https://hdl.handle.net/10481/18091 Wed, 08 Apr 2026 16:06:09 GMT 2026-04-08T16:06:09Z https://hdl.handle.net/10481/91888 Capillary electrophoresis tandem mass spectrometry to determine multiclass cyanotoxins in reservoir water and spinach samples Carmona Molero, Rocío; Aparicio Muriana, María del Mar; Lara Vargas, Francisco Jesús; García Campaña, Ana María; Olmo Iruela, María Monsalud Del Cyanotoxins constitute a group of toxic secondary metabolites, the presence of which in any water body poses a major health risk. Moreover, advanced organisms such as edible plants exposed to these toxins, are a possible pathway for human exposure. Green analytical chemistry is demanding environmentally friendly analytical techniques. In this sense, we propose the use of capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) to determine a mixture of eight cyanotoxins belonging to three different classes: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin and anatoxin-a) and three isomeric non-protein amino acids (β-methylamino-l-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). Separation was achieved by using an acidic background electrolyte consisting of 2 M formic acid and 20% acetonitrile in water. Parameters affecting MS/MS detection and the sheath-liquid interface were also studied. Finally, a combination of pH-junction, field-amplified sample stacking (FASS) and acid barrage as online preconcentration strategies, was employed to improve sensitivity and efficiency. The online preconcentration applied, in combination with a dual cartridge solid-phase extraction (SPE) system, allows to obtain limits of detection in the very low range of µg·L−1 for these multiclass cyanotoxins in reservoir water samples (from 0.005 to 0.10 µg·L−1). Furthermore, for the first time cyanotoxins are analysed in spinach samples through CE-MS/MS using the same SPE procedure, following lyophilisation and solid-liquid extraction with 6 mL 80 % aqueous MeOH.; Las cianotoxinas constituyen un grupo de metabolitos secundarios tóxicos, cuya presencia en cualquier masa de agua supone un importante riesgo para la salud. Además, los organismos avanzados, como las plantas comestibles expuestas a estas toxinas, son una posible vía de exposición humana. La química analítica verde exige técnicas analíticas respetuosas con el medio ambiente. En este sentido, proponemos el uso de electroforesis capilar acoplada a espectrometría de masas en tándem (CE-MS/MS) para determinar una mezcla de ocho cianotoxinas pertenecientes a tres clases diferentes: péptidos cíclicos (microcistina-LR, microcistina-RR y nodularina), alcaloides (cilindrospermopsina y anatoxina-a) y tres aminoácidos isoméricos no proteicos (β-metilamino-l-alanina, ácido 2,4-diaminobutírico y N-(2-aminoetil)glicina). La separación se logró utilizando un electrolito ácido que consistía en ácido fórmico 2 M y acetonitrilo al 20 % en agua. También se estudiaron los parámetros que afectan la detección MS/MS. Finalmente, se empleó una combinación de unión de pH, preconcentración de muestras amplificada en campo (FASS) y bombardeo ácido como estrategias de preconcentración en línea para mejorar la sensibilidad y la eficiencia. La preconcentración en línea aplicada, en combinación con un sistema de extracción en fase sólida (SPE) de doble cartucho, permite obtener límites de detección en el rango de µg/L para estas cianotoxinas multiclase en muestras de agua de embalses (de 0,005 a 0,10 µg/L). Además, por primera vez se analizan las cianotoxinas en muestras de espinacas mediante CE-MS/MS utilizando el mismo procedimiento SPE, tras liofilización y extracción sólido-líquido con 6 ml de MeOH acuoso al 80 %. Este estudio fue financiado por el Proyecto de Investigación PID2021-1278040B-I00 del MCIN/AEI /10.13039/501100011033 y “FEDER Una forma de hacer Europa”, y por el proyecto PROYEXCEL_00195 financiado por la Consejería de Universidad, Investigación e Innovación, Junta de Andalucía. RCM agradece al contrato predoctoral del Proyecto PID2021-1278040B-I00. Financiación para publicación en acceso abierto a cargo de Universidad de Granada / CBUA. https://hdl.handle.net/10481/91888 https://hdl.handle.net/10481/76952 Non-aqueous capillary electrophoresis–time of flight mass spectrometry method to determine emerging mycotoxins Delgado-Povedano, María del Mar; Lara Vargas, Francisco Jesús; Gámiz Gracia, Laura; García Campaña, Ana María Las eniatinas (ENN) y la beauvericina (BEA) son micotoxinas emergentes que tradicionalmente se han determinado mediante cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS). Sin embargo, hasta donde sabemos, hasta el momento no se han publicado métodos analíticos basados ​​en electroforesis capilar (CE)-MS/MS. Debido a su naturaleza no polar, en este trabajo se propone por primera vez un método de CE no acuosa (NACE) acoplada a MS de cuadrupolo-tiempo de vuelo para identificar y cuantificar estas micotoxinas. La determinación se logró en 4 min en condiciones óptimas: acetato de amonio 40 mM en acetonitrilo-metanol 80:20 (v/v) (tampón), 30 kV (voltaje), 80 cm (longitud del capilar), 20 °C (temperatura del capilar) y 50 mbar × 30 s (inyección). Se puede lograr una mayor selectividad en comparación con LC debido a la formación de aductos CE exclusivos como [M + CH3CH2NH3]+. Se seleccionó el modo de adquisición “All Ions” ya que permite la cuantificación de los ENN habituales, así como la identificación de las ENN menos comunes. El método fue validado para muestras de trigo, obteniendo límites de cuantificación de 4,0 a 8,3 μg/kg dependiendo de la micotoxina emergente, valores de recuperación superiores al 87,4 % y valores de precisión intra e interdiarios (RSD) inferiores al 15,1 % en todos los casos. Finalmente, se analizaron 29 muestras de trigo, encontrando 26 muestras con concentraciones de eniatina B superiores al límite de cuantificación (7,5-1480 μg/kg), 20 de eniatina B1 (5,2-550 μg/kg), 7 de eniatina A (10 –55 μg/kg), 4 para eniatina A1 (12,6-77 μg/kg) y 5 para BEA (9,2-16,4 μg/kg). Además, se identificaron tentativamente otras dos ENN. Enniatins (ENN) and beauvericin (BEA) are emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)–MS/MS have been reported so far. Due to their non-polar nature, in this work, a non-aqueous CE (NACE) method coupled to quadrupole time-of-flight–MS is proposed for the first time to identify and quantify these mycotoxins. Determination was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) acetonitrile-methanol (buffer), 30 kV (voltage), 80 cm (capillary length), 20 °C (capillary temperature) and 50 mbar × 30 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M + CH3CH2NH3]+. “All Ions” acquisition mode was selected as it allows the quantification of the usual ENNs, as well as the identity confirmation of less common ENNs. The method was validated for wheat samples, obtaining limits of quantification from 4.0 to 8.3 μg/kg depending on the emerging mycotoxin, recovery values higher than 87.4%, and intra- and inter-day precision values (RSDs) lower than 15.1% in all cases. Finally, 29 wheat samples were analyzed, finding 26 samples with concentrations of enniatin B higher than the limit of quantification (7.5–1480 μg/kg), 20 for enniatin B1 (5.2–550 μg/kg), 7 for enniatin A (10–55 μg/kg), 4 for enniatin A1 (12.6–77 μg/kg) and 5 for BEA (9.2–16.4 μg/kg). Moreover, two other ENNs were tentatively identified. https://hdl.handle.net/10481/76952 https://hdl.handle.net/10481/74305 Sweeping-micellar electrokinetic chromatography with tandem mass spectrometry as an alternative methodology to determine neonicotinoid and boscalid residues in pollen and honeybee samples Carbonell Rozas, Laura; Horstkotte, Burkhard; García Campaña, Ana María; Lara Vargas, Francisco Jesús En este trabajo se propone por primera vez un método electroforético basado en cromatografía capilar electrocinética micelar acoplada con espectrometría de masas en tándem (MEKC-MS/MS) para la determinación simultánea de 9 neonicotinoides (NNI) junto con el pesticida boscalid en muestras de polen y abejas. La separación se llevó a cabo empleando perfluorooctanoato amónico (50 mM, pH 