1. Introduction
Cyanobacteria are a group of primitive microorganisms present in several geographical areas worldwide that are able to grow and proliferate in all aquatic and terrestrial ecosystems, from tropical forests to deserts, oceans, and lakes [
1]. Eutrophication and climate change may enhance the bloom of cyanobacteria, increasing the amount of bioactive secondary metabolites in freshwaters, compounds that might pose a risk to humans, animals, and plants [
2]. Among these secondary metabolites, the alkaloid Cylindrospermopsin (CYN) is an emerging toxin, originally produced by the cyanobacteria
Cylindrospermopsis raciborskii, but now exhibiting a cosmopolitan distribution pattern [
3]. CYN producers belong to several genera, such as
Aphanizomenon,
Anabaena,
Raphidiopsis,
Lyngbya, and
Umezakia [
4,
5,
6,
7,
8], recorded from a great variety of habitats. CYN is a cytotoxin with the structure of a tricyclic guanidine along with a uracil ring [
4].
The mechanisms of the toxic action of CYN in several organs are not totally clarified [
1], although evidence suggests that, both in vitro and in vivo, CYN induced a concentration-dependent protein and glutathione synthesis inhibition as well as genotoxicity due to DNA fragmentation [
9,
10,
11,
12,
13]. Besides, CYN activation by cytochrome P450 seems to be related to its increased toxicity [
14], suggesting that the initial compound and its possible metabolites could act by different mechanisms [
1]. Oxidative stress is also involved as a mechanism of CYN toxicity in both in vitro [
15,
16] and in vivo assays [
17,
18,
19]. For consumers protection and to prevent CYN adverse effects, 0.03 µg kg
−1 of body weight (b.w.) has been settled as the provisional Tolerable Daily Intake (TDI) for this toxin [
20].
In comparison to mammals, the amount of studies that investigated the physiological and biochemical effects of CYN on diverse plants is scarcer. These studies have been reviewed by Corbel et al. (2014) [
21] and, more recently, by Machado et al. (2017) [
2]. These effects include alterations in growth [
22,
23,
24,
25], germination, and development [
26,
27], oxidative stress [
28,
29], decreases in chlorophyll [
30], chromosomal aberrations [
31], changes in mineral content [
25,
29] and in the proteome [
32].
Generally, humans can be exposed to cyanotoxins by the oral route through several pathways: when drinking water contaminated with cyanotoxins, when consuming fish, vegetables, crops and even food supplements susceptible to contain cyanotoxins, or accidentally by ingestion of water while performing recreational activities [
1]. The ability of cyanotoxins such as microcystins (MCs) to accumulate in the tissues of a wide range of agricultural crops has been described and reviewed [
2,
21,
33], although only a few studies have been carried out on the leaves of edible plant species, such as lettuce
(Lactuca sativa) [
34,
35,
36].
However, in spite of the human health risks associated with CYN, less attention has been given to its bioaccumulation in edible agricultural plants compared with MCs [
37], which is considered a public health concern, given CYN's high water-solubility and its ability to transfer to higher trophic levels [
38]. This bioaccumulation may be possible because of the absorption of toxins by plants if surface water contaminated with cyanotoxins is used in agriculture, thus posing risks for food safety. In this sense, field studies have shown a broad range for concentrations of CYN in waters worldwide, although ecologically relevant CYN levels potentially used for the irrigation of vegetables ranged between 5 and 100 µg L
−1 [
19]. At these concentrations, the ability of CYN to enter the food web by this last route has been minimally studied. To fill this gap in knowledge, advances in this field could provide helpful information for public health, concerning the bioaccumulation of CYN in plants and how water resources should be used to minimize crop contamination. White et al. (2005) [
39] suggested that the aquatic macrophyte
Hydrilla verticillata might adsorb CYN in the plant cell walls instead of taking up the toxin through the cells. CYN transdermal absorption could represent an important route of plant uptake because of CYN high presence in dissolved form in the environment [
40,
41]. Silva & Vasconcelos (2010) [
27] observed that the roots of
L. sativa,
Phaseolus vulgaris, and
Pisum sativum accumulated higher concentrations of CYN in comparison to the stems. Additionally, Prieto et al. (2011) [
28] found accumulation of CYN in plants of
Oryza sativa exposed to 2.5 μg CYN L
−1 from an extract, showing a higher amount of the toxin in the roots, compared to the leaves. CYN was also detected in various
Brassica vegetables (roots and leaves) after exposure to a CYN-containing extract, and this accumulation seemed to depend on the concentrations applied to the roots (18–35 μg L
−1), with the levels of CYN ranging from 10 to 21% in the leaves [
42]. Recently, a study showed that the bioaccumulation of CYN in lettuce
(L. sativa) and arugula (
Eruca sativa) depends indirectly on the exposure concentration, and also on time and on the species [
37]. For example, after 7 days of exposure, the CYN concentrations measured in lettuce leaves were 8.29, 4.19, and 3.78 µg CYN kg
−1 for exposure concentrations of 3, 5, and 10 µg CYN L
−1, respectively. In the case of arugula, these values were 11.49, 10.41, and 9.47 µg CYN kg
−1 for the three levels of exposure.
Fresh products such as lettuce, spinach, cabbage, and sprouts are processed minimally. Their consumption has increased over the last decade, maybe because of their high nutritional value, changes in social eating habits, and wider accessibility [
43]. Some recommendations by international organizations and limit values have been set worldwide in order to prevent or manage the potential effects on human health induced by the exposure to CYN under different scenarios [
1]. However, no recommendations or limits have been established so far in the case of vegetables, despite the results demonstrating the risk of ingesting CYN through contaminated vegetables [
37]. Following the recommendations of the European Food Safety Authority (EFSA), there is a need to develop analytical methods for sample preparation and detection of cyanotoxins in complex matrices such as food items [
44]. In this sense, some methods for cyanotoxins analysis in different matrices such as natural blooms, cyanobacterial strain cultures, and biological samples, are reported in the literature and use Liquid Chromatography (LC)–Mass Spectrometry techniques [
36,
45,
46,
47,
48,
49,
50,
51], whose criteria and applications have been recently reviewed by Caixach et al. (2017) [
52]. Concerning CYN, no validation procedures with robustness tests have been developed in vegetables compared to other matrices such as waters, cyanobacterial cultures, or fish tissues [
50,
51,
53]. In fact, and specifically in relation to the matrices assayed in the present study, CYN has been detected in
Brassica vegetables, lettuce, and arugula by ELISA and LC–MS/MS [
37,
42], but these methods have not been validated. Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) allows excellent specificity and sensitivity for cyanotoxins detection and quantification in waters and also in more complicated matrices, becoming the technique of choice for these purposes [
54,
55,
56].
For these reasons, the present work aimed for the first time to develop and validate an analytical method based on UPLC–MS/MS for the extraction and quantification of CYN in edible vegetables samples (
Lactuca sativa L.). The method has been optimized and validated according to international guides [
57,
58]. The present procedure has been designed for the routine determination of CYN in leaves of edible lettuces intended for human consumption, for prevention and risk assessment purposes.
4. Materials and Methods
4.1. Chemicals and Reagents
Cylindrospermopsin standard (95% purity) was purchased from Alexis Corporation (Lausen, Switzerland). CYN standard solutions were prepared in Milli-Q water (100 µg mL−1) and further diluted for their use as working solutions (5–500 µg L−1). All reagents and chemicals employed in this work were of analytical grade. HPLC-grade methanol, acetonitrile, acetic acid, dichloromethane, and trifluoroacetic acid (TFA) were supplied by Merck (Darmstadt, Germany). Deionized water (418 MΩ cm−1 resistivity) was acquired from a Milli-Q water purification system (Millipore, Bedford, MA, USA). BOND ELUT® Carbon cartridges (PGC, 500 mg, 6 mL) were obtained from Agilent Technologies (Amstelveen, The Netherlands, Europe). Reagents for UHPLC–MS/MS were of LC–MS grade; formic acid was supplied by Fluka (Steinheim, Germany) and water and acetonitrile by VWR International (Fontenay-sous-Bois, France). The lettuce samples without CYN were purchased from a local supermarket. In this way, we ensured that they had passed the quality controls required for their human consumption.
4.2. Solvent Extraction and Purification Procedures
Preliminary tests were performed to evaluate the influence of the lyophilization process on CYN concentration. For this, 1 mL of a CYN solution (200 μg L−1) was added to 1 g of lettuce (fresh weight, f.w.) and the sample was lyophilized (−80 °C). In parallel, 1 mL of the same CYN solution (200 μg L−1) was added to 0.05 g of lyophilized lettuce (dry weight, d.w.), corresponding to 1 g of f.w. These procedures were performed in triplicate. Subsequently, CYN was extracted from all the samples to evaluate the influence of these processes. Since no significant differences were observed between the conditions (data not shown), we decided to work with fresh-weight lettuce, once the plants had been exposed to the toxin, as they reach the final consumer.
To develop the extraction and purification procedures, a modified version of the method from Kittler et al. (2012) [
42] was performed, adding a purification stage with graphitized carbon cartridges. Briefly, control fresh lettuce leaves (1.05 ± 0.14 g f.w.) were fortified with CYN standard solutions to obtain three concentration levels: 20, 200, and 500 ng CYN g
−1 f.w. Afterwards, the toxin was extracted with 6 mL of 10% acetic acid; after ultraturrax homogenization for 30 s, the sample was sonicated (15 min) and stirred (15 min). The resulting mixture was centrifuged at 12,000 rpm for 15 min. Once the extract was obtained, a purification step was applied. For this purpose, BOND ELUT
® Carbon cartridges were activated with 10 mL of a solvent mixture of DCM/MeOH (10/90) acidified with 5% formic acid and rinsed with 10 mL of Milli-Q water. Then, the sample was loaded, the column was washed with 10 mL of Milli-Q water, and the analyte was eluted with 10 mL DCM/MeOH (10/90) acidified with 5% formic acid. To concentrate the sample, the extract was evaporated in a rotary evaporator and resuspended in 1 mL Milli-Q water, prior to its UPLC–MS/MS analysis.
