Food Analytical Methods (2023) 16:1227–1236

https://doi.org/10.1007/s12161-023-02485-6

Simultaneous Analysis and Uncertainty of Measurement

for Pymetrozine, Ethoxyquin, and Cycloxydim in Sweet Pepper using
LC–MS/MS
Han Yeol Bang1 · Sung Ho Jang1 · Eun Joo Baek2 · Suel Hye Hur2 · Hyoyoung Kim2 · Ho Jin Kim2

Received: 4 July 2022 / Accepted: 4 May 2023 / Published online: 25 May 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
In this paper, we developed an experimental method via LC–MS/MS that enables simultaneous analysis of pymetrozine,
ethoxyquin, and cycloxydim, which are pesticide substances used in sweet pepper, and we improved the accuracy of the
corresponding experimental method by calculating the uncertainty of measurement. Sweet pepper samples mixed with 5 N
sodium hydroxide were extracted with ACN and 1 g of NaCl and 4 g of M ­ gSO4. Verification was conducted using linearity,
sensitivity, selectivity, and accuracy. As a result, the limits of detection (LOD) were measured between 2.0 and 3.9 µg/kg,
and the limits of quantification (LOQ) were between 6.1 and 11.8 µg/kg. The average recovery rate of the three substances
was 97.39–99.42%. The uncertainty of measurement was calculated after identifying various factors that influence the quan‑
titative analysis. The expanded uncertainties of pymetrozine, ethoxyquin, and cycloxydim were 0.47 μg/L, 0.50 μg/L, and
0.49 μg/L, respectively, at a confidence level of approximately 95%. Additionally, k = 2, which was calculated as 4.6%, 5.0%,
and 4.6% of the result, respectively. Factors such as the sample weighing, temperature, dilution, and calibration curves were
found to affect the uncertainty of measurement. When all the factors were considered, significant results were found, and
thus, we judged that this method is suitable for simultaneous analysis of specific residual substances used in sweet pepper.

Keywords LC–MS/MS · Residue pesticide compounds · Sweet pepper · Validation · Uncertainty of measurement

Introduction diverse areas, including the forestry and livestock indus‑

With the growth of the global population, the demand for
food has sharply risen (Calicioglu et al. 2019). To meet
these demands, agriculture has also been developed. Under
these circumstances, agricultural pesticides have naturally
been used more frequently (Syed et al. 2014). Due to their
high application, agricultural pesticides have been used in
Han Yeol Bang, Sung Ho Jang, and Eun Joo Baek contributed
equally to this work.
* Hyoyoung Kim
hyo02@korea.kr
* Ho Jin Kim
rex7878@korea.kr
1 Agricultural chemicals consist of unique chemi‑
Gyeongnam Provincial Office, National Agricultural
Products Quality Management Service, Busan, Korea
2 lyzed data with the use of a variety of devices have
Experiment Research Institute, National Agricultural
Products Quality Management Service, Gimcheon‑Si 39660,
Gyeongsangbuk‑Do, Korea
tries, and these applications have triggered massive usage
(Aktar et al. 2009). Careless use of agricultural pesticides
has led to hazardous ontogeny with resistance. With the use
of stronger substances to remove them, they have begun to
negatively influence the bioenvironment and the wellbeing
of organisms (Eddleston et al. 2002). For instance, there
are multiple reports that if agricultural pesticides are indi‑
rectly exposed to those of concern (Toumi et al. 2020) or are
absorbed in the human body through various routes, such as
biological concentration based on direct intake (Clasen et al.
2018), it may cause cancer, hormone destruction, asthma,
allergy, fetal congenital anomaly, and even death (Kim et al.
2017). Therefore, it is necessary to thoroughly analyze the
byproducts exposed to agricultural pesticides and secure
their safety.
cals according to their use, and comprehensively ana‑
steadily been reported to date (Da Costa Morais et al.
2018; Rizzetti et al. 2016; Tankiewicz 2019). The

