Determinación de la estructura de secuencias de ADN del tipo A(AT)nT

El ADN es una molécula de importancia vital en los organismos vivos, ya que están directamente relacionados con el material genético de las células. Está formado por una cadena de nucleótidos: adenina (A), citosina (C), guanina (G) y timina (T). Conocer a fondo las posibles estructuras químicas que puede tener el ADN puede ayudar a descubrir su mecanismo de funcionamiento. Este proyecto se centra únicamente en aquellas secuencias de ADN de la forma A(AT)nT, con n igual a 3, 4 y 5; puesto que apenas hay información sobre la estructura de secuencias que contienen únicamente A y T. Para conocer la estructura a nivel atómico de una molécula es preciso utilizar una radiación de longitud de onda comparable a las distancias interatómicas, por lo que la difracción de rayos X es una de las técnicas que permiten un estudio más detallado de la estructura de una molécula. Al interaccionar con los electrones de los átomos dan lugar a diagramas de difracción que, cuando la muestra es un cristal, están formados por manchas más o menos redondas y dispuestas regularmente sobre la imagen. Los diagramas de difracción son analizados mediante programas informáticos (HKL – Denzo y Scalepack) para saber: - celdilla del cristal, grupo espacial y simetría - intensidad de cada difracción Y utilizando un modelo similar y la información anteriormente citada, puede emplearse la metodología del reemplazo molecular para obtener la estructura del cristal. Si la secuencia contiene átomos pesados, puede usarse la técnica MAD para hallar la solución. Se han obtenido múltiples cristales para la secuencia A(AT)4T, pero la resolución de los datos no llegaba para poder conseguir un modelo exacto a través del reemplazo molecular. No obstante, se ha encontrado la celdilla del cristal (44,7x44,7x197,7 Å, 90º 90º 120º) y el empaquetamiento de los dúplex en columnas. El grupo espacial con el que mejores resultados se han obtenido es P3221. Para intentar resolver la secuencia A(AT)4T por MAD, se cristalizó la misma secuencia pero cambiando una T por un bromo-uracilo, ya que tienen un volumen similar, pero el procesado de dichos cristales no ha podido utilizarse por el momento. De las secuencias A(AT)3T y A(AT)5T se han obtenido cristales ya analizados, pero la resolución no era suficiente como para intentar resolver la estructura. Así, se ha calculado la celdilla de cada cristal, dato que servirá para un estudio posterior de patrones de cristalización en secuencias ricas en A-T y debido a similitudes con la secuencia anterior, es posible que también tengan una estructura en columnas

Metabolic study of trimetazidine using ultrahigh performance liquid chromatographytandem mass spectrometry

In the present study, the application of ultra-high performance liquid chromatography-tandem mass spectrometry allowed us to study of known-as well as hitherto unknown-trimetazidine (TMZ) metabolites in human urine and to propose their renal excretion profiles. Urine samples from a healthy volunteer were analyzed at baseline and at 0-4 h, 4-8 h, 8-12 h, and 12-24 h after a single dose of TMZ. A dilute-and-shoot procedure was used as sample treatment before separation. Full-scan spectra of possible metabolites were acquired. Additionally, product ion scan spectra of precursor ions of interest were also acquired at two collision energies. Intact TMZ was a major excretion product, with a maximum concentration at 4-8 h after administration. Moreover, five minor metabolites were observed, namely trimetazidine-N-oxide (M1), N-formyl trimetazidine (M2), desmethyl-trimetazidine O-sulfate (M3), desmethyl-trimetazidine O-glucuronide (M4), and desmethyl-trimetazidine-N-oxide-O-glucuronide (M5). Metabolite M5 has not previously been reported. Excretion curves were constructed based on the chromatographic peak areas of specific mass transitions (precursor ion > product ion) related to each of the detected metabolites

Effects of structural characteristics of (un)conjugated steroid metabolites in their collision cross section value

In this work, the collision cross section (CCS) value of 103 steroids (including unconjugated metabolites and phase II metabolites conjugated with sulfate and glucuronide groups) was determined by liquid chromatography coupled to traveling wave ion mobility spectrometry (LC-TWIMS). A time of flight (QTOF) mass analyzer was used to perform the analytes determination at high-resolution mass spectrometry. An electrospray ionization source (ESI) was used to generate [M+H]+, [M + NH4]+ and/or [M − H]- ions. High reproducibility was observed for the CCS determination in both urine and standard solutions, obtaining RSD lower than 0.3% and 0.5% in all cases respectively. CCS determination in matrix was in accordance with the CCS measured in standards solution showing deviations below 2%. In general, CCS values were directly correlated with the ion mass and allowed differentiating between glucuronides, sulfates and free steroids although differences among steroids of the same group were less significant. However, more specific information was obtained for phase II metabolites observing differences in the CCS value of isomeric pairs concerning the conjugation position or the α/β configuration, which could be useful in the structural elucidation of new steroid metabolites in the anti-doping field. Finally, the potential of IMS reducing interferences from the sample matrix was also tested for the analysis of a glucuronide metabolite of bolasterone (5β-androstan-7α,17α-dimethyl-3α,17β-diol-3-glucuronide) in urine samples.The authors are grateful for the financial support received from Ministerio de Economía y Competitividad (CTQ2015-63968-C2-2-P) and University Jaume I (UJI-B2020-19). The support of Consell Català de l’Esport (Generalitat de Catalunya) and AGAUR (Generalitat de Catalunya) (reference number 2021SGR0040) is also gratefully acknowledged

Determination of polyphenolic profiles by liquid chromatography-electrospray-tandem mass spectrometry for the authentication of fruit extracts

Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12-14 µg/L for 19 of the 26 analyzed polyphenols), linearity (r2 > 0.991), run-to-run and day-to-day precisions (RSD values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone:water:hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods (p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin

L’espectrometria de masses en l’anàlisi de drogues veterinàries

[cat] L'administració continuada d'antibiòtics veterinaris genera recel en el consumidor degut a l'increment de casos de resistència microbiana i d'al·èrgies a medicaments per la presència de residus en aliments d'origen animal. Per aquestes raons, la Unió Europea ha establert límits màxims de residus (MRL) per a les drogues veterinàries permeses en aliments d'origen animal i un nivell de concentració mínim a detectar (minimum required performance limit, MRPL) als mètodes analítics que determinen substàncies prohibides (Reglament 37/2010). Atesa la importància del control de la presència de residus d'antibiòtics en aliments d'origen animal, és necessari disposar de mètodes d'anàlisi ràpids, senzills i amb bona selectivitat i sensibilitat. Les excel·ents prestacions de l'espectrometria de masses tant pel que fa a sensibilitat, selectivitat i a la gran quantitat d'informació estructural que ofereix, especialment acoblada a tècniques de separació, fan que avui dia sigui l'eina de referència en molts laboratoris analítics per a la determinació de compostos orgànics regulats. Per aquest motiu, aquesta tesi està centrada en l'aplicació de l'espectrometria de masses a l'anàlisi de dues famílies d'antibiòtics veterinaris, els fenicols i els aminoglicòsids. En aquesta tesi s'ha estudiat, d'una banda, la separació cromatogràfica dels compostos seleccionats (fenicols i aminoglicòsids) mitjançant cromatografia de líquids acoblada a l'espectrometria de masses amb l'objectiu de desenvolupar mètodes d'anàlisi. L'elevada polaritat dels aminoglicòsids i d'alguns dels fenicols comporta que presentin una baixa retenció en les fases estacionàries convencionals d'octadecilsilà. En aquest context, s'ha avaluat tant la utilització de fases estacionàries alternatives a les d'octadecilsilà per a afavorir la retenció de compostos polars, com els avenços en el desenvolupament de les partícules del rebliment cromatogràfic (partícules sub-2 iim i de nucli sòlid) per a millorar tant les eficàcies com les resolucions obtingudes. Per a la separació dels fenicols, els millors resultats es van obtenir amb la columna de partícula de nucli sòlid fenil-hexil, que va permetre aconseguir una separació ràpida i eficaç i amb una retenció suficient per al metabòlit polar florfenicol-amina. Pel que fa referència als aminoglicòsids, s'ha proposat l'ús d'una columna amb fase estacionària multimodal que proporciona adequada retenció i elució dels anàlits sense necessitat d’emprar formadors de parell iònic o concentracions elevades de sals en la fase mòbil. Els mètodes LC- MS/MS desenvolupats, han demostrat bons paràmetres de qualitat i s'ha aconseguit arribar a la sensibilitat necessària per a determinar els anàlits al nivell de concentració fixat pel MRL de cada matriu. D’altra banda, s’ha estudiat el comportament per espectrometria de masses de les famílies de compostos esmentades, i s’han establert les rutes de fragmentació per poder determinar les transicions més sensibles i selectives. L’estudi de les rutes de fragmentació també ha permès la identificació d’un ió característic per als fenicols, que pot ser emprat en mètodes de cribratge per a la identificació de compostos anàlegs. També s’ha estudiat la formació d'adductes en espectrometria de masses en tàndem amb diferents analitzadors de masses, fenomen que pot distorsionar els espectres de fragmentació i dificultar la identificació de compostos en base a aquests. Així, s’ha determinat que els factors clau en la formació d’aquests adductes són la configuració de l’instrument, especialment pel que respecta a la disposició de la font d’ionització, la humitat del gas de col·lisió emprat i el temps de residència en l’analitzador. Finalment, s’ha aplicat l’anàlisi per injecció en flux (FIA) acoblat a l’espectrometria de masses d’alta resolució pel desenvolupament d'un mètode de cribratge ràpid per a l'anàlisi directa i global de nous productes estupefaents. En aquest cas, s’han avaluat les estratègies d’identificació per espectrometria de masses més adients per a la identificació de compostos desconeguts que han permès identificar la presència de medicaments humans i veterinaris (benzocaïna, levamisol) com a adulterants en mostres d’estupefaents així com la presència de metamizol en una mostra fraudulenta de cocaïna.[eng] The impact of the residues of veterinary drugs on human health is considered one of the biggest problems of the 21st century. For this reason, maximum residue levels (MRLs) for these compounds in foodstuffs of animal origin have been established in many countries. So, there is a need for fast, sensitive and selective methods for the control of the presence of said residues in foods. Liquid chromatography coupled to mass spectrometry is the technique of choice for method development, as it provides high sensitivity and selectivity and high quality spectral information. In this work fast, sensitive and selective LC-MS methods for the determination of two families of veterinary drugs, phenicols and aminoglycosides, in foodstuffs from animal origin have been developed. The liquid chromatography separation of these polar analytes has been evaluated using different stationary phases to achieve good retention and elution for all compounds. A fused-core phenyl-hexyl column has been proposed for the separation of phenicols, whereas a mixed-mode column has been used for the highly polar aminoglycosides. Moreover, the behavior of phenicols and aminoglycosides has been studied using different atmospheric pressure ionization sources to determine the most intense and selective product ions for quantitation and confirmation and to propose their fragmentation pathways. Also, multistage tandem mass spectrometry has been utilized to propose new structures and fragmentation mechanisms for the product ions used for quantitation and confirmation of the studied compounds. Ion-molecule adduct formation inside the mass analyzer has been observed, which can lead to dubious identifications due to the presence of product ions of m/z difficult to explain. So, the importance of several factors such as instrument configuration and CID gas purity in adduct formation has been examined. Finally, the applicability of a direct analysis method such as FIA coupled to high resolution mass spectrometry has been evaluated for the wide-range screening of psychoactive substances. Data dependent analysis has been employed to obtain high quality spectral information that allowed the identification of target, suspect and unknown compounds in the samples. Moreover, the use of in silico fragmenter softwares in combination with the structural information from the product ion scan spectra has been proposed for the identification of unknown compounds

Determinación de la estructura de secuencias de ADN del tipo A(AT)nT

El ADN es una molécula de importancia vital en los organismos vivos, ya que están directamente relacionados con el material genético de las células. Está formado por una cadena de nucleótidos: adenina (A), citosina (C), guanina (G) y timina (T). Conocer a fondo las posibles estructuras químicas que puede tener el ADN puede ayudar a descubrir su mecanismo de funcionamiento. Este proyecto se centra únicamente en aquellas secuencias de ADN de la forma A(AT)nT, con n igual a 3, 4 y 5; puesto que apenas hay información sobre la estructura de secuencias que contienen únicamente A y T.Para conocer la estructura a nivel atómico de una molécula es preciso utilizar una radiación de longitud de onda comparable a las distancias interatómicas, por lo que la difracción de rayos X es una de las técnicas que permiten un estudio más detallado de la estructura de una molécula. Al interaccionar con los electrones de los átomos dan lugar a diagramas de difracción que, cuando la muestra es un cristal, están formados por manchas más o menos redondas y dispuestas regularmente sobre la imagen.Los diagramas de difracción son analizados mediante programas informáticos (HKL – Denzo y Scalepack) para saber:- celdilla del cristal, grupo espacial y simetría- intensidad de cada difracciónY utilizando un modelo similar y la información anteriormente citada, puede emplearse la metodología del reemplazo molecular para obtener la estructura del cristal. Si la secuencia contiene átomos pesados, puede usarse la técnica MAD para hallar la solución.Se han obtenido múltiples cristales para la secuencia A(AT)4T, pero la resolución de los datos no llegaba para poder conseguir un modelo exacto a través del reemplazo molecular. No obstante, se ha encontrado la celdilla del cristal (44,7x44,7x197,7 Å, 90º 90º 120º) y el empaquetamiento de los dúplex en columnas. El grupo espacial con el que mejores resultados se han obtenido es P3221. Para intentar resolver la secuencia A(AT)4T por MAD, se cristalizó la misma secuencia pero cambiando una T por un bromo-uracilo, ya que tienen un volumen similar, pero el procesado de dichos cristales no ha podido utilizarse por el momento.De las secuencias A(AT)3T y A(AT)5T se han obtenido cristales ya analizados, pero la resolución no era suficiente como para intentar resolver la estructura. Así, se ha calculado la celdilla de cada cristal, dato que servirá para un estudio posterior de patrones de cristalización en secuencias ricas en A-T y debido a similitudes con la secuencia anterior, es posible que también tengan una estructura en columnas

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry for the determination of polyphenolic profiles in the characterization and classification of cranberry-based pharmaceutical preparations and natural extracts.

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (UHPLC-APPI-MS/MS) was applied to the analysis and authentication of fruit-based products and pharmaceutical preparations. Two sub-2 µm C18 reversed-phase columns, Syncronis (100x2.1 mm, 1.7 µm) and Hypersil Gold (50x2.1 mm, 1.9 µm) were proposed under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases for the determination of 29 polyphenols, allowing to obtain polyphenolic profiles in less than 13.5 and 23.5 min, respectively. Several atmospheric pressure ionization (API) sources (H-ESI, APCI, APPI) were compared. For dopant-assisted APPI, four organic solvents, toluene, acetone, chlorobenzene and anisole were evaluated as dopants. Both, H-ESI and acetone-assisted APPI were selected as the best ionization sources for the analysis of targeted polyphenols. Acceptable sensitivity (LOD values down to 0.5 µg kg-1 in the best of cases), linearity (r2 higher than 0.995) and good precision (RSD values lower than 15.1%) and trueness (relative errors lower than 10.2%) were obtained with both UHPLC-API-MS/MS methods. A simple extraction procedure, consisting of a sample sonication with acetone/water/hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was used. The proposed UHPLC-ESI-MS/MS and UHPLC-APPI-MS/MS methods with both C18 reversed-phase columns were then applied to the analysis of 32 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic profiles data were then analyzed by principal component analysis (PCA) to extract information of the most significant data contributing to classification of natural extracts according to the type of fruit

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry for the determination of polyphenolic profiles in the characterization and classification of cranberry-based pharmaceutical preparations and natural extracts.

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (UHPLC-APPI-MS/MS) was applied to the analysis and authentication of fruit-based products and pharmaceutical preparations. Two sub-2 µm C18 reversed-phase columns, Syncronis (100x2.1 mm, 1.7 µm) and Hypersil Gold (50x2.1 mm, 1.9 µm) were proposed under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases for the determination of 29 polyphenols, allowing to obtain polyphenolic profiles in less than 13.5 and 23.5 min, respectively. Several atmospheric pressure ionization (API) sources (H-ESI, APCI, APPI) were compared. For dopant-assisted APPI, four organic solvents, toluene, acetone, chlorobenzene and anisole were evaluated as dopants. Both, H-ESI and acetone-assisted APPI were selected as the best ionization sources for the analysis of targeted polyphenols. Acceptable sensitivity (LOD values down to 0.5 µg kg-1 in the best of cases), linearity (r2 higher than 0.995) and good precision (RSD values lower than 15.1%) and trueness (relative errors lower than 10.2%) were obtained with both UHPLC-API-MS/MS methods. A simple extraction procedure, consisting of a sample sonication with acetone/water/hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was used. The proposed UHPLC-ESI-MS/MS and UHPLC-APPI-MS/MS methods with both C18 reversed-phase columns were then applied to the analysis of 32 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic profiles data were then analyzed by principal component analysis (PCA) to extract information of the most significant data contributing to classification of natural extracts according to the type of fruit

Determination of polyphenolic profiles by liquid chromatography-electrospray-tandem mass spectrometry for the authentication of fruit extracts

Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12-14 µg/L for 19 of the 26 analyzed polyphenols), linearity (r2 > 0.991), run-to-run and day-to-day precisions (RSD values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone:water:hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods (p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin