Development and validation of method for

determination of organophosphorus pesticides

traces in liver sample by GC-MS/MS-ion trap

Acta Chromatographica ISKRA BONEVA1, SPASKA YANEVA2 and

DANCHO DANALEV2p
33 (2021) 2, 188–194
DOI: 1
10.1556/1326.2020.00778 Central Laboratory of Veterinary Control and Ecology (CLVCE), Bulgarian Food Safety Agency, 5
© 2020 The Authors Iskarsko Shousse Str, Sofia, Bulgaria
2
University of Chemical Technology and Metallurgy, 8 blvd. Kliment Ohridski, Sofia, 1756, Bulgaria

Received: March 27, 2020 • Accepted: May 03, 2020

Published online: July 17, 2020

ORIGINAL RESEARCH
PAPER ABSTRACT
A method for simultaneous determination of trace of four organophosphorus pesticides residues in
animal liver samples has been developed and validated. This method is based on the preliminary sample
preparation using extraction of target compound with a mixture of toluene-cyclohexane by means of
up-to-date accelerated solvent extraction (ASE), liquid-liquid partitioning with acetonitrile and hexane,
additional clean up step using QuEChERS method. Further the obtained analytes are determined by gas
chromatography with ion-trap detector. The validation of the method is performed in accordance with
the recommendations in Document SANTE/11945/2015 and it meets the acceptability criteria for
precision, mean recovery and limits of quantification. The samples were investigated by analysing blank
liver samples and samples spiked with the target analytes chlorpyrifos-methyl, parathion and pir-
imiphos-methyl at levels of 25, 50, and 75 ng/g and with diazinon at levels of 15, 30, and 45 ng/g. The
recovery for all compounds were in the range from 73 to 104% which perfectly fit with requirements of
documents and European legislations. The repeatability and within-laboratory reproducibility also
reveal acceptable in documents coefficient of variation and uncertainty less than 20 and 18%, respec-
tively. The limits of quantification were less than 3 ng/g for all compounds and allowed determination
of residues below the maximum residue levels (MRLs) set in Regulation (EC) Nº 396/2005.

KEYWORDS
organophosphorus pesticides, GC-MS/MS ion-trap, food analysis, liver samples

INTRODUCTION
Pesticides are organic molecules largely used in agriculture for plant support during the
process of cultivation [1]. They include wide class of compounds but the most prominent for
plant treatment are chlorine and phosphor containing molecules as well as N-methyl-
carbamade. Organophosphorus pesticides (OPPs) are of a large interest because together with
N-methylcarbamates they slowly replace organochlorines due to the established bio-
accumulation of the latter. Typically OPPs are amides, esters or thiol derivatives of phos-
phoric or phosphonic acid. They are easily hydrolysed and thus do not persist in the
environment for a very long time. However, their various toxicity (they act as inhibitors of
*Corresponding author. acetylcholinesterase enzyme) as well as possibility for accumulation in the food chain can
Tel.: þ359 2 8163310. cause a risk for human health [2–4].
E-mail: [email protected]
The presence of these compounds in the food have led to established maximum residue
levels (MRLs) in or on food and feed of plant and animal origin are set at Regulation (EC) Nº
396/2005. All Member States must take and analyse samples for needs of National Moni-
toring Plan according to Council Directive 96/23/EC of 29 April 1996 on measures to

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Acta Chromatographica 33 (2021) 2, 188–194 189