13
Vol.:(0123456789)
1228
development of analyzers makes an accurate analysis
possible (Thammana 2016). In the case of LC, more
sensitivity and selectivity were obtained through an
MS/MS analysis based on diverse ionization methods,
such as ESI or APCI and SRM (Alder et al. 2006).
LC–MS/MS is the best tool for quantitative and qualita‑
tive analysis for targeted analytes due to its high selec‑
tivity and sensitivity (Attwa et al. 2020).
Additionally, extraction steps are also important to
remove matrix constituents’ interferences and target com‑
pounds extraction (Abdelhameed et al. 2019). Using
LC–MS/MS, this study simultaneously developed a method
of efficiently analyzing substances related to residual agri‑
cultural pesticides.
Pymetrozine, ethoxyquin, and cycloxydim are compo‑
nents used for agricultural pesticides, serving as herbicides,
insecticides, and germicides, respectively. These compo‑
nents are highly versatile materials and are registered in the
standards of residual agricultural pesticides in many dif‑
ferent countries. They therefore need to be controlled. It is
important to detect these components, which will thereby
contribute to food safety. Above all, it is necessary to bear in
mind that analytical results can differ depending on the char‑
acteristics of the samples, preprocessing, and the conditions
of the analyzers. In addition, given that the results depend
on a wide range of factors, such as testers and experimental
tools, it is necessary to judge the accuracy of the entire pro‑
cess of the experiment and thereby obtain a result.
Therefore, the uncertainty of measurement is consid‑
ered. One experiment has multiple steps, each of which
includes many factors influencing the results. A fine meas‑
urement error can arise according to a tester’s ability, the
state of experimental tools, and experimental conditions,
which can then influence a final result. Therefore, the reli‑
ability of the experimental result can fall. For this reason,
it is important to calculate uncertainty in each of the steps
and to verify its validity. There is something that fails to
be judged in the measurement of uncertainty. If a matrix
effect is made by the characteristics of samples in the anal‑
ysis process, the result can be negatively influenced. To
establish an accurate analysis method, it is also important
to select appropriate samples.
Finally, the sample selected in consideration of the
aforementioned content was sweet pepper as a hypochlo‑
rous plant. Annually, more than 4 million tons of sweet
pepper are produced (Tridge 2020). This amount seems to
be because of the excellent value of sweet pepper. Sweet
pepper includes useful substances, such as phenolic com‑
pounds and flavonoids (Caruso et al. 2018), and is rich in
antioxidant substances, including vitamin E and carotenoids
(Rao et al. 2011) Sweet pepper is reportedly effective for
particular cancer, delays aging, and shows other kinds of
efficacy (Simonne et al. 1997). In addition, its ascorbic acid
13
Food Analytical Methods (2023) 16:1227–1236
content per 100 g is known to be 60 mg, which is 20%
of the recommended daily allowance (Deepa et al. 2006).
Therefore, sweet pepper is considered to be a highly appli‑
cable fruit and vegetable. The aforementioned substances
of sweet pepper can be used, and it has been reported that
it has almost no matrix effect (Lee et al. 2011). Therefore,
sweet pepper was judged to be appropriate for calculating
the uncertainty of measurement and was therefore selected
as a sample.
This study first selected pymetrozine, ethoxyquin, and
cycloxydim, which are highly applicable as agricultural
pesticides, as analysis subjects, and sweet pepper was
determined to be a medium. For an accurate analysis, the
experimental error factors were analyzed in each step of the
experiment, and then a variety of influences were calculated
based on mathematical and statistical relationships of the
final experimental results. This study is meaningful because
through simultaneous analysis, the results were obtained
quickly and an analysis technique with high reliability was
developed to establish the safety of agricultural products.
Materials and Methods
Samples
In this study, sweet pepper was selected as the medium to
study the uncertainty of measurement in an experimental
method that was developed to measure a specific substance.
Sweet pepper was purchased directly from a local market in
Busan, Korea. All samples were homogenized (Ultra Turrax
t25 basic, IKA®, Korea) using a grinder and stored frozen
at − 4 °C.
Chemicals and Reagents
HPLC-grade water and methanol (MeOH) were purchased
from Merck (Darmstadt, Germany). Acetonitrile (ACN) and
sodium hydroxide (NaOH) used for sample extraction and
purification were purchased and used from Merck (Darm‑
stadt, Germany). Pymetrozine, ethoxyquin, and cycloxy‑
dim standards were purchased from ACCU Standard (New
Haven, USA). Formic acid (98%) and ammonium formate
(99.995%) were purchased from Sigma-Aldrich (St. Louis,
Germany).
Sample and Standard Preparation
The extraction was evaluated and performed by using a
modified QuEChERS-based sample preparation procedure
(Anastassiades et al. 2003). Pymetrozine is stable at pH 7–9
(EFSA 2014), and ethoxyquin and cycloxydim are unstable
at low pH (EURL-SRM 2015; Anastassiades 2007). A 600
Food Analytical Methods (2023) 16:1227–1236 1229

µL of 5 N NaOH was added to 10 g of the weighed sample,
and 10 mL of acetonitrile was used as an extraction sol‑
vent. This was shaken for 1 min, centrifuged at 3000 rpm for
5 min, and then shaken twice using 1 g of NaCl and 4 g of
­MgSO4. The centrifuged supernatant was filtered through a
syringe filter of 0.2 μm or less, and the LC/MS/MS analysis
was performed.
The standard stock solutions of the three pesticide resi‑
dues were prepared in acetonitrile and methanol to make it
1000 mg/L. A standard solution was prepared by properly
mixing the standards of each material, and this solution was
diluted and used in the experiment.
LC–MS/MS Analysis
The analytical conditions are described in Table 1.
LC–MS/MS analysis was performed on a Waters Acquity
UPLC combined with a Xevo™ TQ-Smicro mass spec‑
trometer (Waters, USA). The chromatographic column
used was a Thremo AQUASIL C18, 2.1 × 100 mm,
3.0 μm, and the column temperature was set to 40 °C. The
mobile phase was composed of mobile phases A water
containing 5 mM ammonium formate and 0.1% formic
acid and B MeOH containing 5 mM ammonium formate
and 0.1% formic acid, with the following gradient elu‑
tion: 0 min, 85% A; 0–0.08 min, 85% A; 0.08–1.10 min,
40% A; 1.10–5.35 min, 10% A; 5.35–6.35 min, 10%
A; 6.35–6.40 min, 2% A; 6.40–8.35 min, 2% A;
8.35–8.40 min, 85% A; and 8.40–11.00 min, 85% A. The
sample injection volume was 10 μL, and the flow rate was
set at 0.4 mL/min. The MS conditions were set as fol‑
lows: source temperature: 150 °C, desolvation tempera‑
ture: 350 °C, desolvation gas flow: 800 L/h, and capil‑
lary: 3.00 kV. ESI + showed the best results in terms of
sensitivity, and protonated precursor ions [M + ­H]+ were
selected (Table 1). The ionization conditions and multiple
reaction monitoring (MRM) for the quantitative analysis
are illustrated in Fig. 1.

