Chromatographic analyses were performed in a WATERS ACQUITY UPLC system (Waters, Manchester, UK) consisting of an Acquity UPLC binary solvent manager, an Acquity UPLC sample manager and an Acquity UPLC column heater. Chromatographic separation was performed using a Waters Acquity UPLC BEH C18 column (2.1 mm×100 mm) with 1.7 µm particle size (Milford, MA, USA). The column was maintained at 40 °C, with a mobile phase flow rate of 0.25 mL min⁻¹. The mobile phase contained water (A) and MetOH (B) both with 0.1% formic acid. A gradient elution was employed, starting at 10% B and rising linearly to 90% over 2 min. The composition was held at 90% B for 2min before being returned to the initial conditions of 10% B, followed by re-equilibration for 1.5 min and giving a 5-minute total cycle. The injection volume for each sample was 10 µL in “full loop” modus and the temperature inside the sample manager was maintained at 10 °C.

Mass detection was performed using an Acquity TQD tandem quadruple mass spectrometer (Waters, Manchester, UK), equipped with an electrospray ionization interface (ESI) and operating in the positive ion mode. The ion source was operated at 150 °C with a capillary voltage of 1.0 kV. Nitrogen was employed for both the dissolvent and cone gases at 650 °C and 50 L·hr⁻¹, respectively. The mode of acquisition was multiple reaction monitoring (MRM) at an argon collision gas pressure of 3.5×10⁻³ mBar. MRM conditions were optimized for each pesticide during infusion. Data acquisition and processing were performed using MassLynx 4.1 (Waters, Manchester, UK).

Flight olives (cultivar Picual) were collected from the “Instituto Andaluz de Investigación y Formación Agraria, Pesquera, Alimentaria y de la Producción Ecológica IFAPA Centro ‘Venta del Llano’” (Jaén, Spain). Olive fruits with a maturation degree of 4–5 were directly collected from trees and they were used as a starting material for preparing working samples: blank samples, spiked samples and matrix-matched calibration standards for validation studies.

500 g of olive fruits were arranged on a small tray (25 cm of diameter) for the olives to be divided into, at least, two or three layers. Then, 0.5 ml of multi-compound pesticide spiking solution was sprayed onto the whole sample surface and let stand until the solvents were virtually evaporated. The olives were mixed in order to reach a random distribution and then were sprayed again. This process was repeatedly performed until all the olives were homogeneously sprayed with the pesticide spiking solution using a total volume of 5.0 mL. This process was carried out at two spiking levels. The spiking solution was prepared using a MeCN: acetone (45:55) mixture to ensure both a correct adherence and a rapid evaporation.

The matrix effects in LC-MS with an electrospray ionization source must be studied in complex matrices like olives. This effect is produced when other compounds present in the matrix can interfere in the ionization of the target compound. Interferences can be caused by a decrease or enhancement of the analytical signal, which would cause difficulties concerning the correct detection and quantification of pesticides.

In all the methods previously described in the literature, olives are first crushed and then extracted with an appropriate solvent. These procedures provide a co-extraction of variable amounts of olive oil, which must be later removed as much as possible by applying of different clean up steps.

In the new proposed method, the olives are not crushed so that there should not be a co-extraction of oil. But considering that the liquid extraction surface analysis extract is directly analyzed without any cleaning steps, this potential effect has to be elucidated.

At first, it was assumed that the matrix effect in the surface analysis method would be low. However, other components present on the olive surface (waxes, triglycerides, pigments, etc.) might be co-extracted during extraction and incorporated into the final extract, affecting the correct detection of the target compounds. For this reason, the matrix effect was tested by comparing the slope of the calibration curves in matrix-matched or in the solvent. Then, the matrix/solvent slope ratio for each pesticide was calculated. Calibration curves in the matrix or in the solvent were made with the use of standards prepared at the concentrations of 0, 10, 25, 50, 75, 100, 150 and 300 ng·mL⁻¹.

The potential matrix effect can be quantified by comparing the slopes of the calibration curves prepared with or without the matrix. Taking into account the ratio between them, a classification of the matrix effect can be carried out, so that

Matrix Effect=100 * [1- (slope matrix / slope solvent)]

Based on the classification studied by B. Kmellár (Kmellár, et al., 2008), and depending on the decrease /increase in the slope, different kinds of matrix effects could be considered: soft matrix effect, when the ratio is lower than 20%; moderate, from 20 to 50%; or strong when it is higher than 50% since this could mask the correct detection of the pesticides.

Chromatographic signal suppression was noted in all the pesticides. The matrix effect for some pesticides like Diflufenican, Terbuthylazine or Diuron could be considered as soft matrix effect, and other compounds present in the matrix would have a small impact in the correct determination of these pesticides. However, other pesticides like Dimethoate, Phosmet and Chlorpyrifos should always be calibrated with the matrix, because its matrix effect was greater than 30%. The matrix effects for Oxyfluorfen and Amitrol were extremely strong (more than 60%) and it was very difficult to build a good calibration curve for these pesticides. As a conclusion, all the pesticides were determined from the standard matrix-matched calibrations. The results can be observed in Table 3.

In spite of the fact that MeCN is the most commonly used solvent for pesticide extraction in the analysis of olives, several potential solvents (or mixtures) were evaluated: MeCN, MetOH, MeCN/MetOH (80:20 v/v) and water/ MetOH (1:3 v/v). In order to select the best extraction solvent, the amount (weight) of matrix extracted by each solvent was studied as a first approach. For this purpose, the amount of solvent used for each extraction was 30 mL and the amount of olives selected was 30 g. The extraction was carried out by manual shaking for 5 minutes.

The absolute amount of extract obtained from each solvent was tested from four types of extraction solvents. MetOH extracted 13.2 mg of matrix per g of olives compared to 4.6 mg of matrix that the solvent mixtures extracted or 2.5 mg extracted by MeCN (values calculated as a solid weight after vacuum evaporation of an aliquot of the extract). With these results, MetOH initially could be discarded because the amount of matrix extracted by this solvent was much greater than that found for the rest of solvents. Nevertheless, MetOH was not discarded in order to evaluate its performance from other points of view.

The next parameter evaluated was the recovery of each solvent by extracting samples spiked at 100 µg·kg⁻¹. The best recoveries were obtained when MeCN was used in the extraction. Poor recoveries were obtained using the other three extraction solvents in the study, which were not able to reach 30% and were therefore discarded.

Finally, the optimization of the other variables in the extraction method continued with the use of MeCN as extraction solvent.

The next variable studied was the amount of solvent to extract the analytes, i.e., the solvent/sample ratio. For this purpose, 30 g of spiked samples (100 µg·kg⁻¹) were extracted during 5 min by manual shaking in vessels containing 30, 90 or 150 mL of MeCN (w/v ratio 1/1, 1/3 and 1/5, respectively). A sample/solvent ratio of 1:1 was selected because increasing the solvent volume did not produce a better extraction yield.

In order to automatize the extraction step in order to improve the repeatability of the process, the use of an automatic shaker was studied. Two experimental variables were studied: speed of oscillation and extraction time. For this purpose, samples spiked at the 100 µg·mL⁻¹ level were analyzed, and the obtained recoveries were compared. Firstly, 5 speeds were tested using 5 minutes in each extraction: 100, 250, 500, 750 and 990 oscillations per minute (opm). Recoveries were shown to rise as the speed increased until a maximum speed of 750 opm. Recoveries of the tested pesticides decreased dramatically when the speed of oscillation was higher than 750 opm. Once the speed of 750 opm had been selected, the extraction time was optimized. For this purpose, 4 shaking times were tested: 1, 2, 5 and 10 minutes. It was noted that the recoveries increased according to an increase in time, reaching almost 100% recoveries after 10 minutes.

Finally, the effect of solvent/temperature during extraction was studied as well. Three temperatures were evaluated (25, 30 and 35 °C), while maintaining a constant speed of 750 opm for 10 min, but the respective recoveries did not show significant differences.

The final conditions of the liquid extraction surface analysis method were: MeCN as a solvent extraction in a 1:1 (w/v) ratio, and 10 min of extraction by shaking at 750 opm using a solvent temperature of 25 °C. These results are summarized in Table 4.

In order to know if a single surface extraction was enough, or whether by applying a second extraction on the treated olives a greater amount of pesticides would be extracted, the recoveries of a successive extraction step were evaluated. The results are shown in Table 5. As can be seen, good recoveries were obtained, and reached a range from 70 to 99% for 6 of the 8 pesticides studied when a single extraction was applied. Recoveries between 73–102% were reached when a second extraction was used. It is noteworthy that the second extraction did not reach an appreciable amount of pesticides, and the remaining amount that could not be extracted with a single extraction step was 3%. However, since it is a small and constant value, it could be considered that a single extraction is enough to reach good recoveries for the whole of the pesticides.

Unfortunately, Oxyfluorfen and Amitrole determination were not successful, and this is probably because this extraction method was not effective enough to extract these pesticides from the olive surface.