A protocol for wide-scope non-target analysis of contaminants in small amounts of biota using bead beating tissuelyser extraction and LC-HRMS

This work describes a robust and powerful method for wide-scope target and non-target analysis of xenobiotics in biota samples based on bead beating tissuelyser extraction, solid phase extraction (SPE) clean-up and further detection by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Unlike target methodologies, non-target methods usually aim at determining a wide range of still unknown substances with different physicochemical properties. Therefore, losses during the extraction process were minimised. Apart from that, the reduction of possible interferences showed to be necessary to expand the number of compounds that can be detected. This was achieved with an additional SPE clean-up step carried out with mixed-bed multi-layered cartridges. The method was validated with a set of 27 compounds covering a wide range of physicochemical properties, and further applied to the analysis of krill and fish samples.• The bead beating extraction was efficient for a wide range of organic pollutants in small quantities of biota samples.• Multi-layered solid phase extraction clean-up yield a wide xenobiotics coverage reducing matrix effects.• Method validation with 27 compounds led to a suitable method for non-target analysis of organic pollutants in biota.


a b s t r a c t
This work describes a robust and powerful method for wide-scope target and non-target analysis of xenobiotics in biota samples based on bead beating tissuelyser extraction, solid phase extraction (SPE) clean-up and further detection by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Unlike target methodologies, non-target methods usually aim at determining a wide range of still unknown substances with different physicochemical properties. Therefore, losses during the extraction process were minimised. Apart from that, the reduction of possible interferences showed to be necessary to expand the number of compounds that can be detected. This was achieved with an additional SPE clean-up step carried out with mixed-bed multi-layered cartridges. The method was validated with a set of 27 compounds covering a wide range of physicochemical properties, and further applied to the analysis of krill and fish samples.
• The bead beating extraction was efficient for a wide range of organic pollutants in small quantities of biota samples. • Multi-layered solid phase extraction clean-up yield a wide xenobiotics coverage reducing matrix effects.

Background
The development of non-target methodologies for the determination of organic contaminants that are not covered by existing target methodologies in complex biological samples is an urgent need. Target methods can only cover a small proportion of the compounds that can cause unwanted effects in the environment. Therefore, non-target strategies, where no particular chemicals are being searched for, are necessary to obtain a broader picture and identify new potentially hazardous compounds. In this sense, liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) has dramatically increased the opportunities for the detection of polar organic contaminants in complex samples [3] . However, the development of protocols for the extraction of many compounds covering a large range of physicochemical properties in biological matrices (e.g. biota) is still challenging. Advances in both extraction and clean-up protocols (to reduce interferences) for non-target analysis are needed.
In this work, an extraction method based on bead beating of small biota samples [1] was combined with a non-discriminant clean-up strategy for the non-target analysis of a wide-scope of organic pollutants [2] . In order to test its effectiveness for a wide range of organic contaminants the method was validated with a set of 27 compounds (logP comprised between −1.16 and 6.97) including pharmaceuticals, personal care products, herbicides, food additives and other industrial chemicals.

Sampling and sample pre-treatment
All samples were collected during the Austral Summer International Krill Synoptic Survey on board the RV Kronprins-Haakon and the RV Cabo de Hornos [4][5][6] . Krill and fish samples were collected in the Bransfield Strait (Antarctic Peninsula) and the South Scotia Sea,using a 42 m long macroplankton trawl, with a 36 m 2 mouth opening, and a 3 mm mesh light [4 , 5] . From each catch a subsample of 20-30 krill and 2-3 lantern-fish individuals were collected randomly and wrapped in aluminum foil envelopes before freezing them at −20 °C.
Frozen samples were later transported to laboratory and freeze-dried overnight. Once the constant weight was reached, they were taken with stainless steel tweezers and placed in aluminum foil envelopes until the extraction and chemical analysis.

Analytical protocol
The extraction protocol was adapted from Santos et al. [1] . For both krill and fish, 0.1 g of dried matrix was used.
8. Transfer the supernatant into a glass tube. 9. Repeat steps 4-7 two times and collect the supernatant always in the same tube. 10. Reduce the volume of the extracts (to 50%, approx. 1.5 mL) using a N 2 evaporator in order to eliminate the excess of organic solvent. 11. Place the extract in a glass bottle with 100 mL of HPLC-grade water at pH 6.5 adjusted with ammonia and formic acid. 12. Clean the glass tubes with 3 mL of HPLC-grade water (three times). 13. Stabilize the mixed-mode SPE cartridges (see Note #3 ) with HPLC-grade methanol and HPLCgrade water at gravity. 14. Load the cartridges with the sample volume at 1drop s −1 approx. 15 Note #1 : In order to evaluate proper extraction of the method for each sample, add 20 μL of a solution 1 μg ·mL-1 of selected compounds. Compounds added in the validation are shown in Table 1 . For surrogate addition, a mixture of Clothianidine-d3, Caffeine-d3 and Benzotriazole-d4 in acetone was prepared.
Note #2: In order to prepare the solvents for the extractions, two individual solutions were prepared. For the first one (S1), weigh 19.213 g of citric acid and dissolve them in 10 0 0 mL of HPLC water. For the second one (S2), weigh 14.705 g of tri-sodium citrate 2-hydrate and dissolve them in 10 0 0 mL of HPLC water. Mix 118 mL of S1 with 82 mL of S2 and mix carefully. Then, add 200 mL volume of S1:S2 (59:41) mixture to 200 mL acetonitrile.

Instrumental analysis
After sample extraction, 10 μL of sample extracts are directly injected (avoiding filtration steps in order to minimize compound losses as much as possible) in the UPLC-HRMS instrument under the following chromatographic conditions:  For HRMS, parameters applied for the analysis were:

Quality assurance and quality control
In order to prevent background contamination, all glassware was previously washed with ethanol and acetone and heated overnight at 450 °C. Furthermore, nitrile gloves were worn during the process. For avoiding compounds photodegradation during all the process, solutions were in amber bottles and stored in freezer at −20 °C in the dark.
Additionally, procedural blank samples were processed following all the steps of the extraction protocol (except step 3). These blanks were used for ensuring there has not been any background contamination during the sample treatment procedure.
A mixture of selected compounds ( Table 1 ) prepared in acetone was added directly into the extraction in step 4, allowing solvent evaporation keeping the tube at room temperature for 30-60 min.

Method validation
The method performance was evaluated with a set of compounds including pharmaceuticals, personal care products, herbicides, food additives and other industrial chemicals. The selected compounds comprised a wide range of polarity (log P comprised between −1.16 and 7.03) to ensure a good method performance in both the extraction and the clean-up steps in each sample (see Table 1 ).
The linear dynamic range, based on linear regression calibration curves prepared in each matrix, was studied in standard solution at four different concentration levels, ranging from 25 to 500 μg L −1 . All areas were integrated using the extracted ion chromatogram for the corresponding m/z of the parent ion with a window of 5 ppm. A good linearity range was obtained for almost all compounds. Out of the evaluated 27 compounds, 18 and 20 showed coefficient R 2 > 0.98 in krill and lantern fish, respectively.
In order to evaluate the extraction efficiency absolute recoveries were determined by spiking both krill and lantern fish with a standard mixture at two concentrations (20 and 150 ng g −1 ). Recovery values between 50 and 130% were obtained for > 70% of compounds. Few compounds showed values out of this range indicating poor extraction and/or important matrix effects. Overall results were very satisfactory considering the wide range of physicochemical properties covered by the selected compounds and the complexity of the biologic matrices of interest. The method precision was estimated with repeatability values, in terms of %RSD, for three experimental replicates, showing values below 20% in most cases.
Limits of detection were estimated based on the signal to noise (S/N) ratios of low concentration matrix calibration standards. The method limits of detection (equivalent of S/ N = 3) obtained varied from 0.01 to 10.67 ng g −1 d.w. and from 0.01 to 15.79 ng g −1 d.w. for krill and lantern fish, respectively. Results show the overall good sensitivity of the methodology for the screening of a wide range of xenobiotics in complex biota samples.
Matrix effects (signal suppression or enhancement) were also calculated for the selected compounds in both matrices according to Eq. (1) : Matrix e f f ect ( % ) : ( Area matrix − Area blank ) Area solv ent − 1 x 100 (1) Where Area matrix corresponds to the response given by the instrument for the selected compound in a spiked matrix sample, Area blank is the response given by the instrument in a non-spiked matrix samples and Area solvent is the response given by the instrument in a solvent spiked sample.
Almost all compounds showed ion suppression, with values ranging from −4% to −92% in krill and from −14% to −82% in lantern fish extracts. Only hydroxychloroquine and pilocarpine showed ion enhancement in both matrices.
Overall, the developed methodology showed a very good performance for the determination of a wide range of xenobiotics in biological matrices. This is particularly certain considering the differences in polarity and other physicochemical properties of the selected compounds for the validation step. However, if the final aim of the user is to provide reliable quantitative data of new compounds identified through suspect and non-target approaches, it is advisable to fully validate the methodology for the newly identified substances.