monitor certain substances and residues thereof in live an- residues in liver and determination by GC-MS/MS which to
imals and animal products. On the other hand concerning a reflect to an appropriate final prize of proposed analysis – a
coordinated multiannual control program of the Union the very important part for consumers of this service. The fat
pesticide in food are established in the Commission imple- content in liver is the most important step of sample prep-
menting regulation (EU) 2018/555 [5]. In order to be able to aration appears to be clean up. Liquid-liquid partition is
carry out the monitoring programs, it is necessary for lab- appropriate method for fat removal [16], but additional
oratories to create a really applicable method in laboratory clean step is needed because of remaining fat precipitation in
practice for analysis of OPPs which meets the requirements the defatted extract. The liquid-liquid partition and
of European legislation. In this respect the laboratories QuEChERS methodology, proposed in this paper, is inde-
developed and validate own methods. pendent of special equipment and also makes the handling
Nowadays, rapid methods for determination of pesticides with the samples very easy. QuEChERS methodology (used
are very preferable. Biosensors are widely used as they are clean-up steps) is quick, easy, cheap, effective, but it has been
quick and easy to use. At the same time they offer quick applied with success on several non-fatty and low-fatty food
response and can be validated with appropriate parameters matrices. Recently modified QuEChERS methodology has
according to the legislation, comparable with chromato- been applied in high fatty food as meat and fish as additional
graphic techniques [6, 7]. The main disadvantage of bio- cleanup step with combination with liquid-liquid partition-
sensors is that they report results for entire group of ing [17, 18].
pesticides presented in the sample, but not for individual
target analyte.
The process of analytical method developing include two MATERIAL AND METHODS
main steps: sample preparation (including matrix pretreat-
ment) and analytical determination of target compounds.
Traditionally, the preliminary treatment of complex samples, Solvents and reagents
such as liver, is carried out in a sequence of extraction and Hexane, cyclohexane, acetonitrile, toluene (gradient grade)
clean-up steps. Ledoux summarized that the most widely used and anhydrous sodium sulfate were supplied by Supelco
pesticide extraction technique from foods of animal origin are: (USA). The sodium sulfate was heated overnight at 600 8C
solid–liquid extraction, traditional Soxhlet extraction method, and then 1% distilled water was added. Silica gel (obtain
accelerated solvent extraction (ASE), microwave-assisted from Merck, Germany), 70–30 mesh, was also heated
extraction, matrix solid-phase dispersion and the clean-up overnight at 600 8C, and deactivated with 5% distilled water.
steps are mainly performed by: freezing centrifugation, liquid– Magnesium sulfate (reagent grade, obtain from Merck,
liquid partitioning, gel permeation chromatography (GPC) Germany), DSC-18 (obtain from Merck, Germany), primary
and solid-phase extraction [8]. and secondary amine (PSA) bulk, from Supelco (USA)
Nowadays, QuEChERS (quick, easy, cheap, effective, Helium (99,999% purity), is used as carrier gas.
rugged and safe) methods are developed and widely used for
extraction and clean up procedure of food samples. The
ruggedness characteristics of the QuEChERS approach have
Analytical standards
been thoroughly evaluated in the original [9] and several Chlorpyrifos-methyl (98% purity); Parathion (99% purity);
more publications [10–13]. All these steps during the sample Pirimiphos-methyl (99% purity); and Diazinon (99%. pu-
preparation, aid the instrumental analyses – the cleaner the rity) All of them were purchased from Dr Ehrenstorfer
extract, the lower the determined concentration by the de- (Germany), Mirex- (purity>96%) is obtained from Sigma
tector [14]. Aldrich, Germany. Primary stock solutions of each analyte
Therefore, a modified QuEChERS method, which can be were prepared in hexane at a concentration of 1,000 mg/mL,
applied for liver, would be appropriate alternative for labo- intermediate single standard solutions were prepared in
ratory’s routine work, to save time and money which is hexane at a concentration of 1 mg/mL and the multi-stan-
extremely important for the final prize of proposed analysis. dard solution is also prepared in hexane with concentrations
Also QuEChERS AOAC 2007.01 was selected as the most of all pesticides was prepared by appropriate dilutions.
suitable protocol for routine determination of residues of 16 These mixed working standard solutions with appro-
pesticides most commonly used for fruits treatment during priate OPPs levels were used for making calibration curve,
the citrus production through liquid chromatography studying linearity and spiking blank liver samples. Mirex
coupled to tandem mass-spectrometry (LC–MS/MS) [15]. internal standard solution of 1 mg/mL.
After the extraction and purification procedures, pesticides
need to be separated and further determined. The GC
Equipment
coupled with mass spectrometric detectors, including single
quadrupole, ion trap, and triple quadrupole mass-spec- Gas chromatograph (Varian 431-GC, USA), Ion Trap Mass
trometers, has been adapted for analysis of pesticide residues Spectrometer (Varian 220 MS, USA), capillary column DB-5
in foods of animal origin [8, 16]. MS (Agilent J&W, USA) (length of 30 m, 0.25 mm internal
The aim of this study is focused on the development of diameter, film thickness 0.25 mm), extraction of samples –
easy and quick method for extraction and cleans up of OPPs ASE technique using Dionex ASE 300 extractor., Analytical

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190 Acta Chromatographica 33 (2021) 2, 188–194

Table 1. Identification data of target OPPs by GC-MS/MS

Retention Precursor Excitation
Compounds Mw [g/mol] time [min] ion [m/z] Product ion [m/z] amplitude [V]
chlorpyriphos- 350.59 18,501 286 101; 139; 223 89
methyl
parathion 291.26 21,056 292 136; 144; 233 95
pirimiphos-methyl 305.33 19,786 306 152; 180 100
diazinon 304.34 16,678 305 121; 145 98

balances (Sartorius, PB 221 S and Sartorius, PB 610), Digital with flow rate of 1 mL/min. Transfer line and ion trap tem-
ULTRA-TURRAX (IKA 3565001 T 25). peratures was 280 and 150 8C respectively.

Method validation
ANALYTICAL METHOD
The liver samples free from pesticide residues were used as
blank material for validation procedures. The validation is
Sample preparation performed according to the recommendations in procedure
of SANTE/12682/2019 [19]. The calibration curve was pre-
Extraction. Liver is homogenizing by means of ultra-turrax pared, using blank material spiked at six levels corre-
IKA T25 before extraction. Weigh 10 g of the homogenized sponding to concentrations between 0 and 2 MRLs for all
liver sample, add about 5 g of anhydrous sodium sulfate, analytes. Blank liver samples spiked with OPPs at the levels
comminute thoroughly in a mortar and then transfer into corresponding to 0.5, 1.0 and 1.5 MRLs (25, 50, 75 ng/g for
cell of ASE for the extraction. The total extraction is per- chlorpyrifos-methyl; parathion, pirimiphos-methyl and 15,
formed using 135 mL solvent per sample (toluene/cyclo- 30, 45 ng/g for diazinon) were used for evaluation of re-
hexane 50/50 (v/v) at 100 8C and pressure 1,500 psi. The covery, repeatability, within-laboratory reproducibility and
collected extract are transferred into a flask and rotary- uncertainty. Spiked samples at each concentration level were
evaporated the solvents. analysed in three series, each on the different day, and each
in six replicates. The obtained validation parameters are
Liquid-liquid partitioning with acetonitrile and hexane. The presented in Table 1.
concentrated extract is carefully transferred with 5 ml of Regression curve for all pesticides showed good linearity
hexane to a 50 mL volumetric flask and mixed well. Add 50 with determination coefficients (R2) values of at least 0.98.
mL internal standard (Mirex) from 1 mg/mL stock solution The proportion of analyte remaining at the point of the final
and wait 5 min. Afterwards, a double extraction with determination, following its addition (usually to a blank
acetonitrile is done as follows: add 5 mL of acetonitrile and sample) immediately prior to extraction. Usually expressed
vortex for about 2 min, centrifuge for 2 min at 2,000 rpm as a percentage [19]. Average recovery was calculated by
for better separation of the layers (hexane and acetonitrile). comparing the determined concentrations of spiked samples
Acetonitrile extracts were combined into 15 mL centrifuge to the target level. The reported uncertainty was an
tube and evaporated to dryness under nitrogen at 50 8C expanded uncertainty with a coverage factor of 2 to give a
water bath. level of confidence of approximately 95%, in accordance
with the recommendations of [20]. The limit of quantifica-
Additional purification step with QuEChERS method. The tion (LOQ) is determined as the lowest concentration level
extract after evaporation was dissolved with 2 mL acetoni- that can be validated with acceptable values for recovery and
trile and 100 mg PSA, 400 mg MgSO4, and 100 mg DSC-18 precision. The requirement for LOQ at [19] is LOQ ≤ MRL.
bulk pack (silica gel base material, Polymerically bonded, Results are summarized in Table 2.
octadecyl (18 %C), endcapped) were added into tube for
clean up. The sample was again vortex for 1 min and cen-
trifuges for 3 min at 3,000 rpm. The final eluent was RESULTS AND DISCUSSION
collected and evaporated to dryness under nitrogen and
reconstituted with 100 mL of cyclohexane to determine by
At the beginning of our study several samples were spiked
GC.
with target compounds. The spiked samples were subjected
to preliminary treatment described in the Material and
Chromatographic conditions
Methods section and the obtained chromatograms and
Split/splitless injector was heated at 250 8C and equipped confirmatory spectra are presented on Fig. 1.
with a CP 8400 autosampler (Varian, USA); injection vol- It can be seen that the developed chromatographic
ume was 2 mL. The following oven temperature programme conditions as well as sample preparation procedure do not
was used: 95 8C (1 min), 15 8C/min to 120 8C (2 min), 5 8C/ show any interference peaks of other matrix components in
min to 285 8C (5 min), 5 8C/min. The carrier gas (helium) the retention times of target analytes. In order to confirm

