Kaspofungina antifungikoaren adsortzioaren azterketa beirazko materialean UHPLC-FLUOren bidez

[EUS] Farmakoen analisi egokia egiteko, egonkortasuna eta adsortzioa bezalako parametroak kontuan hartu behar dira. Kaspofungina antifungikoaren analisia erronka handia da farmakoak beirara eta plastikora itsasteko duen joera dela eta. Metodo analitiko egokiak garatzeko eta molekularen aktibitate antifungikoa aztertzeko, ezinbestekoa da adsortzio mekanismo horiek ulertzea eta kontrolatzea. Lan honen helburua da kaspofunginaren adsortzioa aztertzea beirazko materialean eta adsortzioa ekiditen duen silanizatzailea aurkitzea.[ES] Para un correcto análisis de los fármacos es necesario tener en cuenta parámetros como estabilidad y adsorción. El análisis de la caspofungia antifúngica supone un gran reto por la tendencia del fármaco a adherirse al vidrio y al plástico. Para el desarrollo de métodos analíticos adecuados y el análisis de la actividad antifúngica de la molécula es necesario comprender y controlar estos mecanismos de adsorción. El objetivo de este trabajo es analizar la adsorción de la caspofungina en el material de vidrio y encontrar un silanizador que evite la adsorción

Kaspofungina antifungikoaren adsortzioaren azterketa beirazko materialean UHPLC-FLUOren bidez

[EUS] Farmakoen analisi egokia egiteko, egonkortasuna eta adsortzioa bezalako parametroak kontuan hartu behar dira. Kaspofungina antifungikoaren analisia erronka handia da farmakoak beirara eta plastikora itsasteko duen joera dela eta. Metodo analitiko egokiak garatzeko eta molekularen aktibitate antifungikoa aztertzeko, ezinbestekoa da adsortzio mekanismo horiek ulertzea eta kontrolatzea. Lan honen helburua da kaspofunginaren adsortzioa aztertzea beirazko materialean eta adsortzioa ekiditen duen silanizatzailea aurkitzea.[ES] Para un correcto análisis de los fármacos es necesario tener en cuenta parámetros como estabilidad y adsorción. El análisis de la caspofungia antifúngica supone un gran reto por la tendencia del fármaco a adherirse al vidrio y al plástico. Para el desarrollo de métodos analíticos adecuados y el análisis de la actividad antifúngica de la molécula es necesario comprender y controlar estos mecanismos de adsorción. El objetivo de este trabajo es analizar la adsorción de la caspofungina en el material de vidrio y encontrar un silanizador que evite la adsorción

A novel SPE-UHPLC-DAD method for the determination of fumagillin produced by Aspergillus fumigatus in cell culture media

[EN]Fumagillin is a biomolecule produced by Aspergillus fumigatus that is gaining relevance due to its connection with invasive aspergillosis. The determination of this molecule might help to understand the propagation of this disease and study its use as a potential biomarker. In spite of the interest of fumagillin in microbiological research, no quantitative method has been developed so far for its determination in cell culture media. In this work, the first validated method for the quantitative analysis of fumagillin in RPMI-1640 is presented. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled the validation criteria established by EMA and FDA guidelines for bioanalysis (selectivity, carry over, linearity, accuracy, precision, dilution integrity and stability) and offers a limit of quantitation (25 μg·L−1) suitable for its intended use. Indeed, the method was satisfactorily applied to the quantification of the fumagillin produced by three strains of Aspergillus fumigatus with different toxin production capacity.This research was funded by University of the Basque Country (UPV/EHU) (project GIU19/068 and project COLAB20/11) and by Basque Government (grant number IT1362-19). XGP was funded by Basque Government

Fumagilina mikotoxinak aspergilosi inbaditzailearen garapenean duen rola aztertzeko SPE-UHPLC-DAD metodo analitikoa

Invasive aspergillosis caused by Aspergillus fumigatus is a threat for immunocompromised patients. According to recent studies, fumagillin, a mycotoxin produced by this fungus, has been associated with the propagation of the disease. Therefore, this molecule might help to understand the mechanisms of this disease and to study the use of fumagillin as a potential biomarker of invasive aspergillosis. In spite of the relevance of fumagillin analysis in microbiological research, no quanti-tative method has been developed so far for its determination in cell culture media. Here, we present the first validated method for the quantitative analysis of fumagillin in RPMI-1640. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled all the validation criteria established by EMA (European Medicine Agency) and FDA (Food and Drug Administration) guidelines for bioanalysis. Finally, the method was satisfactorily applied to the quantification of the fumagillin produced by different strains of Aspergillus fumigatus and it was observed that they had a different micotoxin production capacity.; Aspergillus fumigatus onddoak sortutako aspergilosi inbaditzailea mehatxua da immunoeskasia duten gai-xoentzat. Azkeneko ikerketa batzuen arabera, fumagilinak, onddoak sortutako mikotoxinak, gaixotasunaren hedapenarekin zerikusia duela ikusi da. Hori dela eta, konposatu honen determinazioa lagungarria izan daiteke bai gaixotasunaren mekanismoak hobeto ulertzeko eta baita aspergilosi inbaditzailearen biomarkatzaile gisa erabili ahal izateko ere. Ikerketa mikro-biologikoetan fumagilinaren analisiak garrantzia izan arren, oraindik ez da haren determinaziorako metodo kuantitatiborik garatu zelula-hazkuntzako inguruneetan. Beraz, lan honetan fumagilinaren determinazio kuantitatiborako lehenengo metodo analitikoa balidatu da RPMI-1640 zelula-hazkuntzako ingurunean. Laginaren tratamendua fase solidoko erauzketarekin egin da, anioi trukatzaile sendoak diren modu mistoko kartutxoak erabiliz. Horrela, egon daitezkeen interferentziak modu eraginkorrean ezabatu dira, eta % 83 ± 7ko berreskurapena lortu da. Analisia fotodiodo detektagailuari akoplaturiko bereizmen oso altuko likido kromatografia erabiliz egin da 336 nm-ko uhin-luzeran. Horrela, metodoak EMA (Europako Medikamentuen Agentzia) eta FDA (Elikagai eta Sendagaien Administrazioa) agentziek balidazio bioanalitikoetarako zehazten dituzten parametro guztien onartze-irizpideak betetzen dituela egiaztatu da. Gero, metodoa A. fumigatus-en lau andui analizatzeko aplikatu da, eta bakoitzak mikotoxinaren kantitate desberdina ekoizteko gaitasuna daukala ikusi da

Study of antifungal agent caspofungin adsorption to laboratory materials

[EN] Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.The authors thank University of the Basque Country (UPV/EHU) (Project GIU19/068 and Project COLAB20/11) and Basque Government (grant number IT1362-19) for financial support. B. Uribe thanks UPV/EHU for the pre-doctoral fellowship in co-supervision with the University of Bordeaux. X. Guruceaga thanks the Basque Government for his predoctoral grant