1. MULTI-RESIDUE PESTICIDE ANALYSIS OF FOOD SAMPLES USING
ACETONITRILE EXTRACTION AND TWO-DIMENSIONAL LIQUID
CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY
Katerina Svobodova1, Ondrej Lacina2, Radim Stepan1, Martin Kubik1, Petr Cuhra1
1
2
Czech Agriculture and Food Inspection Authority (CAFIA), National Reference Laboratory (NRL) for pesticide residues
Za Opravnou 300/6, 150 00 Prague, Czech Republic
HPST, s.r.o., Pisnicka 372/20, 142 00 Prague, Czech Republic
1. Introduction
Czech Agriculture and Food Inspection Authority (CAFIA) is the competent authority for control of pesticide
residues in foodstuffs of plant origin and provides the national and EU co-ordinated monitoring programmes.
CAFIA laboratory is designated as National Reference Laboratory for Pesticide Residues in fruits & vegetables
(NRL-FV), cereals (NRL-CF) requiring Multi Residue Method (MRM) and National Reference Laboratory for Single
Residue Methods (NRL-SRM).
Tandem mass spectrometry coupled with chromatography, such as GC-MS/MS and LC-MS/MS operated in MRM
mode, has become the method of choice for targeted screening of multi-residue analysis in complex food matrix
samples. A fast, easy and efficient preparation of food sample is the key to multi-residue pesticide MS analysis,
which in fact still remains as a challenge.
In the current study easy sample preparation and two dimensional liquid chromatography coupled with tandem
mass spectrometry operated in dMRM mode (2D-LC-MS/MS) was optimized. 2D-LC-MS/MS system seems to be
perspective method of choice for targeted multi-residue analysis of complex food matrix samples. Moreover
both MRM and SRM compounds can be analysed in a single chromatographic run.
The results show benefits of 2D-LC such as improvement of peak shape of highly polar analytes, easy sample
preparation, polarity switching in the same run and time saving.
2. Experimental
Instrument:
• Agilent 6460 Triple Quadrupole System (Agilent Technologies, USA)
• Agilent 1260 Infinity Binary Pump (Agilent Technologies, USA)
• Epics ® - Easy Pesticide Isolation and Concentration System (Jasem, Turkey)
Instrument set up:
• Reverse Phase (RP) column: Poroshell 120 EC-C18, 2,1x100 mm, 2.7 µm
• Heated: at 30°C
• Trap column: Zorbax Stable Bond C8, 4.6x12.5mm, 5µm
• HILIC column: YMC-Pack Diol-NP Narrowbore HPLC column (2.1 mm i.d.) 12 nm S-5 µm 100x2.1 mm
• Heated: at 30°C
• Injection volume: 3 µl
• Mobile Phase:
• Binary pump 1 (RP) A: 5mM NH4COOH/0,1 % FA/H2O
B: MeOH
• Binary pump 2 (HILIC) A: 5mM NH4COOH/0,1 % FA/H2O
B: 90 % MeCN/10 % H2O/ 5mM NH4COOH /0,1 % FA
Ion Mode Positive/Negative switching
Drying gas temperature 230 °C
Drying gas flow 8 L/min
Nebulizer pressure 35 psi
Sheath gas temperature 375 °C
Sheath gas flow 11 L/min
Capillary voltage 2800 V
Tab. 1 MS/MS conditions using Agilent 6460 QqQ LC/MS equipped with Agilent
JetStream ESI source
Tab. 2 LC Gradient used on HILIC column
Time
[min]
A [%] B[%] Flow
[mL/min]
0.0 2.0 98.0 0.200
2.0 2.0 98.0 0.200
2.1 2.0 98.0 0.200
5.0 60.0 40.0 0.200
5.10 60.0 40.0 0.200
13.0 60.0 40.0 0.200
14.0 2.0 98.0 0.200
24.0 2.0 98.0 0.200
Time
[min]
A [%] B[%] Flow
[mL/min]
0.0 95.0 5.0 0.200
1.2 95.0 5.0 0.200
1.3 100.0 0.0 2.000
1.84 100.0 0.0 2.000
1.87 100.0 0.0 0.000
2.5 100.0 0.0 0.000
4.9 95.0 5.0 0.000
5.0 95.0 5.0 0.200
5.5 50.0 50.0 0.300
19.0 2.0 98.0 0.300
21.0 2.0 98.0 0.350
21.10 95.0 5.0 0.350
24.0 95.0 5.0 0.350
Tab. 3 LC Gradient used on RP column Tab. 4 Epics® valve timings
Fig. 1 Epics® interface scheme
Fig. 2 Example of calibration curve obtained for selected pesticides separated on RP column and HILIC column in solvent
*Calibration linearity was evaluated for each analyte. Correlation coefficients R2 >0,995 were obtained for most of target compounds.