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Acta Chromatographica 33 (2021) 2, 188–194 191

Table 2. Limit of quantitation for organophosphorus pesticides in liver by GC-MS/MS

Within-laboratory reproducibility
Compounds MRL [ng/g] LOQa [ng/g] Recovery [%] (n 5 20) (RSDwR) RSD [%], (n 5 20)
chlorpiryphos-methyl 50 1.0 98.3 8.7
parathion 50 2.7 101.7 10.3
pirimiphos-methyl 50 0.7 81.2 7.8
diazinon 30 0.9 82.4 9.1
a
According to criterion of SANTE/12682/2019 limit of quantitation must be ≤ Maximum residue level (MRL).

that the developed method is suitable for its intended use, The quantification of OPPs was obtained using matrix
and to fulfil the aim of this study, the validation process was based calibration curve with organochlorine pesticide mirex
further carried out. as an internal standard. Mirex is chosen as it is not expected
The requirements when MS/MS detector is used for to be present at the sample, has similar retention time and is
determination of pesticides are at least two product ions to easy to extract by means of the used sample preparation. The
be detected for structure confirmation. Data relating to the recoveries of the individual compounds varied from 81 to
identification of target OPPs, are presented in Table 1. 102% which were satisfactory according to document re-
The optimized conditions for molecular ions fragmenta- quirements.
tion for each compound allowed to obtain minimum two Repeatability of measurements and within-laboratory
fragments for target compounds, which meets the require- reproducibility, expressed as relative standard deviation
ment of document SANTE 11945/2015. Three fragments (RSDwR) were lower than 20% for all compounds.
were obtained for chlorpyrifos-methyl and parathion. It is Table 2 summarizes data for MRLs, LOQ, recovery and
important to mention that the molecular ions of pirimiphos- repeatability calculated at LOQ-level. The LOQ is estab-
methyl and diazinon are larger than 300 (m/z 306 and 305, lished between 0.7 and 2.7 ng/g for all compounds,
respectively), which makes it difficult to be break down of considerably lower than the MRLs.
fragments of the ion-trap detector. From the obtained spectra The LOQ was defined as the smallest concentration of
of these two compounds, it can be seen that despite the analyte that can be quantified with acceptable validation
maximum collision energy molecular ions only decreased in parameters (trueness) and precision. In order to determine
intensity, but still present in the spectrum at 100%. the LOQ level, 20 liver samples were spiked with standard

Fig. 1. Chromatograms and MS spectra of target organophosphorus pesticides in liver sample on a LOQ level

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192 Acta Chromatographica 33 (2021) 2, 188–194

