Food Chemistry 405 (2023) 134755

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evaluation of matrix effects for pesticide residue analysis by QuEChERs

coupled with UHPLC-MS/MS in complex herbal matrix
Xiaoli Wu , Zimian Ding *
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Matrix effects (MEs) can heavily affect the accuracy and reproducibility of a pesticide residue analysis method,
Matrix effect especially in complex matrices such as herbs. Therefore, it is of great importance to assess MEs of pesticides in
Pesticide residue analysis herbal matrices. In this research, the MEs of 28 pesticides and their metabolites in five types of herbs repre­
QuEChERs
senting different medicinal parts were evaluated by UHPLC-MS/MS analysis after QuEChERs pretreatment.
UHPLC-MS/MS
Herbal matrix
Suppression MEs were found for most organophosphorus and carbamate pesticides while enhancement MEs were
observed for sulfonylurea. Besides, stronger inhibition effects were observed for early or late elution pesticides
and in matrices of Lonicerae japonicae flos and Perillae folium. Some pesticides were observed with enhancement
MEs in Astragali radix. These results indicated that MEs were mainly affected by the retention time of the ana­
lytes, the ionization mode of the precursor ions, the overall structure of the compounds, and the chemical
composition of the matrices.

1. Introduction residue analysis (Ferrer, Lozano, Agüera, Girón, & Fernández-Alba,

Herbal medicines are applied worldwide as drugs for preventing and
curing diseases (WHO, 2013). Some herbs, such as Perillae folium and
Lonicerae japonicae flos, are also commonly used as food supplements or
food additives due to the special active components they contain (Lu
et al., 2022). But like other plants, herbs can also be contaminated with
pesticides during agricultural practices such as the use of contaminated
water sources and cultivation in contaminated soil (Luo et al., 2021;
Tripathy, Basak, Varghese, & Saha, 2015). Long exposure to low doses of
some pesticides may pose potential health risks such as endocrine
disruption, infertility, immune suppression, carcinogenic, and terato­
genic effects (Tripathy et al., 2015). For example, the previous study
revealed that pesticides were detected in the majority (99.3 %) of
Lonicerae japonicae flos samples and carbofuran may pose a potential
acute health risk to specific population (Wu, Wang, Gu, Xue, & Wu,
2021). Therefore, it is urgent to develop effective quantitative methods
for monitoring pesticide residue in herbs.
Liquid chromatography combined with tandem mass spectrometry
(LC–MS/MS) is a fundamentally analytical technique for pesticide
2011). However, quantification of trace pesticide residues in complex
matrices such as herbs is still challenging because these matrices have
low water content and a high number of undesired components such as
sugars, phenolics, flavonoids, natural pigments, and essential oils
(Rutkowska, Łozowicka, & Kaczyński, 2019). Co-extraction of these
compounds in the sample may lead to fundamental matrix effects (MEs),
causing matrix interference or signal suppression/enhancement of
target analytes. The phenomenon of MEs prevails in both LC–MS/MSand
gas chromatography-tandem mass spectrometry (GC–MS/MS). The
former usually shows inhibition MEs due to the competition between the
non-volatile components in the matrix (such as fatty acids, glycerates,
salts, etc.) and the analytes, which hinders the formation efficiency of
analyte ions (Panuwet et al., 2016). While the latter usually shows
enhancement MEs due to the blockage of active sites in the capillary
column or the injector liner by matrix components, which improve the
mass transfer of the target analyte (Rahman, EI-Aty, & Shim, 2013). But
the precise mechanisms of MEs are still not fully understood.
The approaches to overcome MEs include improvement of sample
preparation, dilution of sample extracts, use of internal standards, and

Abbreviations: ME, matrix effect; UHPLC-MS/MS, ultra-high performance liquid chromatography-tandem mass spectrometry; QuEChERs, quick, easy, cheap,
effective, rugged, and safe; LC-MS/MS, liquid chromatography-tandem mass spectrometry; GC-MS/MS, gas chromatography-tandem mass spectrometry; MRM,
multiple reaction monitoring; TR, retention time.
* Corresponding author.
E-mail address: zmding@implad.ac.cn (Z. Ding).