9'0), que sirvió tanto de surfactante volátil como de tampón electroforético compatible con la detección por espectrometría de masas. Se llevó a cabo la preconcentración en línea de los analitos mediante un procedimiento conocido como "barrido" para mejorar la eficacia de la separación y la sensibilidad. Además, se desarrolló un Quechers miniaturizado como tratamiento de muestra. Ello implicó un menor consumo de disolvente orgánico y el empleo de Z-Sep+ como sorbente dispersante en la etapa de limpieza. La detección se realizó con un triple cuadrupolo en electroespray positivo y mediante monitorización de reacción multiple (MRM). Para conseguir una sensibilidad y precisión satisfactorias se optimizaron los principales parámetros que afectan a la detección como la composición del líquido adicional (etanol, agua, ácido fórmico: 50, 49'5, 0'5 v/v/v) y otras variables relacionadas con el electroespray. Se establecieron curvas de calibrado procedimentales en muestras de polen y abejas. Se consiguieron límites de cuantificación por debajo de 11'6 µg/kg y 12'5 µg/kg, respectivamente. La precisión expresada como desviación estándar relativa (RSD) fue inferior al 15'2 % y las recuperaciones fueron mayores al 70 % en ambos tipos de muestras. Se encontraron dos muestras positivas de polen que contenían imidacloprid y tiametoxam. Imidacloprid se encontró también en una muestra de abejas. Los resultados obtenidos resaltan la viabilidad del método propuesto que supone una alternativa útil, sensible, eficiente y respetuosa con el medioambiente para la determinación de NNI y boscalid en muestras de polen y abejas. In this work, it is proposed for the first time an electrophoretic approach based on micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) for the simultaneous determination of nine neonicotinoids (NNIs) together with the fungicide boscalid in pollen and honeybee samples. The separation was performed using ammonium perfluorooctanoate (50 mM, pH 9) as both volatile surfactant and electrophoretic buffer compatible with MS detection. A stacking strategy for accomplishing the on-line pre-concentration of the target compounds, known as sweeping, was carried out in order to improve separation efficiency and sensitivity. Furthermore, a scaled-down QuEChERS was developed as sample treatment, involving a lower organic solvent consumption and using Z-Sep+ as dispersive sorbent in the clean-up step. Regarding the detection mode, a triple quadrupole mass spectrometer was operating in positive ion electrospray mode (ESI+) under multiple reaction monitoring (MRM). The main parameters affecting MS/MS detection as well as the composition of the sheath-liquid (ethanol/ultrapure water/formic acid, 50:49.5:0.5 v/v/v) and other electrospray variables were optimized in order to achieve satisfactory sensitivity and repeatability. Procedural calibration curves were established in pollen and honeybee samples with LOQs below 11.6 µg/kg and 12.5 µg/kg, respectively. Precision, expressed as RSD, lower than 15.2% and recoveries higher than 70% were obtained in both samples. Two positive samples of pollen were found, containing imidacloprid and thiamethoxam. Imidacloprid was also found in a sample of honeybees. The obtained results highlight the applicability of the proposed method, being an environmentally friendly, efficient, sensitive and useful alternative for the determination of NNIs and boscalid in pollen and honeybee samples. https://hdl.handle.net/10481/74305 https://hdl.handle.net/10481/71025 Multiclass cyanotoxin analysis in reservoir waters: tandem solid-phase extraction followed by zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry Aparicio Muriana, María del Mar; Carmona Molero, Rocío; Lara Vargas, Francisco Jesús; García Campaña, Ana María; Olmo Iruela, María Monsalud Del The presence of cyanobacteria and cyanotoxins in all water bodies, including ocean water and fresh water sources, represents a risk for human health as eutrophication and climate change are enhancing their level of proliferation. For risk assessment and