Table 1  Parameters for analysis

of the 3 compounds by LC MS/ Instrument : Waters Aquity UPLC system/Xevo TQ-S micro
MS Column : Thremo AQUASIL ­C18, 2.1 × 100 mm, 3.0 μm
Mobile phase A : 5 mM ammonium formate and 0.1% formic acid in water
Mobile phase B : 5 mM ammonium formate and 0.1% formic acid in methanol
Gradient program Time (min) A (%) B (%) Flow (mL/min)
Initial 85 15 0.4
0.08 85 15 0.4
1.10 40 60 0.4
5.35 10 90 0.4
6.35 10 90 0.4
6.40 2 98 0.4
8.35 2 98 0.4
8.40 85 15 0.4
11.00 85 15 0.4
Injection volume : 10 μL
Column temperature : 40 ℃
Source Temperature : 150 ℃
Desolvation temperature : 350 °C
Desolvation gas flow : 800 L/h
Capillary voltage : 3.00 kV
Ionization mode : ESI
Scan type : MRM mode
MRM parameter Compounds Q1 Q3 Cone voltage (V) Collision
energy
(eV)
Pymetrozine 218.1 104.9 28 20
218.1 78.9 28 30
Ethoxyquin 218.1 174.05 25 30
218.1 160.05 25 30
Cycloxydim 326.15 280.1 15 15
326.15 180.05 15 20

13
1230
Fig. 1  Multiple reaction monitoring (MRM) conditions for the three pesticide compounds
Method Validation
Method validation has been carried out according to the
guidelines set by the International Union of Pure and
Applied Chemistry (Thompson et al. 2002) and CODEX
(Matthee 2007). This method has been validated for selectiv‑
ity, linearity, sensitivity, precision, and accuracy.
How to Estimate Uncertainty
Based on IUPAC, IUPAP, and OIML, the equation for
obtaining the compound content of sweet pepper was set as
(1). The uncertainty has been estimated from each uncer‑
tainty factor, and the overall uncertainty factors and details
are as follows (Fig. 2).
Vf
Compounds(μg∕kg) = Ce × ×D
We
where Ce is the concentration of the sample in the extraction
solution (μg/L); Vf is the final volume of the sample (mL);
We is the weight of the sample (g); and D is the dilution
factor.
13
Food Analytical Methods (2023) 16:1227–1236
Uncertainty Factor and Calculation
By applying the standard uncertainty to the repeatedly meas‑
ured average value, the average value was evaluated using a
type A standard uncertainty equation (Farrance & Frenkel
2012). For the repeat test uncertainty, the A-type uncertainty
was calculated using Eqs. (3) and (4) according to the standard
deviation method. Moreover, when the results of calibration
reports such as pipettes, flasks, and scales were used, and when
repeated measurements were not performed, the B-type stand‑
ard uncertainty was evaluated. The degree of freedom was
calculated according to the formula contained in the KOLAS-
G-002 Guideline (Korea Laboratory Accreditation Scheme
2016) for expressed the uncertainty of the measurement result.
Therefore, the degrees of freedom for repeated measurements
were calculated as n − 1, and the degrees of freedom for the
standard uncertainty were calculated as n − 2 using n inde‑
(1)
pendent observations to determine the slope of the intercept
and line using the least squares method.
� �
S
u x1 = √ (2)
n
Food Analytical Methods (2023) 16:1227–1236 1231

Fig. 2  Cause-and-effect diagram

� � Sp u4 (Syed et al.)
u x1 = √ (3) ueff = ∑c 4 (6)
n n ui (Syed et al.)
i=1 vi

�∑
n
v s2 ∑n
i=1 i i ueff ≤ v (7)
Sp = �
∑n (4) i=1 i
v
i=1 i
U(y) = kuc (Syed et al.) (8)
The combined standard uncertainty is the standard
uncertainty when the measurement results are obtained where n is the number of measurements; Sp is the pooled
from several different input quantities, which is obtained standard deviation; U is the expanded uncertainty; and k is
using the uncertainty Eq. (5). The extended uncertainty the coverage factor.
was obtained by multiplying the calculated combined
standard uncertainty by the inclusion factor k and using
Eq. (8). If the measurement degree of freedom is greater Results and Discussion
than 10, it is calculated by referring to the uncertainty
calculation guidelines in the EURACHEM/CITAC guide Method Validation
(Moser et al. 2001). Using Eqs. (6) and (7), the coverage
factor k was chosen as 2 at the 95% confidence level, and The developed LC–MS/MS analysis method was verified
if less, the t-distribution was used. through linearity, sensitivity, selectivity, accuracy, and preci‑
� sion measurements.
�n f Selectivity is determined by the presence or absence
uc (y) = ⌊( u(xi ))⌋2 (5) of interference peaks. As shown in Fig. 1, pymetrozine
i=1 x i
appeared at 2.04 min, ethoxyquin appeared at 3.19 min, and
cycloxydim appeared at 4.76 min. The compounds were

13
1232
Table 2  Validation parameters for the developed LC–MS/MS method
Compound RTa (min) Slope Intercept R2b
1 Pymetrozine 2.040 1962.98 4561.06 0.999914 2.0
2 Ethoxyquin 3.195 174.315 526.439 0.999903 2.5
3 Cycloxydim 4.763 785.686 2496.46 0.999885 3.9
a
Retention time (min)
b
Coefficients of correlation
c
Limit of detection, the lowest analyte concentration that produces a response detectable above the noise level of the system
d
Limit of quantification, the lowest level of analyte that can be accurately and precisely measured
e
Relative standard deviation, expressed as %
f
Average of recoveries at two spike levels (high and low)
successfully isolated through a simultaneous analysis, and
the selectivity in LC–MS/MS was demonstrated.
Linearity was verified by setting a solution in which
three substances were mixed as an external calibration
curve. The calibration curve was constructed by integrat‑
ing the dimensions of the peak area and various concentra‑
tions of the mixed standard solution. The concentration of
the mixed standard solution was injected three times, and
then the regression variable was calculated (Table 2). We
have confirmed that a correlation coefficient (R2) close to
0.999 is obtained for all compounds, which allows us to use
an external standard calibration for quantitative purposes.
The limits of detection (LOD) and quantification (LOQ)
were determined to verify the sensitivity of the developed
method, and the LOD and LOQ were calculated using the
following equations.
LOD = 3.3 × 𝜎∕SLOQ = 10 × 𝜎∕S
where σ is the standard deviation and S is the slope.
The range for the LOD was from 2.0 to 3.9 μg/kg, and the
value range for the LOQ was calculated to be between 6.1 and
11.8 μg/kg. The precision was measured by intraday and inter‑
day measurements. The intraday precision was analyzed by 6
repetitions of the mixed standard solution within 1 day, and
the interday precision was analyzed in triplicate for 3 consecu‑
tive days. The precision was calculated as a percentage of the
relative standard deviation (%RSD). Intraday %RSD values
were less than 4.3%, and interday values were less than 3.6%.
Detailed data on all of these are shown in Table 2.
Recovery was evaluated by adding a mixed standard solu‑
tion to the sample. Sweet pepper with a 100 µg/L mixed
standard solution (solution A) and sweet pepper without
the addition of the solution (solution B) were pretreated in
the same manner by the method developed. The recovery
rate was evaluated by comparing the concentration differ‑
ence between the added mixture (solution A) and the non-
added mixture solution B), and the result was in the range of
13
Food Analytical Methods (2023) 16:1227–1236
LODc (μg/kg) LOQd (μg/kg) RSDe (%) Recoveryf (%)
Intraday (n = 6) Interday (n = 9)
6.1 2.4 3.4 99.42
7.6 2.8 3.6 97.39
11.8 4.3 3.2 98.93
approximately 97.39 to 99.42%. Through this, the analysis
method appears to exhibit a high level of accuracy.
Judging from the above results, the developed simultane‑
ous analysis of pesticide residues is considered to be suit‑
able for use in the analysis, as it has shown effective results
in various fields, such as linearity, sensitivity, selectivity,
accuracy, and precision.
Uncertainty in the Measurement
Uncertainty Regarding Weight
The balance certificate, balance stability, and balance resolu‑
tion were used as uncertainty factors. The certificate uncer‑
tainty is 0.0008 g at the approximately 95% confidence
level. Therefore, the standard uncertainty was 0.0004 g.
For the stability of the balance, 10 g was repeatedly meas‑
ured 5 times for 5 days, the pooled standard deviation was
0.000058 g, and the standard uncertainty was 0.000026 g.
The resolution of the balance was 0.0001 g, the standard
uncertainty was 0.000029 g, the combined standard uncer‑
tainty was 0.000402 g, and the relative standard uncertainty
was 0.000040 g.
Uncertainty About Sample Preparation (Final Total Amount)
For sample preparation, a calibration certificate of a 10 mL
hole pipette was used as an uncertainty factor. The certificate
uncertainty is 0.012 mL at the approximately 95% confidence
level. Therefore, the standard uncertainty was 0.006 mL, and
the relative standard uncertainty was 0.000600 mL.
Uncertainty in the Standard Solution
The calibration curve concentration was calculated by divid‑
ing it into stock STD, working STD1, working STD2, and
the calibration curve.
Food Analytical Methods (2023) 16:1227–1236 1233