Fig. 3 Example of improvement of peak shape using 2D-LC Epics® system; matrix-matched standard (conc. 0,05 mg/kg)
These chromatograms show the
improvement of peak shape of highly
polar compounds when 2D-LC
separation is used. The first
chromatogram shows behaviour of
these polar compounds on RP column
(RP separation is widely used in multi
residue pesticide analysis).
Fig. 4 Chromatogram of matrix-matched calibration standard (conc. 0,05 mg/kg)
A 10 g sample is weighed into a 10 mL
conical-bottom polypropylene centrifuge
tube with a screw cap; water and 100
μL of internal standard (conc. 10 mg/L)
is added. Then 10 mL of 1% formic acid
in acetonitrile is added and mixture is
shaken in mechanical shaker for 30 min.
To separate solid particles of mixture
the tube is centrifuged at 4500 rpm for
7 min. Then 6 ml of extract is
transferred into conical-bottom
polypropylene centrifuge tube (15 mL)
filled with 1g NaCl. Tube is shaken in
mechanical shaker for 1 min and then
centrifuged at 4500 rpm for 7 min.
Experiments were performed on
samples of the two matrix (apple and
oat). Samples were spiked with the
appropriate amount of mixture of 300
pesticides including i.e. Quaternary
Ammonium Compounds (QAC) (BAC-n,
Quats) and Organotins. Samples were
extracted by the method described
above. Target concentration of analytes
was 0,05 mg/kg.
3. Results & Discussion:
Reporting limits i.e. practical limits of
quantification corresponds to the
concentration of target analytes at the
lowest calibration level. Reporting limit for
79,67 % of all analytes was 1 ppb
(including QAC, Quats and Organotins), at
2 ppb 8,47 % , 8 ppb 9,15 % and 2,71 %
at the 40 ppb.
Fig. 5 Recovery of the method expressed as the average of the recovery evaluated for each analyte (n=6) in apple
and oat matrix
Precision for each analyte expressed as
relative standard deviation (n=6, RSD %)
doesn´t exceed 10 %. Trueness expressed
as recovery (%) is evaluated for each
analyte too. Presented method showed
lower recoveries (<60 %) for 6,21 %
analytes (e.g. Cyromazine, Chlormequat
etc.)
4. Conclusions:
The presented method based on two-
dimensional liquid chromatography enable to
(i) improve chromatographic separation i.e.
peak shape of highly polar compounds due to
HILIC separation, (ii) improve sensitivity (for
79,67 % of analytes was achieved 1 ppb
reporting limit), (iii) analyse MRM and SRM
compounds in the same run and (iv) save time
due to easy sample preparation and polarity
switching. The recovery of some analytes from
SRM group is below 60 % (e.g. Quats –
Chlormequat) because of extraction with NaCl
addition. For these compounds the presented
method gives screening information and SRM
method need to be used for confirmation and
precise quantification.
Valve Positio
n
Time
[min]
1 B 0.0
2 B 0.0
2 A 1.2
1 A 1.2
2 B 1.87
1 B 5.5
2 B 24.05
2 B 24.05
Fig. 6 Reproducibility of 2D-LC separation of selected
compounds separated on HILIC and C18 column (matrix-
matched standard conc. 0,05 mg/kg)
Sample preparation: