Jurnal Akademika Kimia, 11(1): 6-13, February 2022
ISSN (online) 2477-5185 | ISSN (print) 2302-6030
http://jurnal.fkip.untad.ac.id/index.php/jak/

OPEN ACCESS

1

Development of Hollow Fiber Liquid Phase Microextraction Method for
Determination of Diazinon Residues in Vegetable Samples
*Eviomitta R. Amanda1, Yanuardi Raharjo2, & Usreg S. Handajani2
1

Program Studi Teknologi Laboratorium Medis - STIKES Rumah Sakit Anwar Medika, Sidoarjo – Indonesia 61263
Program Studi Kimia/FST - Universitas Airlangga, Surabaya – Indonesia 60115

2

Received 09 August 2021, Revised 14 December 2021, Accepted 07 February 2022
doi: 10.22487/j24775185.2022.v11.i1.6-13

Abstract
An extraction method based on a combination of hollow fiber liquid-phase microextraction with a high-performance
liquid chromatography diode array detector (HF-LPME HPLC-DAD) has been developed and demonstrated to analyze
pesticide residues in vegetables. This study aims to determine the optimum extraction conditions and validation
performance of this method. Diazinon pesticide was selected as the target model analyte. HF-LPME is performed by
stacking microliter organic solvent droplets through an HPLC syringe coated with polypropylene hollow fiber by directly
dipping it into the sample solution and stirring it during the extraction process. Finally, the organic solvent was put into
an HPLC syringe at the end of the extraction. Then, it was injected into the HPLC-DAD at the wavelength of 247 nm.
Several important extraction parameters have been optimized. The optimization results showed the type of organic solvent
of n-hexane, the length of the hollow fiber of 1.5 cm, the volume of the sample solution of 20 mL, and the stirring
speed of 600 rpm. The validation performance obtained a limit of detection (LoD) of 0.10 mgL-1, limit of
quantification (LoQ) of 0.33 mgL-1, percent recoveries of 99.88%, a coefficient of variation of 3% (n=15), and the
enrichment factor of 19,982 times. Under optimal conditions, the developed method was applied to extract diazinon
in vegetable matrix samples using the spiking method. Mustard green was selected as a model matrix sample. From
the research, the percentage recoveries of diazinon obtained in the mustard green matrix sample are 98.80% 100.41%.

Keywords: Diazinon, hollow fiber liquid-phase microextraction, high-performance liquid chromatography,
pesticide residue, vegetable

mgL-1. However, in several agricultural products,
Introduction
including meat, beef liver, and beef fat, in Bogor and
Organophosphate pesticides are a group of
Lampung, diazinone residues exceeded the
chemical compounds widely used in agriculture to
permissible BMR (0,9 mgL-1) (Indraningsih & Sani,
prevent agricultural products from being attacked
2004). It may be due to the limitation of the
by pests (Pundir et al., 2019). Although the
instrument detector because the analysis of pesticide
pesticides used have many advantages, these
residues is very challenging due to trace
pesticides residues are very harmful to the
concentration and difficulties in isolating analytes
environment and are toxic to human health
from complex samples. Therefore, the development
(Menezes et al., 2016). Some health problems of a sensitive and selective analytical method to
caused by exposure to pesticide residues include
preconcentrate and enhance the instrument’s signal
pancreatic disorders, tumors, seizures, and death
is necessary.
(Cai et al., 2016; Jokanović, 2018). Based on these
The sample preparation step plays an essential
negative impacts, the European Union has set role in the analytical method before instrumental
guidelines for the maximum permissible level of
analysis. This step aims to clean, concentrate, and
organophosphate residue in vegetables, fruits, and
isolate the analyte from the complex matrix.
-1
meats, 20-500 ngL (Hasan et al., 2017). There are However, this step is very complicated and takes a
several compounds derived from organophosphate
much longer time (Kamaruzaman et al., 2017).
pesticides, one of which is diazinon. According to Therefore, a method of determining the trace
the Ministry of Health and the Ministry of the
concentration of pesticide residues that is fast, easy,
Agriculture Republic of Indonesia, the maximum
inexpensive, and effective is urgently needed.
residue limit (MRL) for diazinon in vegetables is 0.5
⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
*Correspondence:
Eviomitta R. Amanda
e-mail: eviomittarizki@gmail.com

