Analyzing avocado: How to deal with lack of water and keep the fats out

Avocados are a popular produce item. In fact, the consumption of avocados rose by 450% since 2000! Avocado may be delicious, but it is difficult to deal with in the lab. The good news – avocados are ranked number 1 on the clean 15 list, meaning they have the least amount of pesticide residues. With so many avocados on the market, the testing is as important as ever, which may not be that easy. Avocados are a commodity that consists of approximately 70% water and 15 % fat. High-fat content and a lack of water mean adjustments must be made in order to ensure accurate pesticide residue results. Here, we explore several QuEChERS extraction and cleanup options and present a recommended approach.  For GC-MS/MS analysis, most of the fat needs to be removed because excess fat can lead to poor analyte recoveries and an increase in instrument maintenance.

Image source: https://daily.jstor.org/the-illustrious-history-of-the-avocado/

Fats can be removed during extraction by using a non-polar solvent such as hexane. This approach can be used in junction with QuEChERS, but there is a caveat. Unfortunately, using hexane with QuEChERS may lead to analyte loss since some of the pesticide residues are also non-polar and could be dissolved in the hexane with the fats. Because I’m analyzing avocados by GC-MS/MS, this would be a problem, so that leaves us with “just” regular QuEChERS. Due to its low water content, avocado needs to be hydrated before the extraction. There are two ways to accomplish this: the first is to add enough water to already homogenized avocados to get 10 mL of total water content, i.e. 3 mL for every 10 g of avocados; the second approach is taken from EN method 15662:2018 and is for commodities with intermediate water content (40-80%) and high matrix load or high oil content (>5%). This method samples 5 grams of commodity and adds 6 mL of water, which is then extracted with 10 mL of acetonitrile (ACN). As in my previous blogs, I used the QuEChERS performance mix to compare these two methods. For this comparison I used the EN method salts (Fig 2).

Figure 2: Comparison of pesticide recoveries (area/area of internal standard) with two extraction methods. Method 1: 10 g avocados and 3 mL water; method 2: 5 g avocados and 6 mL water.

While the first method (10 g of avocados and 3 mL water) had better recoveries for some pesticide residues, the EN method 15662:2018 (5 g  avocados and 6 mL water) method had better overall recoveries (Fig 2). Next, I compared the EN, Original, and AOAC salts following the same ratio of water and avocados, i.e. 6 ml and 5 g for the EN and original unbuffered methods and 9 ml and 7.5 g for the AOAC method (Fig 3).

Figure 3: Comparison of pesticide recoveries (area/area of internal standard) with different extraction salts.

The recoveries (area/area of internal standard) were comparable, however, if we normalize them to the EN salts results, the AOAC salts have the highest maximum and mean recovery (Table 1).

Table 1: Comparison of pesticide recoveries with different extraction salt (normalized to EN salts)

Because we still need to remove the oils, the selection of dSPE is crucial. The end-capped C18 (EC-C18) containing dSPE is designed to remove the remaining fats. With this in mind, I compared four different dSPE tubes which varied by their PSA, EC-C18, and GCB (graphitized carbon black) content (Table 2, Fig 4).

Table 2: Description of dSPE used for the optimization

Figure 4: Comparison of pesticide recoveries (area/area of internal standard) with different dSPE clean-up. See Table 4 for dSPE content.

From the comparison of the recoveries in figure 4, we can immediately notice that dSPE #26219 has significantly higher recoveries for 2-phenylphenol, malathion, and myclobutanil, but, has much lower recoveries for cyprodinil, chlorpyrifos, and thiabendazole. This means #26219 is not a suitable candidate for this analysis. The other dSPE tubes have much more similar responses, with slightly better recoveries for #26125. The dSPE #26242 has similar recoveries to dSPE #26125, but the absence of PSA (Table 2) may cause trouble with a wider range of pesticides.

To summarize, for my further pesticide work I chose to work with AOAC salts with an adjusted ratio of avocados to water (7.5 g to 9 mL) using #26125 dSPE.