https://doi.org/10.1016/j.foodchem.2022.134755
Received 30 June 2022; Received in revised form 9 October 2022; Accepted 23 October 2022
Available online 29 October 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
X. Wu and Z. Ding
performance of matrix-matched calibration (Raposo & Barceló, 2021).
But the available research is mainly involved in fruits and vegetables,
research on complex matrices such as herbs is limited. Rutkowska et al
established a series of methods for minimizing MEs in dried complex
matrices. By using the combination of PSA, ENVI-Carb, and MgSO4 as
cleanup sorbents, MEs were reduced for most pesticides in dried herbs
(Rutkowska, Łozowicka, & Kaczyński, 2017). Under the optimal com­
bination of clean-up sorbent, matrix-matched calibration was proved to
be a feasible method to compensate for MEs of pesticides in complex
seed matrices (Rutkowska, Łozowicka, & Kaczyński, 2020). Besides,
three approaches were also applied to minimize MEs of pesticides in
dried herbs and fruits, and the injection of analyte protectants at the
beginning of the sequence was found to be the most promising (Rut­
kowska et al., 2019). These studies are all focused on MEs by GC–MS/MS
analysis. Since the effects and causes of the two techniques are different,
more research is needed on LC-MS/MS aspect. Moreover, it should be
stressed that MEs can only be reduced to some extent but cannot be
eliminated completely, especially in complex matrices (Rutkowska
et al., 2019). Therefore, it is necessary to explore the contributing pa­
rameters that affect MEs.
MEs are associated with many factors such as the sample preparation
method, the chemical nature of the analytes, the sample matrix, the
elution conditions, and so on (Trufelli, Palma, Famiglini, & Cappiello,
2011). It was found that MEs were more pronounced for early and late
eluting pesticides, the possible reasons may be due to the chromato­
graphic separation or ionization efficiency of the analytes and the loss of
analytes by adsorption in the operating system, respectively (Li, Liu,
Duan, Saint, & Mulcahy, 2015). Matrix fingerprinting showed that
difficult matrices commodities provided a higher number of co-
extracted compounds than other commodities (Gómez-Ramos, Rajski,
Lozano, & Fernández-Alba, 2016). However, it was unfortunate that the
clean-up step showed a slight reduction for commodities with strong
MEs (Kittlaus, Schimanke, Kempe, & Speer, 2012). Another extremely
important factor that affects MEs is the properties of analytes. For
example, nonpolar regions and oxygenated groups in the analytes may
lead to an increase in ionization efficiency in the presence of the co-
eluting matrix and the corresponding signal enhancement (Moham­
med et al., 2020). Although the above-mentioned studies show that ME
depends on many factors such as the conditions of analysis, the types of
matrices, and the properties of analytes, they are seldom involved in
complex matrices such as herbs and lack systematical investigation on
the chemical composition of the pesticides.
The aim of the current study was to evaluate the influence factors of
MEs for pesticide analysis in complex herbal matrices. For the research,
28 kinds of pesticides (including sulfonylurea, organophosphorus,
carbamate, and their metabolites) and five herbs representing different
medicinal parts (root, rhizome, stem, leaf, flower) were selected as the
test subjects. QuEChERs (quick, easy, cheap, effective, rugged, and safe),
the most popular method for pesticide residue analysis in herbs (Rut­
kowska et al., 2017, 2019), was also developed for sample preparation.
The pesticides were determined by an ultra-high performance liquid
chromatography-tandem mass spectrometry (UHPLC-MS/MS). Both
matrix-matched calibration curves and solvent calibration curves were
established for the assessment of MEs.
2. Materials and methods
2.1. Chemicals and reagents
The pesticide mixture standard solutions (2–20 µg/mL), N-propyl
ethylenediamine (PSA), octadecylsilane bonded silica gel (C18), silica
gel, and graphitized carbon black (GCB) were acquired from ANPEL
Laboratory Technologies Inc. (Shanghai, China). HPLC grade acetoni­
trile and formic acid were purchased from Fisher Scientific (Thermo
Fisher Scientific, USA). Analytical reagents of anhydrous sodium acetate
(CH3COONa) and anhydrous magnesium sulfate (MgSO4) were
2
Food Chemistry 405 (2023) 134755
obtained from Sinopharm Chemical Reagent Beijing Co., ltd. (China).
Pinelliae rhizome, Astragali radix, Dendrobii offcinalis caulis, Lonicerae
japonicae flos, and Perillae folium were collected from local commercial
herbal markets and stored at − 18 ◦ C.
2.2. UHPLC-MS/MS determination
Pesticides were detected by a LC-20AD Shimadzu liquid chroma­
tography system (Shimadzu, Tokyo, Japan) coupled with an Applied
Biosystems 5500 triple quadrupole tandem mass spectrometer equipped
with electrospray ionization (ESI) (AB SCIEX, CA, USA). Chromato­
graphic separation was performed on a Hypersil GOLD C18 column
(100 mm, 2.1 mm i.d., 1.9 μm) (Thermo Scientific, CA, USA) with a flow
rate of 0.2 mL/min at 30 ◦ C. The elution solvent was acetonitrile (A) and
0.1 % formic acid in water (B). The gradient elution was shown in
Fig. 1A and performed as follows: 30 % of acetonitrile at 0–0.5 min;
change to 90 % of acetonitrile at 0.5–9 min; maintain 90 % of acetoni­
trile at 9–13 min; return to 30 % of acetonitrile at 13–14 min and hold
for 4 min. The pesticides were analyzed in the positive mode of ESI and
the scheduled multiple reaction monitoring (MRM) parameters are
shown in Table 1.
2.3. Sample preparation
The herbs were fully ground and homogenized before analysis.
Samples were prepared by the following QuEChERs method. Firstly, 3 g
of the sample was weighed into a centrifuge tube (50 mL) and 15 mL of
acetic acid solution (1 %) was added. After soaking for 30 min, aceto­
nitrile (15 mL) was added to the mixture and vortexed for 5 min (500 r/
min). Then a mixture of MgSO4 (6 g) and CH3COONa (1.5 g) was added
to the above mixture. After immediately shaking, cooling in an ice bath
for 10 min, and centrifuging for 5 min (4000 r/min), 9 mL of the su­
pernatant was transferred to a dispersed solid phase extraction purifi­
cation tube containing MgSO4 (900 mg), PSA (300 mg), C18 (300 mg),
silica gel (300 mg), and GCB (90 mg). The purification tube was oscil­
lated and centrifugated, then 5 mL of supernatant was carefully pipetted
and concentrated on a nitrogen blower and diluted to 1.0 mL with
acetonitrile. Finally, 2 μL of test solution was injected into the system for
analysis.
2.4. Evaluation of matrix effect (ME)
Both matrix-matched calibration curves and solvent calibration
curves were established for the assessment of MEs. Solvent standards
were prepared by diluting the stock mixture standard solutions (0.1–1
µg/mL) with acetonitrile to 1–10 ng/mL, 2–20 ng/mL, 5–50 ng/mL,
10–100 ng/mL, and 20–200 ng/mL, respectively. The matrix-matched
standards were obtained to the same concentrations by diluting with
the blank matrix solution.
ME was calculated by the following equation: ME (%) = (slope in
matrix/slope solvent in matrix − 1) × 100 %. Signal suppression would
occur if ME < 0 while signal enhancement would occur if ME > 0. |ME|
> 50 % means strong ME and 20 % <|ME|≤ 50 % indicates medium ME.
|ME|≤ 20 % is considered negligible due to the shift between repeat­
ability values (Ferrer et al., 2011; Raposo & Barceló, 2021).
3. Results and discussion
3.1. Effect of retention time
The gradient elution method is usually used for the separation and
determination of multiple pesticides. It was reported that MEs of pesti­
cides were strongly affected by the elution conditions since co-
extractives were separated within the same chromatographic gradient
(run) (Kittlaus et al., 2012). Therefore, the effect of retention time (TR)
of the analytes was also discussed in this research.
X. Wu and Z. Ding Food Chemistry 405 (2023) 134755

Fig. 1. (A) Gradient elution for the separation and determination of the pesticides. (B) elution time and MEs of the pesticides in five matrices throughout the
UHPLC run.

Table 1
Chemical class, retention time (TR), Molecular mass, parent ion, quantification ion pair, confirmation ion pair of the pesticides.
No. Pesticide Chemical class TR Molecular Parent ion Quantification ion pair CE Confirmation ion pair CE DP
(min) mass (m/z) (V) (m/z) (V) (V)

1 Methamidophos Organophosphate 1.40 141.13 [M + H]+ 142.0＞94.0 19 142.0＞125.0 18 54

2 Aldicarb-sulfoxide Carbamate 1.42 206.26 [M + H]+ 207.0＞132.0 10 207.0＞89.0 20 51
3 Monocrotophos Organophosphate 1.64 223.16 [M + H]+ 224.1＞193.0 14 224.1＞127.0 21 71
4 Aldicarb-sulfone Carbamate 1.81 222.26 [M + H]+ 223.0＞148.0 11 223.0＞76.0 15 60
5 Carbofuran-3- Carbamate 2.48 237.25 [M + H]+ 238.1＞181.1 14 238.1＞163.0 20 50
hydroxy
6 Phosfolan Organophosphate 3.30 255.3 [M + H]+ 256.2＞61.0 4 256.2＞139.9 22 51
7 Fenamiphos- Organophosphate 3.84 319.36 [M + H]+ 320.1＞233.0 34 320.1＞292.0 21 80
sulfoxide
8 Phosphamidon Organophosphate 4.07 299.69 [M + H]+ 300.0＞174.0 19 300.0＞127.0 36 75
9 Aldicarb Carbamate 4.27 190.26 [M + 208.1＞116.0 11 208.1＞89.0 20 34
NH4]+
10 Fenamiphos-sulfone Organophosphate 5.14 335.36 [M + H]+ 336.1＞266.0 28 336.1＞308.1 20 80
11 Metsulfuron-methyl Triazinylsulfonylurea 5.14 381.36 [M + H]+ 382.1＞167.1 18 382.1＞141.0 22 60
12 Carbofuran Carbamate 5.46 221.26 [M + H]+ 222.1＞165.0 17 222.1＞123.0 28 40
13 Chlorsulfuron Triazinylsulfonylurea 5.55 357.77 [M + H]+ 358.0＞141.0 20 358.0＞167.0 13 60
14 Ethametsulfuron- Triazinylsulfonylurea 5.71 410.41 [M + H]+ 411.1＞196.1 23 411.1＞168.1 35 49
methyl
15 Phorate-sulfoxide Organophosphate 5.78 276.38 [M + H]+ 277.0＞199.0 10 277.0＞97.0 25 55
16 Terbufos-sulfoxide Organophosphate 6.88 304.43 [M + H]+ 305.1＞187.0 10 305.1＞153.0 15 60
17 Phorate-sulfone Organophosphate 7.18 292.22 [M + H]+ 293.0＞247.0 10 293.0＞115.0 30 50
18 Isocarbophos Organophosphate 7.18 288.0 [M + 312.1＞270.0 20 312.1＞236.1 25 90
Na]+
19 Fenamiphos Organophosphate 7.58 303.36 [M + H]+ 304.1＞217.1 21 304.1＞234.0 18 77
20 Ethoprophos Organophosphate 7.87 242.3 [M + H]+ 243.0＞131.0 25 243.0＞173.0 16 51
21 Terbufos-sulfone Organophosphate 8.00 320.43 [M + H]+ 321.1＞143.0 20 321.1＞171.0 18 75
22 Isazafos Organophosphate 8.74 313.74 [M + H]+ 314.1＞120.0 25 314.1＞162.1 18 55
23 Cadusafos Organophosphate 9.26 270.39 [M + H]+ 271.0＞159.0 19 271.0＞215.0 11 65
24 Coumaphos Organophosphate 9.26 362.62 [M + H]+ 363.0＞227.0 35 363.0＞307.0 22 80
25 Isofenphos-methyl Organophosphate 9.30 331.37 [M + H]+ 332.0＞273.0 10 332.0＞231.0 30 50
26 Fonofos Organophosphate 9.46 246.3 [M + H]+ 247.0＞109.1 25 247.0＞137.0 20 50
27 Sulfotep Organophosphate 9.51 322.32 [M + H]+ 323.0＞171.1 21 323.0＞115.0 33 70
28 Phorate Organophosphate 9.60 260.4 [M + H]+ 261.0＞75.0 19 261.0＞47.0 49 50

Fig. 1A shows the gradients for infusion experiments and the elution
zone of the analytes. The mobile phase was with 30 % acetonitrile at the
beginning, reached high organic concentrations (90 % acetonitrile) at
the stage of gradient elution, and adjusted to the starting conditions (30
% acetonitrile) at the final stage. It can be seen from Fig. 1A that the
pesticides are eluted in the region of 1.4 ~ 9.6 min and the retention
time (TR) for the pesticides is listed in Table 1.
Fig. 1B shows TR distribution of the pesticides and their corre­
sponding ME in the five matrices throughout the gradient run. It is
known that compounds with greater polarity eluted earlier while that
with less polarity eluted later during LC separation on a C18 column.
Strong signal suppression (ME > 50 %) was observed for meth­
amidophos (TR = 1.40 min), aldicarb-sulfoxide (TR = 1.42 min),
monocrotophos (TR = 1.64 min), isocarbophos (TR = 7.18 min),
isofenphos-methyl (TR = 9.3 min), fonofos (TR = 9.46 min), sulfotep (TR
= 9.51 min), and phorate (TR = 9.6 min) in most matrices. This indi­
cated that both early and late eluting pesticides were observed with
strongly signal decrease, which was identical with that in the literature
(Li et al., 2015). The suppression effects of the early-eluting pesticides
(methamidophos, aldicarb-sulfoxide, and monocrotophos) may be due
to the co-eluting of highly polar coexistence compounds on the reversed-
phase column, which may affect the ionization efficiency of the target
analyte (Kittlaus et al., 2012). Another reason could be that lower
organic content at the initial eluting time may affect the ESI ionization
(Li et al., 2015). The decrease effects of late eluting pesticides (iso­
fenphos-methyl, fonofos, sulfotep, and phorate) may be due to certain