First, for the stock STD, a 0.1 g sample was sampled using
a standard product with a purity of 98%, and 1000 mg/L was
prepared in a 100 mL volumetric flask, which was diluted
100 times to prepare working STD1 (10 mg/L). Working
STD2 (100 μg/L) was then made. The calibration curves
were 1 μg/L, 2.5 μg/L, 5 μg/L, 10 μg/L, and 20 μg/L by
diluting working STD2.
In the case of the stock STD, the standard uncertainty in
the purity of the standard product was 0.011547 g. Next,
for a 0.1 g sample, the balance certificate, balance stability,
and balance resolution were used as uncertainty factors. The
standard uncertainty in the certificate was 0.0004 g, and as
a result of the repeated measurement of 0.1 g five times
for 5 days for the stability of the balance, a pooled stand‑
ard deviation of 0.000075 g was obtained, and the standard
uncertainty was 0.000034 g. The standard uncertainty in the
resolution of the balance was 0.000029 g, and the combined
standard uncertainty in all factors was 0.000402 g.
For a 100 mL volumetric flask, tolerance, tempera‑
ture, and repeated measurements were used as uncer‑
tainty factors. The volumetric flask used was expressed as
100 ± 0.08 mL at 20 °C, and the standard uncertainty in the
tolerance was 0.046188 mL. Since the temperature is 4 °C
different from the standard temperature (20 °C) based on
the current temperature of 24 °C, the standard uncertainty
for a 100 mL flask temperature is 0.048497 mL. The repeat‑
ability was measured by the mass and weighing using 10
repetitions with water in a 100 mL flask, with a standard
deviation of 0.038418 mL and a standard uncertainty of
0.012149 mL. When all conditions were considered, the
combined standard uncertainty for a 100 mL volumetric
flask was 0.068066 mL.
Therefore, the uncertainty in the stock standard solution
is 12.469570, taking the standard material purity, the bal‑
ance, and the uncertainty in the flask into account.
Uncertainty in the Working Standard Solution
As mentioned above, since working STD1 must be made by
diluting it based on the stock STD, the standard uncertainty in
the stock STD must be considered. The standard uncertainty in
stock STD is 12.469570, the 1 mL pipette uncertainty is used
as the certificate value (0.0015 mL), and the standard uncer‑
tainty is 0.000750 mL. The standard uncertainty for a 100 mL
volumetric flask was 0.068066 mL, so taking this together, the
uncertainty for working STD1 was 0.125106.
Since working STD2 is also manufactured by diluting work‑
ing STD1, the standard uncertainty in working STD1 must be
used. The standard uncertainty in working STD1 is 0.125106,
and the standard uncertainties of the 1 mL pipette and 100 mL
volumetric flask are 0.000750 mL and 0.068066 mL, respec‑
tively, which are the same as the previous conditions. Taken
together, the uncertainty in working STD2 is 1.255156. The
above details are summarized in Table 3.

Table 3  Uncertainty values for determining the standard solution

Uncertainty factors Description Uncertainty Combined
standard uncer‑
tainty
√
Stock solution (1000 mg/L) 1000 × uPurity 2 + uWeight 2 +uFlask 2 12.469570 0.231534
ucStock=
√
Pymetrozine Working solution 1 (10 mg/L) 10 × uStock 2 + uPipette 2 +uFlask 2 0.125106
ucW1=
√
Working solution 2 (100 μg/L) 100 × uW1 2 + uPipette 2 +uFlask 2 1.255156
ucW2=
Calibration (9)-(12) formula reference 0.062371
√
Ethoxyquin Stock solution (1000 mg/L) 1000 × uPurity 2 + uWeight 2 +uFlask 2 12.469570 0.247271
ucStock=
√
Working solution 1 (10 mg/L) 10 × uStock 2 + uPipette 2 +uFlask 2 0.125106
ucW1=
√
Working solution 2 (100 μg/L) 100 × uW1 2 + uPipette 2 +uFlask 2 1.255156
ucW2=
Calibration (9)-(12) formula reference 0.115290
√
Cycloxydim Stock solution (1000 mg/L) 1000 × uPurity 2 + uWeight 2 +uFlask 2 12.469570 0.240852
ucStock=
√
Working solution 1 (10 mg/L) 10 × uStock 2 + uPipette 2 +uFlask 2 0.125106
ucW1=
√
Working solution 2 (100 μg/L) 100 × uW1 2 + uPipette 2 +uFlask 2 1.255156
ucW2=
Calibration (9)-(12) formula reference 0.073556

13
1234 Food Analytical Methods (2023) 16:1227–1236

Table 4  Measurement uncertainties of pymetrozine, ethoxyquin, and cycloxydim

Compounds Uncertainty Standard uncer‑ Relative stand‑ Relative com‑ Combined stand‑ Extended Measurement
factor tainty ard uncertainty posite standard ard uncertainty uncertainty uncertainty
(ur) uncertainty (urc) (uc) (U) (confidence level
about 95%, k = 2)

Pymetrozine Sampling 0.000402 g 0.000040 0.022509 0.231 μg/L 0.47 μg/L 10.27 ± 0.47 μg/L
Total quantity 0.006000 mL 0.000600
Calibration curve 0.231534 μg/L 0.022501
Ethoxyquin Sampling 0.000402 g 0.000040 0.024502 0.247 μg/L 0.50 μg/L 10.08 ± 0.50 μg/L
Total quantity 0.006000 mL 0.000600
Calibration curve 0.247271 μg/L 0.024494
Cycloxydim Sampling 0.000402 g 0.000040 0.022764 0.241 μg/L 0.49 μg/L 10.57 ± 0.49 μg/L
Total quantity 0.006000 mL 0.000600
Calibration curve 0.240852 μg/L 0.022756

Uncertainty in the Calibration Curve
For the calibration curves, 5 concentrations were measured in
triplicate by LC–MS/MS. The correlation coefficient (R2) was
0.9999 for pymetrozine, 0.9998 for ethoxyquin, and 1.0000 for
cycloxydim, and the calibration curve was calculated by the
least squares linear method. The calibration curve uncertainty
was calculated using Eqs. (9)–(12).
Ri = C1 × B1 + B0
and using Eqs. (9)–(12), the standard uncertainties of the
calibration curves were 0.062371 µg/L, 0.115290 µg/L, and
0.073556 µg/L, respectively. The combined standard uncer‑
tainties of the previously calculated stock STD, working
STD1, working STD2, and calibration curve concentrations
were 0.231534 µg/L, 0.247271 µg/L, and 0.240852 µg/L,
respectively, for pymetrozine, ethoxyquin, and cycloxy‑
dim, and the relative standard uncertainties were 0.022501,
0.024494, and 0.022756, respectively.
(9)

√ Combined Standard Uncertainty

2
( ) s 1 1 (x − x)
u C0 = + + (10)
B1 p m Sxx The relative combined standard uncertainties reflecting all
of the samplings, the final quantity, and the calibration curve
�
∑n � �2 standard uncertainties are 0.022509 µg/L, 0.024502 µg/L,
i=1
[Ri − B0 + Bi Ci ] (11) and 0.022764 µg/L, respectively. The combined standard
s=
n−2 uncertainties of pymetrozine, ethoxyquin, and cycloxydim
were 0.231 μg/L, 0.247 μg/L, and 0.241 μg/L, respectively.
∑n 2 The expanded uncertainties were 0.47 μg/L, 0.50 μg/L, and
Sxx = (Ci − C) (12)
i=1 0.49 μg/L, respectively, at a confidence level of approxi‑
where Ri is the measurement of the area of the calibration mately 95%. Additionally, k = 2, which was calculated as
standard; Ci is the concentration of the calibration standard; 4.6%, 5.0%, and 4.6% of the result, respectively. Table 4
B1 is the slope of the calibration curve; B0 is the intercept summarizes the uncertainty of measurement in pymetrozine,
of the calibration curve; p is the number of measurements ethoxyquin, and cycloxydim using the developed analysis
of the determination; m is the number of measurements for method.
the calibration; x is the concentration of compounds for the
calibration; x is the mean value of the different calibration
standards; and i is the index for the number of calibration Conclusion
standards to obtain the calibration curve.
To calculate the uncertainty of measurement, 10 µg/L LC–MS/MS method using ESI + in MRM mode was devel‑
of a standard substance was added to the analysis sam‑ oped for the fast and simultaneous determination of pymetro‑
ple and applied to the development test method, and the zine, ethoxyquin, and cycloxydim in paprika and improved
results were utilized. The calibration curve concentration the accuracy of the corresponding experimental method by
values of pymetrozine, ethoxyquin, and cycloxydim were calculating the uncertainty of measurement. Sweet pepper
10.290 μg/L, 10.095 μg/L, and 10.584 μg/L, respectively, samples mixed with 5 N sodium hydroxide were extracted

13
Food Analytical Methods (2023) 16:1227–1236
with ACN and 1 g of NaCl and 4 g of M ­ gSO4. Verification
was conducted using linearity, sensitivity, selectivity, and
accuracy. As a result, the LOD was measured between 2.0
and 3.9 µg/kg, and the LOQ was between 6.1 and 11.8 µg/
kg. The average recovery rate of the three substances was
97.39–99.42%.
The sampling, total quantity, and calibration curves
were selected as uncertainty factors and measured
for each item. The sampling and total quantity were
0.000402 g and 0.006000 mL for all substances, respec‑
tively, and for the calibration curves, pymetrozine was
0.231534 μg/L, ethoxyquin was 0.247271 μg/L, and
cycloxydim was determined to be 0.240852 μg/L. The
relative standard uncertainties for the three factors were
also the same at 0.000040 and 0.000600 for the sam‑
pling and total quantity, respectively, and for the calibra‑
tion curve, pymetrozine was 0.022501, ethoxyquin was
0.024494, and cycloxydim was 0.022756. The relative
combined standard uncertainties of pymetrozine, ethox‑
yquin, and cycloxydim were 0.022509, 0.024502, and
0.022764, respectively, the combined standard uncertain‑
ties were 0.231 µg/L, 0.247 µg/L, and 0.241 µg/L respec‑
tively, and the expanded uncertainties were 0.47 μg/L,
0.50 μg/L, and 0.49 μg/L respectively. The measure‑
ment uncertainties calculated by combining all of these
were calculated as 10.27 ± 0.47 μg/L for pymetrozine,
10.08 ± 0.50 μg/L for ethoxyquin, and 10.57 ± 0.49 μg/L
cycloxydim.
In conclusion, when the analysis method developed for
pymetrozine, ethoxyquin, and cycloxydim was analyzed, it
was possible to analyze the three substances at the same
time, thus reducing time, and it was judged that the analysis
method was effective through a high accuracy and recovery
rate. In addition, since the test method was conducted in an
appropriate environment through the uncertainty of meas‑
urement calculation and since the results were also signifi‑
cantly calculated, it is expected that the use of this method
to analyze residual pesticide substances in sweet pepper can
contribute to improving food safety. In the generally used
EURL method, pymetrozine, ethoxyquin, and cycloxydim
were individually analyzed. However, in this study, it was
possible to simultaneously analyze three components of
sweet pepper with one pretreatment.
Author Contribution Conceptualization: Hyoyoung Kim and Ho Jin
Kim; methodology: Hyoyoung Kim; software: Sung Ho Jang; valida‑
tion: Sung Ho Jang and Han Yeol Bang; data curation: Ho Jin Kim;
writing—original draft preparation: Sung Ho Jang and Han Yeol Bang;
writing—review and editing: Hyoyoung Kim, Suel Hye Hur, and Eun
Joo Baek; project administration: Ho Jin Kim. All authors have read
and agreed to the published version of the manuscript.
Data Availability All data generated or analyzed during this study are
included in this published article.
1235
Declarations
Ethics Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication Not applicable.
Conflict of Interest Han Yeol Bang declares that he has no conflict of
interest. Sung Ho Jang declares that he has no conflict of interest. Eun
Joo Baek declares that he has no conflict of interest. Suel Hye Hur
declares that he has no conflict of interest. Hyoyoung Kim declares
that he has no conflict of interest. Ho Jin Kim declares that he has no
conflict of interest.
References
Abdelhameed AS, Kadi AA, Attwa MW, AlRabiah H (2019) Validated
LC-MS/MS assay for quantification of the newly approved tyros‑
ine kinase inhibitor, dacomitinib, and application to investigating
its metabolic stability. PLoS One 14(4):e0214598. https://d​ oi.o​ rg/​
10.​1371/​journ​al.​pone.​02145​98
Aktar W, Sengupta D, Chowdhury A (2009) Impact of pesticides use
in agriculture: their benefits and hazards. Interdiscip Toxicol
2(1):1–12. https://​doi.​org/​10.​2478/​v10102-​009-​0001-7
Alder L, Greulich K, Kempe G, Vieth B (2006) Residue analysis of 500
high priority pesticides: better by GC–MS or LC–MS/MS? Mass
Spectrom Rev 25(6):838–865. https://​doi.​org/​10.​1002/​mas.​20091
Anastassiades M, Lehotay SJ, Štajnbaher D, Schenck FJ (2003) Fast
and easy multiresidue method employing acetoni-trile extrac‑
tion/partitioning and “dispersive solid-phase extraction” for the
determination of pesticide residues in pro-duce. J AOAC Int
86(2):412–431. https://​doi.​org/​10.​1093/​jaoac/​86.2.​412
Anastassiades M (2007) QuEChERS validation method for acidic pes‑
ticides. https://w
​ ww.e​ url-​pestic​ ides.e​ u/l​ ibrar​ y/d​ ocs/f​ v/2​ ndtr2​ 007_​
Acidi​cKost​elac.​pdf. Accessed 07 september 2022
Attwa MW, Kadil AA, Abdelhameed AS, Alhazmi HA (2020) Meta‑
bolic stability assessment of new PARP inhibitor talazoparib using
validated LC-MS/MS methodology: in silico metabolic vulner‑
ability and toxicity studies. Drug Des Dev Ther 14:783–793.
https://​doi.​org/​10.​2147/​DDDT.​S2394​58
Calicioglu O, Flammini A, Bracco S, Bellù L, Sims R (2019) The
future challenges of food and agriculture: an integrated analysis
of trends and solutions. Sustainability 11(1):222. https://​doi.​org/​
10.​3390/​su110​10222
Caruso G, Stoleru VV, Munteanu NC, Sellitto VM, Teliban GC, Bur‑
ducea M, Tenu I, Morano G, Butnariu M (2018) Quality per‑