© 2022 the Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons
Attribution-NonCommercial-ShareAlike 4.0 International, which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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Volume, 11, No. 1, 2022, 6-13

Jurnal Akademika Kimia
purchased from Sigma Aldrich (Singapore). Doubledistilled deionized water of at least 18 MΩ was
purified by a nano ultrapure water system
(Barnstead, USA). Diazinon 250 mg (98,5%) was
purchased from Sigma Aldrich (Singapore) as the
selected pesticide. A standard stock solution of
diazinon (1000 mgL-1) was prepared in methanol
and stored in a freezer at about 4 ˚C. Polypropylene
hollow fiber membrane (600 μm id, 200 μm wall
thickness, and 0,2 μm pore size) was purchased from
Membrane (Wuppertal, Germany). The mustard
green sample was obtained from agricultural land in
Sidoarjo, East Java.

Miniaturization and simplification of analytical
methods for convenience and pre-concentration are
popular trends in analytical chemistry (Rozaini et
al., 2019). It also supports green analytical
chemistry by minimizing the small volume of
organic solvents during the sample preparation step
(Rozaini et al., 2017). Several pre-concentration
methods based on the liquid-phase microextraction
technique have been developed to analyze
organophosphate residues, including single drop
microextraction (SDME) (Tang et al., 2018). This
method uses microliters of organic solvent as the
acceptor phase. Drops of organic solvent hang up on
the tip of the microsyringe needle. However, it has
a lot of problems, like the unstable droplet that
hangs from the stirrer during the process. Therefore,
the droplets are protected by using hollow fibers to
stabilize them droplets to overcome these
drawbacks. The hollow fiber also acts as a filter for
the separation of analytes in complex matrices. This
liquid extraction method is hollow fiber liquidphase microextraction (HF-LPME) (Cai et al.,
2016). HF-LPME has also been successfully applied
to extract carbamate pesticide residues (Ma et al.,
2014).
In this research, the performance of HF-LPME
combined
with
high-performance
liquid
chromatography was determined to detect diazinon
in mustard green. Several critical parameters such as
the type of organic solvent, the length of the hollow
fiber, the sample volumes, and the stirring speed
were optimized. Method validation parameters were
evaluated, including a limit of detection (LOD), the
limit of quantifications (LOQ), % recoveries,
decisions, and enrichment factors.

HPLC conditions
The HPLC system used, Agilent type 1100
(United Kingdom), diode array detector, and
consists of a pump. The column used a Bondapack
C-18 reverse phase microcolumn, particle size 10
μm, column size 3.9 × 300 mm, and column
temperature 30 ˚C. ACN-water mixture of 70:30
(v/v) was used as the mobile phase at a flow rate of
0.8 mL/min under an isocratic pump. The standard
solution and the prepared sample were injected in
triplicate into the HPLC/DAD instrument and
detected at 247 nm. The magnetic stirrer was
obtained from Daihan Labtech LMS-1003, with a
magnetic stirrer (Snipbar) size 12 × 5 mm.
Hamilton syringe for HPLC 25 μL was received
from Switzerland.
Hollow fiber liquid-phase microextraction
The setup of the HF-LPME procedure is shown
in Figure 1. Several important extraction
parameters, such as the types of organic solvent, the
hollow fiber lengths, the sample volume, and the
stirring speeds, were optimized. Under optimal
extraction conditions, this developed method was
applied to extract diazinon in mustard green matrix
samples.

Methods
The materials used in this study are HPLC
grade Acetonitrile was obtained from Merck
(Germany). Organic solvents (methanol, toluene, nhexane, and carbon tetrachloride), p.a were

Figure 1. Setup of HF-LPME

7

Eviomitta R. Amanda et al.
The polypropylene hollow fiber membrane is
cut in various sizes (e.g., 1 cm) and sealed at one edge
with a sealer machine. The hollow fibers were
cleaned with acetone before application in the HPLPME to remove some contaminants. A Hamilton
microsyringe 25 μL for HPLC was used to transfer
the acceptor phase into the sample solution. A
microsyringe needle containing an acceptor phase
(e.g., n-hexane 3 μL) was inserted into the rubber
septum of the sample vial. The tip of the
microsyringe needle is then inserted into the hollow
fiber segment. The hollow fiber is immersed in the
acceptor phase (e.g., n-hexane) for 5 s to impregnate
the pores of the hollow fiber before being inserted
into the needle tip. The assembly was immersed in
the sample solution (a standard solution of 6 ppm
diazinon was used). The extraction process begins
by stirring the sample solution for a specific time
(e.g., 10 min) using a magnetic stirrer. Finally, the
extraction, the acceptor phase was inserted into a
microsyringe and then directly injected into the
HPLC-DAD at a wavelength of 247 nm. Several
important extraction parameters such as the type of
the organic solvent (toluene, n-hexane, and carbon
tetrachloride), the lengths of the hollow fiber (1,
1.5, 2, and 2.5 cm), the volume of the sample
solutions (15, 17.5, 20, 22.5, and 25 min), and the
stirring speeds (300, 400, 500, 600, and 700 ppm)
have been optimized. The other variables are kept
constant in optimizing one of the extraction

parameters. For example, optimization of the types
of organic solvents, other variables were held regular
(length of hollow fiber of 2 cm, the volume of
organic solvent of 3 μL, stirring speed of 500 rpm,
extraction time of 15 minutes, and volume of
standard diazinon solution (6 ppm) of 20 mL). The
optimum extraction conditions were evaluated and
applied to extract diazinone in vegetable matrix
samples. Green mustard was chosen as vegetable
matrix samples to assess the performance of the
proposed method in the actual sample condition.
Results and Discussion
Peaks separation
Diazinon analysis was carried out at optimum
separation and quantification conditions in HPLCDAD. The best conditions were found at 247 nm
when the flow rate was 0.8 mL/min, and the
mixture of ACN and water was 70:30 (v/v). The
reversed phase column C-18 was chosen to separate
diazinon. For non-polar analytes, like diazinon (Log
Kow = 3.81), this column is the best choice for this
type of test. Not only the kind of column but the
composition of the mobile phase also plays a vital
role during the separation process. ACN-water
mixture 70:30 (v/v) showed a good separation peak
for diazinon analysis. The separation of the diazinon
peak in the solvent n-hexane is illustrated in Figure
2.

Figure 2. Chromatogram of sample separation: 1. acetonitrile, 2. methanol, 3. n-hexane, 4. diazinone (6
ppm)
on the Log Kow value of diazinone (3.81), it is
closest to the Log Kow of n-hexane (3.9) compared
to the Log Kow of toluene (2.73) and carbon
tetrachloride (2.04), this causes diazinone to be
easier to extract in n-hexane. The most important
thing to keep in mind when choosing organic
solvents is that the organic solvent should be able to
stick to the hollow fibers, dissolve well with analytes,
become insoluble in water, and not evaporate
quickly during extraction (Esrafili et al., 2018).
Then, n-hexane is used for more parameter
optimization. Figure 3 shows the types of organic
solvent optimization that worked best.

Optimization of the types of organic solvent
There were three kinds of organic solvents
optimized as extractor phase for extraction of
diazinons, such as toluene, n-hexane, and carbon
tetrachloride. Each type of organic solvent was
replicated 3 times. HPLC-DAD then analyzed the
extracted solution. The organic solvent n-hexane
showed optimal results. n-hexane has the lowest
solubility in water at 25 ˚C (9.5 mg/mL) compared
to toluene and carbon tetrachloride. It causes the
extracted diazinon in n-hexane to be not quickly
released back to the donor phase. In addition, based

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Jurnal Akademika Kimia

Peak area

1500
1000
500
0
Toluena

n-heksana

karbon
tetraklorida

Type of organic solvent

Figure 3. Optimization of the type of organic solvents
perforated fiber and the volume capacity of the
perforated fiber is regulated by the size of the bottle
used for extraction (Gjelstad, 2019; Mlunguza et al.,
2020). The more extended length of the hollow
fiber, the more organic solvents will spread to the
pores of the hollow fiber, but at the time of
extraction, it cannot be reached by the microsyringe
needle. For that, it causes the extracted diazinon to
be smaller. The hollow fiber length of 1.5 cm was
used to optimize the analytical parameters further.
At the size of the hollow fiber is more than 2.5 cm,
the hollow fiber will contact with magnetic stirrer so
that it will be disturbed by the kinetic force and
separated from the microsyringe needle. The
optimization of the length of the hollow fiber is
shown in Figure 4.

