology and isotherm study,
Anal. Methods Environ. Chem. J., 2020.
3 (2020) 44-53. https://doi.org/10.24200/
amecj.v3.i03.110
[2] M.R.R. Kahkha, Magnetic bentonite
nanocomposite for removal of amoxicillin
from wastewater samples using response
surface methodology before determination
by high performance liquid chromatography,
Anal. Methods Environ. Chem. J., 3 (2020)
25-31. https://doi.org/10.24200/amecj.
v3.i03.108
[3] E. Peterson, P. Kaur, Antibiotic resistance
mechanisms in bacteria: relationships
between resistance determinants of antibiotic
producers, environmental bacteria, and
clinical pathogens, Front. Microbiol., 9
(2018) 2928. https://doi.org/10.3389/
fmicb.2018.02928
[4] F. Huang, Recognition of typical antibiotic
residues in environmental media related
to groundwater in China (2009− 2019), J.
Hazard. Mater., 399 (2020) 122813. https://
doi.org/10.1016/j.jhazmat.2020.122813
[5] S.M. Zainab, Antibiotics and antibiotic
resistant genes (ARGs) in groundwater: A
global review on dissemination, sources,
interactions, environmental and human health
risks, Water Res., 187 (2020)116455. https://
doi.org/10.1016/j.watres.2020.116455
[6] P. Dhiman, G. Rana A. Kumar , G. Sharma,
Nanostructured magnetic inverse spinel Ni–
Zn ferrite as environmental friendly visible
light driven photo-degradation of levooxacin,
Chem. Eng. Res. Des., 175 (2021) 85-101.
https://doi.org/10.1016/j.cherd.2021.08.028
Table 2. Validation of proposed method in real hospital wastewater based
on Co-MOF adsorbent by pipette tip solid-phase extraction procedure (PT-SPE)
Wastewater Sample # Added
(µg.L-1)
Found
(µg.L-1)
Recovery
(%) RSD % (n=7)
Sample 1
0 25.0 - -
50 89.1 98.2 3.5
100 124.3 74.3 4.2
Sample 2
0 41.0 - -
50 89.2 98.7 1.3
100 140.1 98.1 2.5
Sample 3
0 55.0 - -
50 104.01 98.7 4.1
100 154.2 98.5 5.6
095.5 - -
Sample 4 50 147.4 103.8 2.8
100 192.1 96.6 3.3
0 0.58 - -
Sample 5 0.5 1.06 96.0 2.7
1.5 2.11 102.0 3.9
Solid-Phase Extraction for determination of levooxacin Mohammad Abbaszadehbezi et al
58 Anal. Methods Environ. Chem. J. 5 (2) (2022) 51-59
[7] S.M. Alwan, Al-Akidi Therapeutic Protocol
for Severe Infections Associated with
Covid-19: Potential and Effective Treatment
by Levooxacin and Vitamin D3 and Zinc
(Part 1), J. Pharm. Pharmacol., 10 (2022)
105-118. https://doi.org/10.17265/2328-
2150/2022.04.001
[8] G. Huang, M. Su, Y. Liu, W. Zhang, Comparative
Study of Hyper-crosslinked Polymer-Solid
Phase Microextraction and Stir Bar Fabric
Phase Sorptive Extraction for Simultaneous
Determination of Fluoroquinolones in Water,
Chromatogr., 85 (2022) 539-549. https://doi.
org/10.1007/s10337-022-04165-9
[9] E. Tsanaktsidou, C. Markopoulou, P. D.
Tzanavaras, C. K. Zacharis, Homogeneous
liquid phase microextraction using
hydrophilic media for the determination
of uoroquinolones in human urine
using HPLC-FLD, Microchem. J., 172
(2022)106906. https://doi.org/10.1016/j.
microc.2021.106906
[10] M. Shamsipur, N. Mafakheri, N. Babajani,
A natural deep eutectic solvent–based
Ultrasound-vortex-assisted dispersive liquid–
liquid microextraction method for ligand-less
pre-concentration and determination of traces
of cadmium ions in water and some food
samples, Food Anal. Methods, 15 (2022)
1203-1213. https://doi.org/10.1007/s12161-
021-02222-x
[11] J. Hu, , S. Zhu , S.-E. Chen , R. Liu , J.
Sun , X.-En Zhao , H. Liu, Multiplexed
derivatization strategy-based dummy
molecularly imprinted polymers as sorbents
for magnetic dispersive solid phase extraction
of globotriaosylsphingosine prior to UHPLC-
MS/MS quantitation, Microchim. Acta, 187
(2020) 1-11. https://doi.org/10.1007/s00604-
020-04341-4
[12] L. P. Zhang, X.-X. Tan, Y.-P. Huang , Z.-S.
Liu, Floating liquid crystalline molecularly
imprinted polymer coated carbon nanotubes
for levooxacin delivery, Eur. J. Pharm.
Biopharm., 127 (2018) 150-158. https://doi.
org/10.1016/j.ejpb.2018.02.012
[13] J. Meng, X. Wang, Microextraction by
packed molecularly imprinted polymer
combined ultra-high-performance liquid
chromatography for the determination
of levooxacin in human plasma, J.
Chem., 2019 (2019) 4783432. https://doi.
org/10.1155/2019/4783432
[14] W. Ma, K.H. Row, Simultaneous determination
of levooxacin and ciprooxacin in human
urine by ionic‐liquid‐based, dual‐template
molecularly imprinted coated graphene
oxide monolithic solid‐phase extraction, J.
Sep. Sci., 42 (2019) 642-649. https://doi.
org/10.1002/jssc.201800939
[15] H. Liu, Pian Jin, Fucheng Zhu, A review
on the use of ionic liquids in preparation
of molecularly imprinted polymers for
applications in solid-phase extraction, TRAC
Trends Anal. Chem., 134 (2022) 116132.
https://doi.org/10.1016/j.trac.2020.116132
[16] M.P.G. de Llasera, M. Hernández
Camarillo , A. Rodrigo García Cicourel,
Semi-continuous monitoring of HMWPAH
in microalgae cultures by PT-SPE/HPLC/FD-
UV: Estimation of the degradation constant,
Anal. Biochem., 633 (2021) 114415. https://
doi.org/10.1016/j.ab.2021.114415
[17] H. Shirkhanloo, M. Osanloo, M. Ghazaghi, H.
Hassani,Validation of a new and cost-effective
method for mercury vapor removal based on
silver nanoparticles coating on micro glassy
balls, Atm. Pollut. Res., 8 (2017) 359-365.
https://doi.org/10.1016/j.apr.2016.10.004
[18] H. Shirkhanloo, A. Khaligh, HZ. Mousavi,
A. Rashidi, Graphene oxide-packed micro-
column solid-phase extraction combined wif
ame atomic absorption spectrometry for
determination of lead (II) and nickel (II) in
water samples, Int. J. Enviro. Anal. Chem.,
95 (2015) 16-32. https://doi.org/10.1080/030
67319.2014.983437
[19] M. Ghazaghi, HZ. Mousavi, H. Shirkhanloo,
A. Rashidi, Ultrasound assisted dispersive
micro solid-phase extraction of four
59
tyrosine kinase inhibitors from serum and
cerebrospinal uid by using magnetic
nanoparticles coated with nickel-doped
silica, Microchim. Acta, 183 (2016) 2779-
2789. https://doi.org/10.1007/s00604-016-
1927-z
[20] S. Golkhah, H. Zavvar Mousavi, H.
Shirkhanloo, A. Khaligh, Removal of Pb (II)
and Cu (II) Ions from Aqueous Solutions
by Cadmium Sulde Nanoparticles, Int. J.
Nanosci. Nanotechnol., 13 (2017) 105-117.
http://www.ijnnonline.net/article_25609.
html
[21] H. Shirkhanloo, SD. Ahranjani, A lead analysis
based on amine functionalized bimodal
mesoporous silica nanoparticles in human
biological samples by ultrasound assisted-
ionic liquid trap-micro solid phase extraction,
J. Pharm. Biomed. Anal. 157 (2018) 1-9.
https://doi.org/10.1016/j.jpba.2018.05.004
[22] B. Paknejad, H. Shirkhanloo, M. Aliomrani,
Is their any relevance between serum heavy
metal concentration and BBB leakage in
multiple sclerosis patients, Biol. Trace
Elem. Res., 190 (2019) 289-294. https://doi.
org/10.1007/s12011-018-1553-1
[23] I. E. Uyand, V. A. Zhinzhilo, Recent strategies
to improve MOF performance in solid phase
extraction of organic dyes, Microchem. J.,
168 (2021) 106387. https://doi.org/10.1016/j.
microc.2021.106387
[24] S. Tajik, Y. Orooji, F. Karimi, Z. Ghazanfari,
H. Beitollahi, High performance of screen-
printed graphite electrode modied with Ni–
Mo-MOF for voltammetric determination of
amaranth, J. Food Meas. Charact., 15 (2021)
4617-4622. https://doi.org/10.1007/s11694-
021-01027-0
[25] Z. Wang, S. Wang, A. Wang, X. Liu, J.
Chen, Q. Zeng, L. Zhang, W. Liu,
Covalently linked metal–organic framework
(MOF)-polymer all-solid-state electrolyte
membranes for room temperature high
performance lithium batteries, J. Mater.
Chem. A, 6 (2018)17227-17234. https://doi.
org/10.1039/C8TA05642K
[26] M.R.R. Kahkha, M. Kaykhaii, Fast
determination of bisphenol a in spiked juice
and drinking water samples by pipette tip
solid phase extraction using cobalt metal
organic framework as sorbent, Bull. Chem.
Soc. Ethiopia, 32 (2018) 595-602. https://doi.
org/10.4314/bcse.v32i3.17
Solid-Phase Extraction for determination of levooxacin Mohammad Abbaszadehbezi et al