formances of sweet pepper under farming management. Notu‑
lae Botanicae Horti Agrobotanici Cluj-Napoca 47(2):458–464.
https://​doi.​org/​10.​15835/​nbha4​71113​51
Clasen B, Loro VL, Murussi CR, Tiecher TL, Moraes B, Zanella R
(2018) Bioaccumulation and oxidative stress caused by pesticides
in Cyprinus carpio reared in a rice-fish system. Sci Total Environ
626:737–743. https://​doi.​org/​10.​1016/j.​scito​tenv.​2018.​01.​154
da Costa Morais EH, Collins CH, Jardim ICSF (2018) Pesticide deter‑
mination in sweet peppers using QuEChERS and LC–MS/MS.
Food Chem 249:77–83. https://​doi.​org/​10.​1016/j.​foodc​hem.​2017.​
12.​092
Deepa N, Kaur C, Singh B, Kapoor HC (2006) Antioxidant activity
in some red sweet pepper cultivars. J Food Compos Anal 19(6–
7):572–578. https://​doi.​org/​10.​1016/j.​jfca.​2005.​03.​005
Eddleston M, Karalliedde L, Buckley N, Fernando R, Hutchinson G,
Isbister G, Konradsen F, Murray D, Piola JC, Senanayake N,
13
1236
Sheriff R, Singh S, Siwach S, Smit L (2002) Pesticide poison‑
ing in the developing world—a minimum pesticides list. Lancet
360(9340):1163–1167. https://​doi.​org/​10.​1016/​S0140-​6736(02)​
11204-9
EFSA (2014) Conclusion on the peer review of the pesticide risk
assessment of the active substance pymetrozine. EFSA J
12(9):3817, 102. https://​doi.​org/​10.​2903/j.​efsa.​2014.​3817
EURL-SRM (2015) Improvement of ethoxyquin recoveries by QuECh‑
ERS through the addition of ascorbic acid, version 2. http://​www.​
crl-​pesti​cides.​eu/​userf​i les/​file/​EurlS​RM/​EurlS​rm_​Obser​vatio​ns_​
Ethox​yquin_​V2.​pdf. Accessed 07 september 2022
Farrance I, Frenkel R (2012) Uncertainty of measurement: a review of
the rules for calculating uncertainty components through func‑
tional relationships. Clin Biochem Rev 33(2):49–75
Kim K-H, Kabir E, Jahan SA (2017) Exposure to pesticides and the
associated human health effects. Sci Total Environ 575:525–535.
https://​doi.​org/​10.​1016/j.​scito​tenv.​2016.​09.​009
Korea Laboratory Accreditation Scheme (2016) Guide to the evaluation
and expression of uncertainty in measurement. KOLAS-G-002.
http://​www.​kolas.​go.​kr.
Lee SW, Choi J-H, Cho S-K, Yu H-A, Abd El-Aty AM, Shim J-H
(2011) Development of a new QuEChERS method based on dry
ice for the determination of 168 pesticides in paprika using tan‑
dem mass spectrometry. J Chromatogr A 1218(28):4366–4377.
https://​doi.​org/​10.​1016/j.​chroma.​2011.​05.​021
Matthee M (2007) The codex alimentarius commission and its stand‑
ards. Maastricht Univ. https://d​ oi.o​ rg/1​ 0.1​ 007/9​ 78-​90-​6704-​515-5
Moser J, Wegscheider W, Sperka-Gottlieb C (2001) Quantifying the
measurement uncertainty of results from environmental analytical
methods. Fresenius J Anal Chem 370(6):679–689. https://d​ oi.o​ rg/​
10.​1007/​s0021​60100​836
Rao TVR, Gol NB, Shah KK (2011) Effect of postharvest treatments
and storage temperatures on the quality and shelf life of sweet
pepper (Capsicum annum L.). Sci Hortic 132:18–26. https://​doi.​
org/​10.​1016/j.​scien​ta.​2011.​09.​032
Rizzetti TM, Kemmerich M, Martins ML, Prestes OD, Adaime MB,
Zanella R (2016) Optimization of a QuEChERS based method
by means of central composite design for pesticide multiresidue
13
Food Analytical Methods (2023) 16:1227–1236
determination in orange juice by UHPLC–MS/MS. Food Chem
196:25–33. https://​doi.​org/​10.​1016/j.​foodc​hem.​2015.​09.​010
Simonne AH, Simonne EH, Eitenmiller RR, Mills HA, Green NR
(1997) Ascorbic acid and provitamin A contents in unusually
colored bell peppers (Capsicum annuumL.). J Food Compos Anal
10(4):299–311. https://​doi.​org/​10.​1006/​jfca.​1997.​0544
Syed JH, Alamdar A, Mohammad A, Ahad K, Shabir Z, Ahmed H,
Ali SM, Sani SGAS, Bokhari H, Gallagher KD, Ahmad I, Eqani
SAMAS (2014) Pesticide residues in fruits and vegetables from
Pakistan: a review of the occurrence and associated human health
risks. Environ Sci Pollut Res 21(23):13367–13393. https://d​ oi.o​ rg/​
10.​1007/​s11356-​014-​3117-z
Tankiewicz M (2019) Determination of Selected priority pesticides
in high water fruits and vegetables by modified QuEChERS and
GC-ECD with GC-MS/MS confirmation. Molecules 24(3):417.
https://​doi.​org/​10.​3390/​molec​ules2​40304​17
Thammana M (2016) A review on high performance liquid chromatog‑
raphy (HPLC). Res Rev J Pharm Anal 5(2):22–28
Thompson M, Ellison SLR, Wood R (2002) Harmonized guidelines
for single-laboratory validation of methods of analysis (IUPAC
Technical Report). Pure Appl Chem 74(5):835–855. https://​doi.​
org/​10.​1351/​pac20​02740​50835
Toumi K, Joly L, Vleminckx C, Schiffers B (2020) Biological monitor‑
ing of exposure to pesticide residues among Belgian florists. Hum
Ecol Risk Assess Int J 26(3):636–653. https://​doi.​org/​10.​1080/​
10807​039.​2018.​15288​60
Tridge. Global production of other sweet pepper (2020) https://​www.​
tridge.c​ om/i​ ntell​ igenc​ es/o​ ther-​sweet-​pepper/p​ roduc​ tion (accessed
June 20, 2022)
Publisher's Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds
exclusive rights to this article under a publishing agreement with the
author(s) or other rightsholder(s); author self-archiving of the accepted
manuscript version of this article is solely governed by the terms of
such publishing agreement and applicable law.