studies on occurrence, the development of reliable and sensitive analytical approaches able to cover a wide range of cyanotoxins is essential. This work describes the development of an HILIC-MS/MS multiclass method for the simultaneous analysis of eight cyanotoxins in reservoir water samples belonging to three different classes according to their chemical structure: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin, anatoxin-a) and three non-protein amino acids isomers such as β-methylamino-L-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). A SeQuant ZIC-HILIC column was employed to achieve the chromatographic separation in less than 12 min. Previously, a novel sample treatment based on a tandem solid-phase extraction (SPE) system using mixed cation exchange (MCX) and Strata-X cartridges was investigated with the aim of extracting and preconcentrating this chemically diverse group of cyanotoxins. The Strata-X cartridge, which was configured first in the line of sample flow, retained the low polar compounds and the MCX cartridge, which was at the bottom of the dual system, retained mainly the non-protein amino acids. The optimization procedure highlighted the importance of sample ion content for the recoveries of some analytes such as the isomers β-Nmethylamino-L-alanine and 2-4-diaminobutyric acid. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and precision in terms of repeatability and intermediate precision. This work represents the first analytical method for the simultaneous analysis of these multiclass cyanotoxins in reservoir water samples, achieving LOQs in the very low range of 7·10-3 – 0.1 µg·L-1. Despite high recoveries obtained at the LOQ concentration levels (101.0-70.9%), relative standard deviations lower than 17.5% were achieved. https://hdl.handle.net/10481/71025 https://hdl.handle.net/10481/67955 Monitoring of cyanotoxins in water from hypersaline microalgae colonies by ultra high performance liquid chromatography with diode array and tandem mass spectrometry detection following salting-out liquid-liquid extraction Hemmati, Maryam; Tejada-Casado, Carmen; Lara Vargas, Francisco Jesús; García Campaña, Ana María; Rajabi, Maryam; del Olmo-Iruela, Monsalud In this study two different analytical approaches have been developed to determine the presence of sev-eral cyanotoxins in saline water samples from a continental salt marsh. A salting-out assisted liquid-liquidextraction (SALLE) has been used in combination with ultra-high performance liquid chromatography-tandem mass spectrometry and UV-diode array detection (UHPLC-MS/MS and UHPLC-DAD). The targetanalytes are eight microcystins named MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, MC-LFand nodularin (NOD), covering a wide range of polarities. The separation was achieved using a ZorbaxEclipse Plus RRHD C18 column (50 × 2.1 mm, 1.8 m) in less than 7.5 and 5.5 min for UV and MS/MSdetection, respectively. The mobile phase used consisted of water (solvent A) and acetonitrile (MeCN)(solvent B), both containing 0.01% of formic acid for DAD and 0.4% of formic acid for MS/MS detection,at a flow rate of 0.4 mL min−1. The temperature of the column was set at 25◦C and 20 L of samplewere injected. The main parameters affecting the SALLE procedure were studied and the following opti-mum values were obtained: neutral pH, 2 mL of acetonitrile as extraction solvent and 1.2 g of ammoniumsulfate as salting-out agent for 4 mL of water sample. The validation protocols for both methods wereaccomplished with real water samples obtaining LODs ranging from 1.0 to 3.4 g L−1and 0.02 to 0.11 gL−1for DAD and MS/MS respectively. Although the SALLE-UHPLC-DAD methodology is easier and cheaperthan UHPLC-MS/MS significantly better detection limits were achieved with tandem mass spectrometryas well as allowing for unambiguous identification. Extraction recoveries were higher than 77.0% (exceptfor MC-RR and NOD which were 53.2% and 54.3, respectively) with satisfactory inter-day and intra-dayprecisions (RSD below 13.3%). https://hdl.handle.net/10481/67955