LOS SEGUROS AGRÍCOLAS Y SUS EFECTOS EN EL AGRO DE ESTADOS UNIDOS

Diclofenac was extracted from water samples using a filter absorption method as

described in detail in Chapter 3. Water samples of 100 mL volume were transferred to Schott

bottles, acidified to a pH of 2 using approximately 3 drops of H2SO4, and had a Sartorius

Biolab 4.8 cm diameter, 0.2 µm nylon membrane filter added. The sample was then covered

in aluminium foil to protect against photo-degradation and put on a shaker table for 24 hours

at medium speed. The filter was removed and transferred to a 15 mL amber vial, which was

then placed on a heating block at 65-100°C to dry. The filter was rinsed three times using 4

mL of MeOH, and was vortexed during every rinse to ensure maximum extraction of

diclofenac from the filter and the aliquots of MeOH were combined in a 15 mL amber glass

vial. The MeOH was then evaporated off under nitrogen at 65°C until dry. The vial was

rinsed three times with MeOH (500 µL, 250 µL, 250 µL) and the aliquots of MeOH were

combined in a 15 mL amber glass vial..

Each extraction batch (n = 24 samples) included a spiked sample and a Milli-Q blank.

Two spike concentrations (0.1 and 200 µg/L) were used dependent on the expected sample

concentration.

2.6.2. High performance liquid chromatography analysis

HPLC is an analytical method that separates compounds dissolved in a solvent, based

on their structure and size due to their interactions with a solid adsorbent column (Kupiec,

2004). HPLC was used in this project as it is an affordable method of sample detection that

works well for high environmental concentrations of diclofenac (Nováková et al. 2006).

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Samples were analysed on a Dionex UltiMate 3000 HPLC fitted with a photodiode

array (PDA) detector, quaternary pump, mobile phase degasser, and autosampler. The

column used was a Phenomenex Luna 5u C18 (2) column (150 x 4.60 mm). The mobile

phase was 60:40 NaH2PO4 buffer, pH 3: acetonitrile, run isocratically at 1 mL per minute.

Injection volume of the test solution was 20 µL with a total analysis time of 31 minutes per

sample. Ultra-violet PDA detection of diclofenac was at 240 nm with a retention time of 13.2

minutes.

A calibration curve of diclofenac standards was prepared for each run with a

concentration range of 0 to 2000 µg/mL. The calibration standards were run before and after

each batch of samples. The 100 and 1000 µg/mL standards, and a blank of AcN were

analysed after every 10 samples, while every 20 samples a sample was injected in duplicate

to monitor variability. These samples confirmed the stability and reproducibility of the

calibration. Calibration curves were linear (R2 = 0.9995) over the calibration concentration

range.

2.6.4. Gas chromatography–mass spectrometry analysis

GC-MS is an analytical method that combines two commonly-used techniques to

successfully separate volatile and semi-volatile compounds while also selectively detecting

them. Compounds are separated via GC, before being pumped into the MS where they are

ionised. Upon ionisation, the individual compounds are exposed to a beam of electrons that

induces fragmentation which results in a determination of mass that can be divided by the

charge (Fialkov et al. 2007). This allows determination of the molecular weight. Because of

this specificity, the GC-MS detects compounds at levels as low as sub-nanograms (Sneddon

et al. 2007). The GC-MS is a popular technique for qualitative and quantitative determination

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2.6.5. Derivatisation

The samples prepared in Section 2.6.1 were evaporated until dry under nitrogen at

80°C and then had 100 µL of AcN added. Standards were made, ranging in concentration

from 0 to 100 µg/L. To both the standards and the samples, 25 µL of hydroxypyrene (internal

standard) and 100 µL of MSTFA (derivatisation agent) were added, before being vortexed

and placed on a heating block at 80°C for 1 hour (Farré et al. 2007).

2.6.6. Analysis of derivatised samples

The derivatised sample extracts and calibration standards were analysed by GC-MS

using a Shimadzu GC-2010 Gas Chromatograph, interfaced to a Shimadzu AOC-20i Auto

Injector and a Shimadzu GCMS-QP2010Plus detector. Instrumental control, data acquisition

and data processing were performed using the Shimadzu GCMS Solution software (Version

2.70). Analytes were separated on a Rxi-5Sil column (5% diphenyl/95% dimethyl

polysiloxane) 30 m x 0.25 mm ID, 0.25 µm film thickness, with an integrated guard column

(10 m, Integra-Guard) (Restek, Belleftone USA). Derivatised samples and calibration

standards were injected into the injection port in splitless mode at a temperature of 280°C in

1 µL volumes. The initial oven temperature of 80°C was held for 1 minute, then increased at

a rate of 10°C/min to 150°C, then increased at 8°C/min to 215°C, and then increased by

10°C/min until it reached the final temperature of 280°C. The total run time was 32.63

minutes. Helium was used as the carrier gas at a flow rate of 5.5 mL/min.

The ion source was held at 230°C and the GC-MS interface at 280°C. Electron Impact

Spectra (EIS) were obtained at 70 eV in selected ion mode (SIM). The MS was calibrated

against perfluorotributylamine (PFTBA) before each sample run using the mass spectrometry

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Retention times and mass-to-charge ratios used for detection and quantification of the

individual compounds were as follows: diclofenac quantifying ion 214, qualifying ions 73,

367, and 352, retention time 20.91 minutes; hydroxypyrene (the internal standard)

quantifying ion 290, qualifying ions 275 and 259, retention time 22.65 minutes.

2.6.7. Quality assurance/quality control

To give quality control, controls were run with every exposure. This was to ensure

there was no contamination of the water used and to make sure the cleaning of the tanks was

being carried out properly. To provide quality assurance, exposure samples were run in

duplicate for the first exposure and in triplicate for the following two exposures. Blanks were

also carried out in duplicate or triplicate respective of the exposure week.

A water sample spiked with diclofenac was included in every batch to determine

method recovery. Water blanks were spiked with either 2 mL or 1 mL of diclofenac standard

(Section 2.4.1) depending on the expected concentration (200 µg/L and 0.1 µg/L

respectively). These samples then underwent the same extraction process as experimental

samples.

Duplicates of samples, high and low standards, and a blank were run during both

HPLC and GC-MS analyses to ensure stability and reproducibility of results. Standards were

run before the samples to ensure there was no interference with the readings such as early

elution or detection of the compound, or excess background noise. The blanks were used to

detect contamination from the machines and to act as a background reference when analysing

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