3
X. Wu and Z. Ding
specific surface adsorption in the system (such as sample loop, sample
vial, and flow patch) during the detection process rather than electro­
spray or ionization efficiency (Li et al., 2015). In addition to the above
results, most of the pesticides in the middle elution region (2.48 ~ 9.26
min) were observed with moderate MEs except isocarbophos, which
indicates it may be strongly influenced by other factors.
Similar trends were observed in diverse herbs except for that stron­
ger suppression MEs were observed in Lonicerae japonicae flos and Per­
illae folium than in other matrices, especially for early or late elution
pesticides. The results indicated that MEs of the pesticides may be sus­
ceptible to these two matrices. Besides, Astragali radix was found to have
apparent signal enhancement effects on metsulfuron-methyl (TR = 5.14
min), chlorsulfuron (TR = 5.55 min), and ethametsulfuron-methyl (TR =
5.71 min), which may be attributed to some special components it
contains.
Although the retention time of the pesticides has a significant impact
on their MEs, modification of a well-established chromatographic con­
dition is not recommended. It is because slight modification may lead to
time drift of the analytes and co-eluting substance, which may also result
Fig. 2. Chemical structures of representative pesticides.
4
Food Chemistry 405 (2023) 134755
in completely new intensities (Kittlaus et al., 2012).
3.2. Effect of the chemical structure of the analytes
The chemical nature of the compounds, such as molecular polarity,
molecular weight, and functional groups may be another key factor that
affects the ME (Trufelli et al., 2011). 28 kinds of pesticides and their
metabolites, including organophosphorus pesticides, carbamate pesti­
cides, and sulfonylurea herbicides, were investigated in this study. The
structures of representative compounds are presented in Fig. 2 and
categorized into four main groups: (A) primary amine (–NH2), (B) sul­
fonylurea, (C) carbamate, and (D) organophosphorus.
It can be seen from Fig. 1B that methamidophos (TR = 1.40 min) and
isocarbophos exhibit stronger inhibitory effects (ME < − 50 %) in all
matrices and the value of isocarbophos (TR = 7.18 min) in Perillae folium
is even up to − 86.9 %. Coincidentally, only the two pesticides have the
group of –NH2 (Fig. 2A). Although amino was considered to be a func­
tional group that interacted with the active surfaces and caused signal
enhancement/suppression in GC system (Saka et al., 2013), it may not
X. Wu and Z. Ding
be the major factor in LC-MS analysis. As concluded in 3.1, the stronger
depression effect of methamidophos is related to its earlier elution time
but that of isocarbophos may be attributed to other factors. It is observed
in Table 1 that the parent ion of isocarbophos is [M + Na]+ (m/z =
312.1) instead of [M + H]+. It may be because the occurrence of sodium
adducts is susceptible to sample composition since small changes may
derive from the major analyte signal drifting and differences in the mass
spectrum (Lara-Ortega, Robles-Molina, Brandt, Schütz, & Franzke,
2018). In conclusion, the presence of –NH2 has effects on the polarity
and ionization mode of the analytes in LC-MS/MS analysis, which may
result in a strong matrix suppression effect.
It was worth noticing that 3 sulfonylurea herbicides (mesulfuron,
aminosulfuron, and chlorsulfuron) (Fig. 2B) showed rare signal
enhancement effects, with the mean value of 14.5 %, 17.6 %, and 3.3 %,
respectively. This phenomenon has also been reported by other authors,
but the hypotheses to explain the signal enhancement are still lacking
(Mohammed et al., 2020; Trufelli et al., 2011). One reason may be that
the presence of non-polar regions in the molecular structure, such as
phenyl and triazinyl, oxygenated group, and carbonyl group, increase
the surface activity in resulting ESI + droplets and the ionization effi­
ciency (Chawla et al., 2017; Mohammed et al., 2020). The other reason
may be that the matrix components can act as dopants and increase the
ionization efficiency of analytes with high ionization energy (Steiner,
Krska, Malachova, Taschl, & Sulyok, 2020). In addition, chlorsulfotron
showed less signal enhancement compared with methyl sulfotron and
aminosulfotron, and even signal depression in Dendrobii offcinalis caulis
and Perillae folium. It may because that the substituent group of –COO- in
the molecular structure is displayed by a highly electronegative group of
Cl, which decreases the ionization efficiency (Chawla et al., 2017;
Mohammed et al., 2020).
Carbamate pesticides (Fig. 2C), which contain the group of -O-CO-
NH-, were observed with weak or moderate matrix inhibition effect in
the five herbs except for aldicarb sulfoxide. Both aldicarb-sulfoxide (TR
= 1.42 min) and aldicarb-sulfone (TR = 1.81 min) are oxidation me­
tabolites of aldicarb (TR = 4.27 min). The change in eluting time indi­
cated that oxidation greatly increased the polarity of the analytes. As
already mentioned in 3.1, the strong signal inhibition of aldicarb sulf­
oxide may be due to its short retention (TR = 1.42 min). However,
aldicarb-sulfone, which has a similar eluting time (TR = 1.81 min), was
observed with a medium inhibition ME. It may be because co-eluting
matrix compounds were not a broad band but narrow peaks in the
chromatography (Kittlaus et al., 2012). It was worth noting that
although aldicarb-sulfone and aldicarb differ in TR, they presented
similar inhibition percentages of MEs. In addition, carbofuran-3-
hydroxy exhibited a larger signal inhibition effect than carbofuran. It