Table 3. The characteristics of linearity for studied substances in legislation. Recoveries exceeding 100% are mainly obtained
investigated range due to the matrix effect. Kiljanek and co-authors developed a
Regression working range method for analysis of OPPs in liver by GC-NPD, using
Name equation a 2
R [ng/g] GPC followed by silica gel column to purify the samples
[21]. Despite the application of the GPC system, which is
chlorpiryphos-methyl 61.4 3 þ0.08 0.99 1.0–75.0
expensive and solvent and time consuming, their reported
parathion 17.5 3 þ0.02 0.97 2.7–75.0
pirimiphos-methyl 85.6 3 þ0.13 0.99 0.7–75.0
results for recoveries are in the ranged from 76% to 105%
diazinon 55.5 3 0.04 0.97 0.9–45.0 [21]. These results are comparable to those obtained in the
a
present work, using a much faster and easier sample prep-
According to Document SANTE/12682/2019 the criteria is aration method. All repeatability and reproducibility results
R2 ≥ 0.95.
are below 20% and even below 10%, fully meeting the cri-
terion. Reproducibility is related to the applicability of the
solution and then analysed. The results are summarized in method under different conditions, such as different oper-
Table 2. All LOQ values are below the MRLs, which fully ator, different time or place of analysis. The data shows that
covers the requirement. The lowest LOQ is for pirimiphos- the method is stable and changing the external conditions
methyl – 0.9 ng/g, which is more than 30 times less than the does not affect the results. The measurement uncertainty
MRL. value in document SANTE/12682/2019 is calculated as the
The linear range is determined as function between the average of the uncertainties reported by all laboratories in
concentration of the pesticides and the ratio of its area to the the European Union, participating in inter-laboratory tests
internal standard (IS). The calibration method is a proce- for the analysis of pesticides in food, organized by the Eu-
dural calibration curve with spiked samples using the in- ropean Reference Laboratory.
ternal standard method for calculation. Five increasing The presented method allowed a simultaneous determi-
concentrations of the pesticides were determine as a func- nation of four organophosphorus pesticides in animal liver
tion of the MRL and a blank sample was used for zero level – by GC-MS/MS ion trap. European Regulations required the
0.0; LOQ; 0.5 multiplied by MRL; 1,0 multiplied by MRL; 1,5 monitoring programs for control of pesticide residues based
multiplied by MRL; 2.0 multiplied by MRL. The concen- on effective methods with high level of quality control at the
tration of IS in each sample is equal. The data for linearity is same time. Therefore, we have sought a new sample prep-
summarized in Table 3. aration procedure with low time and solvent consumption.
The data presented in Table 3 shown very good linearity We have used extraction with ASE – technique and have
for all target pesticides. The range from LOQ up to 2.0 times implemented liquid-liquid partitioning to remove fat con-
MRL is covered with determination coefficient R2 ≥0.95. tent in extracts followed by QuEChERS purification step.
Further the investigation for recovery, accuracy and There are several solvents used for liquid-liquid partitioning
uncertainty were realized. The obtained data are in Table 4. described in the literature. Our experiments included two
Recovery rate is related to the selection of suitable sample possible extraction systems acetonitrile:hexane 1:1 (v/v) or
preparation. The lowest recovery was obtained for parathion acetonitrile:acetone 1:1 (v/v) by liquid-liquid partitioning.
at a validation level 1–73%, and the highest recovery was for The acetonitrile:hexane combination was preferred because
diazinon in the highest concentration of validation – 104%, of better separation between the two solvents and easily
but all obtained results cover the criteria of European removal of hexane layer. Acetonitrile is polar enough to

Table 4. Recovery, repeatability, reproducibility and uncertainty obtained for target OPPs in liver by GC-MS/MSa
Within laboratory
Spike level Recovery [%] Repeatability reproducibility Uncertainty
Pesticide [ng/g] (n 5 18) RSDr [%], (n 5 6) RSDwR [%] (n 5 18) [%]
chlorpiriphos-methyl 25 97.8 4.31 6.12 6.07
50 99.0 7.32 7.64 6.27
75 101.2 6.63 6.51 4.46
parathion 25 72.6 6.61 8.81 7.41
50 99.8 8.55 8.87 7.23
75 97.4 7.80 9.54 5.55
pirimiphos-methyl 25 99.9 3.02 5.90 6.10
50 98.4 4.41 6.27 5.55
75 99.3 2.56 2.54 2.50
diazinon 15 78.2 5.33 7.46 6.95
30 101.8 7.99 8.05 6.69
45 103.5 5.43 5.80 3.75
a
Specified criteria that must be met by Document № SANTE/12682/2019 are: for recovery: 70–120%; for precision (repeatability and
reproducibility): ≤20% and for uncertainty: ≤ 50%.

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Acta Chromatographica 33 (2021) 2, 188–194 193

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