Page 25 - Application Notebook - Solution for Food Safety

Application No. C154
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„ Experimental
Pesticide spiked samples, extracted using established
Table 2 MS/MS methods used to acquire data in MRM Spectrum
QuEChERS based methods, were provided by Scientific Mode and a conventional MRM method with 2 MRM transitions
Analysis Laboratories, UK. In order to test the per compound. As part of the comparative study, the same LC
performance of the MRM Spectrum Mode database conditions were used for both methods.
and library searching a number of matrices were tested LC-MS/MS Mass MRM Spectrum 2 MRM method
including turmeric, plum, peppermint, parsnip, cherry, spectrometry Mode: generating
lime, pumpkin, tomato and potato. Final extracts were library searchable
prepared in acetonitrile without any dilution and spectra
directly injected into the LC-MS/MS. A water co- Target number of 193 193
injection method, performed automatically in the auto- compounds
sampler, was used to improve early eluting peak Total number of MRM 1,291 transitions 386 (374 in ESI+ and
shapes in addition to a sub 2 micron particle size transitions (1,229 in ESI+ and 62 12 in ESI-)
column to improve peak capacity (Table 1) . in ESI-)

Pause time/dwell 1 msec./3 msec. 1 msec./3 msec.
Calibration curves were prepared in the range 0.01 to time
0.2 mg/kg. Repeatability of the method was tested Ionisation mode ESI +/- ESI +/-
using avocado matrix at 0.1 mg/kg. In the final method
samples were analysed in ESI +/- using a polarity Polarity switching 5 msec 5 msec
switching time of 5 msec. time
Interface 350 °C 350 °C

On average 7 MRM transitions were applied to each temperature
compound, with more than 10 MRM transitions applied
Heat bl°Ck 300 °C 300 °C
to 34 compounds. All MRM transitions were acquired temperature
throughout the MRM window without the need for
Desolvation line 150 °C 150 °C
triggering thresholds. The method includes a total of temperature
1,291 MRM transitions for 193 pesticides in a run time
of only 15 minutes. A dwell time of 3 msecs was applied Nebulising gas 3 L/min 3 L/min
to every MRM transition. In order to evaluate the data Heating gas 10 L/min 10 L/min
quality from the MRM Spectrum Mode method, the
same method was set up with 2 MRMs applied to each Drying gas 10 L/min 10 L/min
compound (386 MRMs in total) using the same
acquisition method (Table 2).
„ Results and Discussion

LabSolutions software was used to automatically In developing monitoring programs for chemical
optimize the fragmentation for all pesticides and contamination methods are designed to determine a
generate a MRM Spectrum mode method. The MRM list of known analytes with a focus on delivering a rapid,
Spectrum Mode method for library searching and cost-effective analysis that generates no false-negative
compound verification could be simply and quickly set or false-positive results. Guidelines for compound
up using the Shimadzu pesticide database. This identification have been published by the EU in
database contains more than 6,000 MRM transitions for directive SANTE/11945/2015 . This identification
over 750 pesticides. criteria requires at least two MRM transitions with an
ion ratio and retention time within defined tolerance

LabSolutions Insight v3.0 software was used to review limits.
quantitative data and MRM Spectrum mode library
searching with advanced filtering tools to review by To help reduce false detect reporting in pesticide
exception and to reduce false detect reporting. monitoring programs, a MRM method was developed
with a higher number of MRM transitions for each

Table 1 LC acquisition parameters target pesticide to increase the level of confidence in
assay specificity. By combining multiple MRM
Liquid chromatography
transitions for a compound into a product ion
UHPLC Nexera LC system spectrum, pesticide identification can be verified and
confirmed against a MS/MS reference spectral library.
Analytical column HSS T3 (100 × 2.1, 1.7 μm)
Using MRM Spectrum mode can help markedly reduce
Column temperature 40 °C false detect reporting without affecting the data
quality for optimized quantitation or identification.
Flow rate 0.4 mL/minute

Solvent A 5 mmol/L ammonium formate and Fig. 2, shows the MRM chromatogram for all 193
0.004 % formic acid
pesticides spiked at 0.010 mg/kg measured with MRM
Solvent B 5 mmol/L ammonium formate and Spectrum mode. Using this mode 1,291 MRM
0.004 % formic acid in methanol transitions were measured for 193 pesticides. Despite
Binary Gradient Time (mins) %B the high data density acquired with MRM Spectrum
Mode (for example, 151 MRM transitions were
1.50 35
registered in the same time window during the analysis,
11.50 100 see Fig. 3) sensitivity was not affected by the high data
acquisition rate.
13.00 100
13.01 3
15.00 Stop
Injection volume 0.1 μL (plus 30 μL water)