may be because the presence of the –OH group in the molecular struc­
ture affected the ionization efficiency of the analytes (Trufelli et al.,
2011).
Most organophosphorus pesticides show moderate inhibition MEs in
the five herbs. Three types of pesticides were selected to investigate the
relationship between MEs and chemical structure: phorate, terbufos,
fenamiphos, and their metabolites (Fig. 2D). It can be drawn from TR
that, similar to aldicarb and its metabolites, oxidation also greatly in­
creases the polarity of organophosphorus pesticides. Signal depression
effects were all observed for phorate (TR = 9.6), phorate-sulfone (TR =
7.18 min), and phorate-sulfoxide (TR = 5.78 min), with average values
of − 44.8 %, − 23.6 %, and − 28.0 %, respectively. As referred to in 3.1,
the stronger inhibiting effect of phorate was mainly owing to its longer
TR. Terbufos-sulfone (TR = 8.00 min) and terbufos-sulfoxide (TR = 6.88
min) are the homologs of phorate-sulfone (TR = 7.18 min) and phorate-
sulfoxide (TR = 5.78 min), respectively. The average depression MEs of
the former two were − 20.3 % and − 23.4 %, and the latter were − 23.6 %
and − 28.0 %. This indicated that depression MEs decreased as the car­
bon chain increased. It may be due to the alkyl group has electron-giving
group capacity and the electron-giving capacity of -C(CH3)3 is greater
than that of –CH3, which increases the nonpolarity of the molecular
5
Food Chemistry 405 (2023) 134755
structure. Phenylphosphos and its metabolites showed relatively weak
depression MEs with the mean value of − 22.2 % (fenamiphos), − 10.5 %
(fenamiphos-sulfone), and − 4.7 % (fenamiphos-sulfoxide), respectively.
This, like sulfonylurea herbicides, may also be ascribed to the presence
of the non-polar region of phenyl in the analytes.
After the upwards analyzing, the following conclusions can be
drawn: (1) excluding the effects of elution time, the majority of carba­
mate and organophosphorus are observed with weak to moderate in­
hibition MEs, while sulfonylurea herbicides are observed with
enhancement MEs; (2) the presence of nonpolar groups in the molecular
structure such as phenyl, carbon chain may reduce the degree of sup­
pression MEs of the analytes, while the polar groups such as the group of
–OH and -Cl increase suppression MEs; (3) the occurrence of strong
signal suppression is not only due to the early or late elution time of the
analytes but also the ionization mode of parent ion since sodium adduct
[M + Na]+ is susceptible to the matrix.
3.3. Effects of herbal matrices
In HPLC-MS/MS analysis, matrix components can affect the ion in­
tensity of a target analyte by interfering with its ionization or competing
with the target analyte for the available charges (Panuwet et al., 2016).
Therefore, the matrix is one of the principal factors that affect the MEs of
pesticides. Dried herbs have more significant MEs due to their low water
content, high number of co-extractives, and special active components
such as sugars, phenolics, flavonoids, natural pigments, and essential
oils (Rutkowska et al., 2019). MEs in five representative herbs were
investigated in this study and the results are presented in Fig. 3. The
herbs are Pinelliae rhizome, Astragali radix, Dendrobii offcinalis caulis,
Lonicerae japonicae flos, and Perillae folium and their medicinal parts are
rhizome, root, stem, flower, and leaf, respectively.
Fig. 3A indicates that the majority of the pesticides are influenced by
signal suppressions (ME < 0) in all tested matrices. The boxplot of the
ME data shows that Pinelliae rhizome, Astragali radix, and Lonicerae
japonicae flos have relatively low dispersion and inhibition level with
average value of –22.8 %, − 17.6 %, and − 25.5 %, while Lonicerae
japonicae flos and Perillae folium have relatively high dispersion and in­
hibition level with the average value of − 36.7 % and − 41.8 %.
Fig. 3B shows that the weak, medium, and strong MEs are in the
range of 14 ~ 46 %, 46 ~ 61 %, and 7 ~ 39 %, respectively. The per­
centages of moderate ME in Pinelliae rhizome, Astragali radix, and Perillae
folium are all 46 %. Of the five evaluated matrices, Pinelliae rhizoma was
the one with the least ME, as 46 % of the tested pesticides presented
weak ME and only 7 % of the pesticides presented strong ME. On the
other hand, Perillae folium exhibited the highest ME, with 14 % of
compounds having weak ME and 39 % having strong ME. Similar to
Pinelliae rhizoma, Astragali radix also presented smaller ME with 43 % of
pesticides exhibiting weak ME and 11 % exhibiting strong ME. In Den­
drobii offcinalis caulis, 61 % of pesticides showed medium ME, 32 %
showed weak ME, and the rest showed strong ME (7 %). In Lonicerae
japonicae flos, half of the pesticides (50 %) are affected by medium ME,
weak ME (21 %), and strong ME (29 %) expected the other half.
In comparing the matrices, it can be concluded that stronger MEs
were observed in Lonicerae japonicae flos and Perillae folium. It is known
that signal suppression may be associated with the co-elute components
such as volatiles, pigments, sugars, and other interfering species, which
affect the chromatographic separation and ionization process of target
analytes (Chawla et al., 2017; Mohammed et al., 2020). It can be seen
from Fig. 3C that the extracts of Perillae folium and Lonicerae japonicae
flos are rich in pigment compared with Astragali radix and Pinelliae rhi­
zoma. Therefore, pigments in the matrices may be one factor that results
in matrix suppression effects. But this is just a speculation and still needs
to be proved by more tests.
X. Wu and Z. Ding Food Chemistry 405 (2023) 134755

Fig. 3. (A) box plot of MEs of the pesticides in herbal matrices (B)Number and percentage of pesticide for weak, medium, and strong MEs in herbal matrices (C)
Acetonitrile extract of Pinelliae rhizome (a), Astragali radix (b), Dendrobii offcinalis caulis(c), Lonicerae japonicae flos (d), and Perillae folium (e).

3.4. Relative MEs of analytes in herbal matrices

As can be concluded that MEs were rather different in diverse

matrices, and this led to the variability of analytical results between
different types of matrices. However, the value of ME near zero is not
necessary to obtain reliable results. In addition to the above mentioned
that an absolute ME of<20 % represents a lack of significance, analytical
response variability in samples from different donors/sources below 15
% represents a lack of significant ME (Kasperkiewicz, Lendor, & Paw­
liszyn, 2022; Panuwet et al., 2016; Raposo & Barceló, 2021). Therefore,
relative MEs are expressed as relative stand deviation (RSD) of slopes of
matrix-matched calibration curves constructed in tested herbs spiked
with pesticide standards. The calibration curves of representative pes­
ticides in solvent and five herbs are shown in Fig. 4.
It can be seen from Fig. 4 that 43 % of the pesticides (12 out of 28)
resulted in RSD values of slopes<15 % in the five matrices. The orders
are: phorate-sulfoxide (7.8 %), phosphamidon (10.0 %), chlorsulfuron
(10.6 %), terbufos-sulfoxide (10.9 %), ethametsulfuron-methyl (12.2
%), cadusafos (12.8 %), carbofuran (13.0 %), fenamiphos-sulfone (13.0
%), phorate-sulfone (13.4 %), ethoprophos (14.0 %), terbufos-sulfone
(14.2 %), and metsulfuron-methyl (14.5 %). These pesticides are
considered not affected by intra-matrix variations. It was a coincidence
that lower relative MEs were mainly concentrated amid in the elution
zone (from 4 min to 8 min). Therefore, elution time may be one of the
major factors that affect relative MEs. Special consideration is required
for the other pesticides which have larger relative MEs. Variability be­
tween matrices or batches from different origins should not be negligible Fig. 4. Relative matrix effects of pesticides in five herbs.
for these pesticides. For example, the higher variability is observed for
isofenphos-methyl (41.8 %, Pesticide No. 25) and fenamiphos (38.8 %,
The matrix-matched calibration curves and solvent calibration
Pesticide No. 19), which indicates that special attention should be paid
curves of four typical pesticides (isofenphos-methyl, phosphamidon,
for the analysis of them in different matrices.
fenamiphos, and isazafos) are shown in Fig. 5. As for isofenphos-methyl

6
X. Wu and Z. Ding
Fig. 5. Matrix-matched calibration curves in herbal matrices of (A) isofenphos-methyl, (B) phosphamidon, (C) fenamiphos, and (D) isazafos.
(Fig. 5A), the biggest inhibiting effect was observed in Perillae folium and
the smallest inhibiting effect was observed in Pinelliae rhizome. Similar
result was also observed for phosphamidon (Fig. 5B), but with negligible
intra-matrix variations (RSD < 15 %).
Both fenamiphos (Fig. 5C) and chlorsulfuron (Fig. 5D) were observed
with enhancement MEs in Astragali radix. And this phenomenon was also
observed for other pesticides such as ethametsulfuron-methyl, metsul­
furon-methyl, and fenamiphos-sulfone. This indicated that some special
components in Astragali radix may affect the MEs of these pesticides.
Calycosin, which was reported to be one of the main active compounds
in Astragali radix, may be one of the components due to the non-polar
region such as phenyl it contains (Lu et al., 2022). However, more
research is needed to confirm the speculation.
4. Conclusion
In this study, five herbs representing different medicinal parts were
taken as complex matrices to evaluate the MEs of 28 pesticides and their
metabolites by UHPLC-MS/MS analysis. Inhibition MEs were observed
for most pesticides. Stronger inhibitions were observed for pesticides of
early or late elution time and matrices of Lonicerae japonicae flos and
Perillae folium. On the other hand, enhancement MEs were observed for
pesticides of sulfonylurea and matrices of Astragali radix. The presence
of nonpolar groups such as phenyl and alkyl in chemical structure may
reduce the inhibition MEs while polar groups such as hydroxy and
chlorine increase inhibition effects. Primary and secondary metabolites
in herbs may also lead to the increase or decrease of the inhibition MEs,
7
Food Chemistry 405 (2023) 134755
but further studies are required to confirm the composition and probable
cause. We do hope this study can help explore the occurrence mecha­
nisms of ME and develop approaches for reducing or compensating ME.
CRediT authorship contribution statement
Xiaoli Wu: Methodology, Resources, Investigation, Writing – orig­
inal draft, Funding acquisition, Conceptualization. Zimian Ding:
Methodology, Resources, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This work was funded by the CAMS Innovation Fund for Medical
Sciences (CIFMS, 2022-I2M-1-017) and the National Natural Science
Foundation of China (No. 81803721).
X. Wu and Z. Ding Food Chemistry 405 (2023) 134755

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