Optimization of the length of the hollow fiber
The optimization of the length of the hollow
fiber is done by varying the length by 1; 1.5; 2; and
2.5 cm. Each variation of perforated fiber length was
repeated 3 times. HPLC-DAD then analyzed the
extracted solution. The optimum hollow fiber
length for diazinon extraction using the HF-LPME
method is 1.5 cm. The shorter the hollow fiber, the
less spread of the acceptor phase on the surface of
the hollow fiber, so that the diazinon that enters
through the pores of the hollow fiber is also less and
produces a smaller peak area. The longer the hollow
fiber, the more phase acceptor (organic solvent)
dissociated on the surface of the hollow fiber so that
more diazinon is extracted. The length of the

Peak area

1000
800
600
400
200
0
0.5

1.5

2.5

3.5

Length of hollow fiber (cm)

Figure 4. Optimization of the length of the hollow fiber
results of the optimization of the sample volume are
shown in Figure 5.

Optimization of the sample solution volume
By adjusting the sample volume to 15, 17.5,
20, 22.5, and 25 mL, the sample solution volume is
optimized. Each modification in the volume of the
sample solution was repeated three times. The
extracted solution was then evaluated using HPLCDAD. The optimal volume of sample solution for
diazinon extraction using the HF-LPME method is
20 mL. The larger volume of the sample solution
contains more analytes (Salvatierra-stamp et al.,
2018; Venson et al., 2019). Combining a small
volume of acceptor solution and a large volume of
donor solution causes a high concentration factor
(Raharjo et al., 2009; Salvatierra-stamp et al., 2018).
The sample solution volume of 20 mL was used to
optimize the analytical parameters further. The

Optimization of the stirring speed
The optimization of stirring speed was carried
out with variations of 300, 400, 500, 600, and 700
rpm. Each variation of mixing speed was repeated 3
times. HPLC-DAD then analyzed the extracted
solution. Stirring can increase the mass transfer rate
during the extraction process (Zuluaga et al., 2021).
Stirring the sample solution with the HF-LPME
method can shorten the time to reach
thermodynamic equilibrium, especially for analytes
with a larger molecular mass. In the HF-LPME
method, the organic solvent is covered and
protected by a hydrophobic hollow fiber, making it
easier to control the higher stirring speed (Zuluaga
et al., 2021).
9

Eviomitta R. Amanda et al.

Peak area

1500
1000
500
0
10

15

20

25

30

Sample volume (mL)

Figure 5. Optimization of the sample volume
Based on Figure 5, it can be seen that the
optimal stirring speed is 600 rpm. The higher the
stirring speed, the higher the transfer rate of the
analyte to the organic solvent. However, a higher
stirring speed can result in the release of the
extracted diazinon on the surface of the hollow fiber
to be released back into the sample solution (Cai et
al., 2016). The increasing stirring speed can
generate bubbles of air within the pores of the fiber,
making it difficult mass transfer from the donor

phase to the lumen of the thread (Zuluaga et al.,
2021). Higher stirring speed also increases the
kinetic force so that it will disturb the position of
the hollow fiber immersed vertically in the sample
solution. The stirring speed of 600 rpm is the
optimal result for diazinon extraction by the HFLPME method and is used for standard curve
extraction. The optimization of the stirring speed
results is shown in Figure 6.

Peak area

1500
1000
500
0
200

400

600

800

Stirring speed (rpm)

Figure 6. Optimization of the stirring speed
LoD and LoQ values of the developed method are
0.10 mgL-1 and 0.33 mgL-1, respectively. The
proposed method offers excellent repeatability with
%CV less than 3%. Meanwhile, the relative
recovery was 99.88%. The detailed results are
presented in Table 1.
The enrichment factor (EF) plays a vital role in
the microextraction technique. The enrichment
factor described the pre-concentration phenomenon
that occurs by the distribution of the analyte from
the sample solution (Vaq) to the acceptor phase (Vorg)
in a smaller volume. It is also closely related to the
distribution constant as a fundamental principle in
liquid-liquid extraction. Determination of EF can
be defined by equations (1), (2), and (3) (Raharjo et
al., 2009; Zuluaga et al., 2021).

Validation method
After obtaining optimal conditions, several
standard solution concentrations were prepared in
deionized water. The stock solutions were treated
using the HF-LPME method and directly injected
into the HPLC-DAD. Extraction replication using
the HF-LPME method at each concentration of the
standard solution was carried out three times.
Linearity range, detection limit (LoD),
quantification limit (LoQ), % recovery (%R),
precision or coefficient of variation (% CV), and
enrichment factor (EF) were evaluated.
A good linearity range was achieved at 0.20010.000 mgL-1 with a coefficient correlation (r2) of
0.9999, which indicated that the analyte showed
good linearity in each concentration range. The

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Jurnal Akademika Kimia

Table 1. Analytical performance of HF-LPME HPLC-DAD
Correlation
Enrichment
Linearity
LoD
LoQ
Recovery
coefficient
Factor
range
-1
2
-1
(%)
(times)
(mgL )
(r )
(mgL )
(mgL-1)
0.20099.88
19,982
0.9999
0.10
0.33
10.000

Analyte
Diazinon

𝐸𝐸 =

𝑛
𝑛

, 𝐸𝐸 =

𝐸𝐸 =

𝐶
𝐶

, 𝐸𝐹 =

𝐸𝐹 =

𝐸𝐸 . 𝑉
𝑉

𝐾

.𝑉

/

𝐾

/

𝑛
𝑛

.𝑉
𝑉
.

.𝑉

+𝑉

Coefficient
of variation
(%)
3.00

Vorg are the volume of the sample solution and the
volume of the acceptor phase, respectively.
a. Application of the proposed method in
vegetable matrix sample
The vegetable sample matrix was prepared by
diluting 10 gr of crushed mustard green in 100 mL
of deionized water. The solution was then allowed
to stand for 3 hours. The filtrate and sedimentation
were separated using a Buchner filter. The filtrate
was treated using the HF-LPME method under
optimal extraction conditions and directly injected
into the HPLC-DAD. Each of the three different
standard solution concentrations was added to the
sample matrix by spiking. This way, we could see
how much of the sample was recovered. The
performance of the proposed method in the
vegetable matrix sample is presented in Table 2.

(1)

(2)

(3)

where EE is the extraction efficiency and EF is the
enrichment factor. Corg and norg are the concentration
and mass of the extracted analyte in the organic
solvents, respectively. Co and no are the
concentration and mass of the analyte initially
present in the sample solution, respectively. Vaq and

Table 2. Recoveries of diazinon in vegetable sample matrix using HF-LPME HPLC-DAD
Analyte
Diazinon

0 ppm
ND

Recovery on spiking method (%)
2 ppm
6 ppm
98.90
100.41

10 ppm
99.63

b. Comparison of the proposed method with
other previous methods for determination
diazinon residue
The comparison of HF-LPME-HPLC-DAD
with other reported methods for the analysis of
diazinon residue in the vegetable appeared in Table
3.
Table 3. Comparison of the proposed method with other published methods for the determination of
diazinon
Extraction techniqueSample
%R
LoQ
Ref.
Instrument
DSPE-GC-NPD
Potato
95.760.06 mgkg-1 (Saraji et al., 2021)
99.87
LLE-GC-FID
Lavender and
100.90
0.085 mgL-1 (Rezk et al., 2018)
Rosemary leaves
HF-LPME-HPC-DAD
Green Mustard
99.88
0.33 mgL-1
Present work

The analyte recovery is excellent, with a value
greater than 98.90%. The results showed that the
proposed method was suitable for the determination
of trace amounts of pesticide residues in vegetables,
especially diazinon.

11

Eviomitta R. Amanda et al.
Animal and Veterinary Sciences (Wartazoa),
14(1), 1–13.
Jokanović, M. (2018). Neurotoxic effects of
organophosphorus pesticides and possible
association with neurodegenerative diseases in
man: A review. Toxicology, 410(December),
125–131.
Kamaruzaman, S., Sanagi, M. M., Yahaya, N., Wan
Ibrahim, W. A. W., Endud, S., & Wan
Ibrahim, W. N. W. (2017). Magnetic microsolid-phase extraction based on magnetiteMCM-41 with gas chromatography–mass
spectrometry for the determination of
antidepressant drugs in biological fluids.
Journal of Separation Science, 40(21), 4222–
4233.
Ma, X., Wang, J., Wu, Q., Wang, C., & Wang, Z.
(2014). Extraction of carbamate pesticides in
fruit samples by graphene reinforced hollow
fibre liquid microextraction followed by high
performance liquid chromatographic detection.
Food Chemistry, 157(August), 119–124.
Menezes, H. C., Paulo, B. P., Paiva, M. J. N., &
Cardeal, Z. L. (2016). A simple and quick
method for determination of pesticides in
environmental water by HF-LPME-GC/MS.
Journal of Analytical Methods in Chemistry,
2016(September), 1–16.
Mlunguza, N. Y., Ncube, S., Mahlambi, P. N.,
Chimuka, L., & Madikizela, L. M. (2020).
Optimization and application of hollow fiber
liquid-phase microextraction and microwaveassisted extraction for the analysis of nonsteroidal anti-inflammatory drugs in aqueous
and plant samples. Environmental Monitoring
and Assessment, 192(557), 1–14.
Pundir, C. S., Malik, A., & Preety. (2019). Biosensing of organophosphorus pesticides : A
review.
Biosensors
and
Bioelectronics,
140(September), 111348.
Raharjo, Y., Sanagi, M. M., Ibrahim, W. A. W.,
Naim, A. A., & Aboul-Enein, H. Y. (2009).
Application of continual injection liquid-phase
microextraction method coupled with liquid
chromatography
to
the
analysis
of
organophosphorus pesticides. Journal of
Separation Science, 32(4), 623–629.
Rezk, M. R., El-Aleem, A. E. B. A., Khalile, S. M.,
& El-Naggar, O. K. (2018). Determination of
residues of diazinon and chlorpyrifos in
lavender and Rosemary leaves by gas
chromatography.
Journal
of
AOAC
International, 101(2), 587–592.
Rozaini, M. N. H., Semail, N., Saad, B.,
Kamaruzaman, S., Abdullah, W. N., Rahim, N.
A., Miskam, M., Loh, S. H., & Yahaya, N.
(2019). Molecularly imprinted silica gel
incorporated with agarose polymer matrix as
mixed matrix membrane for separation and pre-

Generally, each published method has
advantages and disadvantages. Dispersive solidphase extraction (DSPE) method combination with
gas chromatography-nitrogen phosphorous detector
(GC-NPD) for analysis diazinon in potato needs
solid sorbent. Therefore, to minimize carryover
analyte in the porous sorbent need more treatment
and time to desorb the analyte completely (Saraji et
al., 2021). While liquid-liquid extraction (LLE)
method, combination with gas chromatographyflame ionization detector (GC-FID), needs a larger
volume of organic solvent, so it produces more
organic waste (Rezk et al., 2018). LoQ in this work
is different from other methods because of the
instrument detector. However, another validation
parameter of the present work with the previous
method is not significantly different, so it can be
used as a simple extraction method to remove
diazinon residue from a vegetable sample.
Conclusions
The HF-LPME combined with HPLC-DAD
provides good performance for determining
diazinon pesticides in vegetable matrix samples.
High percent recovery and extraction efficiency are
achieved. The low value of LoD, LoQ, and %CV
indicates that the proposed method has excellent
potential for residue pesticide analysis.
Acknowledgments
The authors are grateful to the Department of
Chemistry, Faculty of Science and Technology,
Airlangga University, Surabaya, and Anwar Medika
Health College for the facilities and support in this
research.
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