Jonathan R. Beck and Charles Yang, Thermo Fisher Scientific, San Jose, California, USA.
Pesticides are used throughout the world to control pests that are harmful to crops, animals, or people. Because of the danger
of pesticides to human health and the environment, regulatory agencies control their use and set pesticide residue tolerance
levels. The limits of detection (LODs) for many of these substances are at the parts-per-trillion (ppt) level. To achieve
this level of detection, off-line sample pre-concentration is often performed. However, these sample preparation procedures
can be time consuming, adding as much as one to two days to the total analysis time. Therefore, a method for on-line sample
pre-concentration that bypasses the off-line sample pre-concentration provides a significant time savings over conventional
methods.
We describe a method for on-line sample clean-up and analysis using the EQuan system. This method couples a fast-HPLC system
with two Hypersil GOLD LC columns (Thermo Scientific, Bellefonte, Pennsylvania, USA) — one for pre-concentration of the sample
(1 mL), the second for the analytical separation — and an LC–MS–MS instrument. Using this configuration, run times of six
minutes are achieved for the analysis of a mixture of pesticides.
Experimental Conditions Sample preparation: Bottled drinking water was spiked with a mixture of the following pesticides: carbofuran, carbaryl, diuron, daimuron, bensulphuron-methyl,
tricyclazole, azoxystrobin, halosulphuron-methyl, flazasulphuron, thiodicarb and siduron. Concentrations were prepared at
the following levels: 0.5, 1, 5, 10, 50, 100, 500 and 1000 pg/mL (ppt).
HPLC: Fast-HPLC analysis was performed using the Accela High Speed LC System (Thermo Scientific, San Jose, California, USA). A
1 mL water sample was injected directly onto a 20 mm × 2.1 mm i.d., 12 μm Hypersil GOLD loading column in a high aqueous mobile
phase at a flow-rate of 1 mL/min. After approximately one minute, a 6-port valve on the mass spectrometer was switched via
the instrument control software. This enabled the loading column to be back flushed onto the analytical column (Hypersil GOLD
50 × 2.1 mm i.d., 1.9 μm), where the compounds were separated prior to introduction into the mass spectrometer. The total
run time for each analysis was six minutes. The mobile phases for the analysis were water and acetonitrile, both containing
0.1% formic acid.
MS: MS analysis was performed on a TSQ Quantum Access triple stage quadrupole mass spectrometer with a heated electrospray ionization
(H-ESI) probe in the positive ESI mode.
Results and Discussion
Figure 1
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Chromatograms for the calibration standard at a concentration of 500 pg/mL are shown in Figure 1. In the fast-HPLC run, all
11 of the individual analytes were eluted before three minutes. In contrast, none of the analytes in the standard HPLC run
were eluted until nearly eight minutes into the run. Further optimization of the chromatography for the Fast-HPLC would produce
even shorter run times.
Calibration curves for all 11 compounds were generated using LCQUAN 2.5 software (Thermo Scientific, San Jose, California,
USA). Excellent linearity was achieved for all of the compounds analysed in this experiment. All of the compounds were detected
and quantified at levels well below the regulatory requirements set by the Japanese Ministry of Health, Labour and Welfare.1