4.3. Chromatographic Conditions
The chromatographic separation was carried out with a UPLC Acquity (Waters, Milford, MA, USA) coupled to Xevo TQS-micro (Waters, Milford, MA, USA) consisting of a triple quadrupole mass spectrometer equipped with an electrospray ion source operated in positive mode. UPLC analyses were performed on a 50 × 2.1 mm Acquity BEH C18 1.7 µm column, at a flow rate of 0.45 mL min−1. For chromatographic separation, a binary gradient was used with (A) water and (B) methanol as mobile phases, containing, both of them, 0.1% formic acid (v/v). The injection volume was 5 µL. The profile for elution was: 0% B (0.8 min), linear gradient to 90% B (2.2 min), 90% B (1 min), and finally 100% B (1 min). Multiple Reaction Monitoring (MRM) was applied, by which the parent ions and fragments ions were monitored at Q1 and Q3, respectively. The transitions for the analyte CYN are 416.2/194.0 and 416.2/176.0. The transition 416.2/194.0 (quan) was chosen for quantitation of CYN, and the transition 416.2/176.0 (target) as confirmatory. The ion ratio were measured in the CYN standard solution and in the lettuce samples as target area/quan area × 100, giving a mean ion ratio of 38.8% with SD of 1.2% for the CYN standard solution (n = 10), and a mean ion ratio of 39.6% with SD of 5.1% for CYN in the lettuce samples (n = 5) exposed to the toxin. For UPLC–ESI–MS/MS analyses, the mass spectrometer was set to the following optimised tune parameters: Capillary voltage: 3.0 kV, Source Temperature: 500 °C, and source Gas flow: 1000 L h−1.
4.4. Experimental Exposure of Vegetables and Application of the Validated Method
The lettuce plants (
Lactuca sativa) were purchased from a local market (Porto, Portugal) as sprouts. The roots were washed with deionized water to remove all remaining soil and prepare the plants for hydroponic cultivation. The plants were introduced into opaque glass jars, and the roots were completely immersed in culture medium [
70] at pH 6.5, as described by Freitas et al. (2015) [
29]. The plants were acclimated for one week with white fluorescent light (light–dark period of 14–10 h), at a controlled temperature of 21 ± 1 °C until the start of the experiment.
The
Chrysosporum ovalisporum (LEGE X-001) cyanobacterial CYN-producing strain (CYN+) was isolated from Lake Kinneret, Israel [
6] and was grown for biomass production in the Interdisciplinary Centre of Marine and Environmental Research, CIIMAR (Porto, Portugal) as described in Guzmán-Guillén et al. (2014) [
71]. The extraction of CYN from the culture was performed according to Guzmán-Guillén et al. (2012) [
50] with modifications described in Guzmán-Guillén et al. (2017) [
55]. CYN retention time was 9.55 min, and the concentration obtained was 2.14 μg CYN mg
−1 of lyophilized cells.
After acclimation, five specimens of lettuce were exposed to a solution of CYN extracted from the culture at a concentration of 10 µg L−1. In order to do this, the culture medium was changed three times a week for 21 days, adding to the medium the volume of C. ovalisporum culture necessary to reach the exposure level (10 μg L−1). Moreover, five lettuce plants were included in the experiment as a control group without exposure to the toxin. When the experiment was over, the plants were washed with distilled water, frozen, and lyophilized (Telstar Lyoquest) for CYN extraction, following the validated method proposed in the present study.
4.5. Method Validation
The proposed method was validated taking into account the Eurachem (2016), the AOAC (2016), and González and Herrador (2007) guides [
57,
58,
63] for linearity, sensitivity, precision, recovery, and robustness. Three CYN validation standards were employed, performing the measures in triplicate each day for three consecutive days, and these concentrations tried to cover the optimal working range.
One mL of solutions at three different CYN concentrations (20, 200, and 500 µg CYN L
−1) was added to the plant matrix to obtain 20, 200, and 500 ng g
−1 f.w. Precision and recovery were obtained by applying a one-factor analysis of variance (ANOVA), as explained in the Results and Discussion section, and then they were compared with tabulated reference values. Moreover, a robustness (ruggedness) study was performed by spiking the matrices with an intermediate validation standard of 200 µg CYN L
−1 (equivalent to 200 ng g
−1 f.w.), according to Youden's procedure (1967) [
67]. Small, deliberate variations in the following parameters were tested with Student’s t test in order to evaluate the ruggedness of the method: (1) time for the sample to pass through the cartridge; (2) water volume for washing the cartridge; and (3) volume of DCM/MeOH employed for CYN elution.