Eduarda M. P. Silva

Photooxidation of glycated and non-glycated phosphatidylethanolamines monitored by mass spectrometry.

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications.

Cationic β-vinyl substituted meso-tetraphenylporphyrins: synthesis and non-covalent interactions with a short poly(dGdC) duplex

Several cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenylporphyrin derivatives were synthesized starting from 2-formyl-meso-tetraphenylporphyrin, and the corresponding Zn(II) complex, and different N-alkyl derivatives of 2- and 4-methylpyridine and 2- and 4-methylquinoline. The new compounds were obtained in a one-step process via base catalyzed aldol-type condensation reactions. Electrospray ionization mass spectrometry (ESI-MS) and ultraviolet-visible (UV-vis) spectroscopy were used to investigate the binding mode of the synthesized cationic beta-vinyl-pyridinium and beta-vinyl-quinolinium-meso-tetraphenylporphyrin derivatives with a short GC duplex oligonucleotide. Analysis of the obtained mass spectrometry results indicates the probable occurrence of outside binding. UV-vis spectroscopy data also points to non-intercalation. The potential photosensitizing capacity of these compounds was also ascertained from preliminary photophysical studies.

Structural effects of the β-vinyl linker in pyridinium porphyrins: spectroscopic studies in organic solvents and AOT reverse micelles.

Two isomeric β-vinylpyridinium porphyrins, 2-[2-(2-methylpyridinium)vinyl]-5,10,15,20-tetraphenylporphyrin (1, ortho isomer) and 2-[2-(4-methylpyridinium)vinyl]-5,10,15,20-tetraphenylporphyrin (2, para isomer), which have shown different photodynamic behavior were investigated in organic solvents and sodium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles. In organic systems, the absorption spectra present a red-shifted band that is more intense in the para isomer, in addition to the usual Soret band. This new band presents interesting solvatochromic effects which obey the multiparametric Kamlet-Taft equation. In AOT reverse micelles, the ortho isomer exhibits a strong dependence with the parameter ω0 = [H2O]/[AOT] which indicates that the molecule resides at the interface toward the organic phase. By contrast, no evidence was detected for the encapsulation of para isomer 2 in AOT reverse micelles. The hypothesis of two ground state isomers with different contributions of trans and quinoid structures is advanced on the basis of the overall data collected from electronic absorption, steady-state, and transient-state fluorescence emission. A charge transfer state in which an electron is fully transferred from the porphyrin to the pyridinium moiety is associated to a quinoid structure in isomer 2. The trans/quinoid relative proportions may be accounted for by the orientation of the ortho-/para-pyridinium isomers relatively to the porphyrin core.

Tricationic Porphyrin Conjugates: Evidence for Chain-Structure-Dependent Relaxation of Excited Singlet and Triplet States

Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from approximately 0.03 in pH 7 buffer to approximately 0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding (1)S(1) lifetimes are approximately 5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)(n)) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-alpha-d-galactopyranosyl or a alpha/beta-d-galactopyranosyl group occurs in ethanol (k approximately 0.15 ps(-1)) after (1)S(1) state solvation (approximately 700 fs). Relaxation of bulky P-(Lys)(n) polypeptide chains takes place on a longer time scale in all solvents (k <or= 0.01 ps(-1)) with enhanced internal conversion. Triplet state ((3)T(1)) transient spectra of all derivatives in PBS, SDS, TX100, and ethanol exhibit a strong absorbance with a broad maximum in the 460-475 nm region and minor maxima at approximately 540, 630, and 690 nm. In ethanol, energy transfer from the P-H (3)T(1) state to beta-carotene provides an estimate of epsilon approximately 40,000 M(-1) cm(-1) at 460 nm for the P-H (3)T(1) state. Using triplet meso-tetraphenylporphyrin as an actinometer, the P-H triplet quantum yield (Phi(T)) is estimated to be approximately 0.50 in all solvents. This high Phi(T) leads to effective singlet oxygen production in buffered solutions.

Electrospray Tandem Mass Spectrometry of β-Nitroalkenyl Meso-Tetraphenylporphyrins

β-Nitroalkenyl meso-tetraphenylporphyrins [β-TPPCHC(NO2)R)], as free-bases and Zn(II) complexes, were studied by electrospray mass spectrometry (ESI-MS). Under this ionisation condition the [M + H]+ ions are formed. The fragmentation pattern of the resulting [M + H]+ ions were studied by electrospray tandem mass spectrometry (ESI-MS/MS). The ESI-MS/MS of β-nitroalkenylporphyrins, either as free-bases or as Zn(II) complexes, show several interesting features, distinct from the typical behaviour of nitro compounds. For the studied compounds, common main fragmentation patterns are observed, namely characteristic losses of NO2•, HNO2, 2OH•, RNO2, RCNO, RCNO2, RCH2NO2, C6H5• plus NO2• and the formation of the protonated macrocycle, [TPP + H]+ or [ZnTPP + H]+. However, depending on the presence or absence of the metal and the nature of the R substituent, important differences are observed on the relative abundances of the ions formed by the same fragmentation pathway. The presence of bromine in the alkenyl group leads to a peculiar behaviour, since the main fragmentation pattern corresponds to the combined elimination of the bromine atom with the typical nitro group fragments. When R = Br, the loss of the nitro group occurs in low relative abundance (11-16%). However, when R = CH3, the relative abundance of the ion due to the loss of HNO2 changes drastically from 100%, observed for the free-base porphyrin, to 29% in the case of the Zn(II) complex. These variations of the relative abundance of the fragment corresponding to the loss of the nitro moiety (typically considered as a diagnostic fragment) can induce to an erroneous interpretation of their MS/MS spectra. Some fragmentations are observed only for the free-base porphyrins, namely the loss of •CH(NO2)R and HNO2 plus C2H2, while the loss of OH•, H2O, OH• plus H2O and RCCH plus H2O is observed only for the complexes. Unusual and unexpected fragmentations are also observed, namely the losses of RCNO, RCNO2 and HNO2 plus C2H2. This work demonstrates that valuable structural information about the β-nitroalkenyl substituents linked to meso-tetraarylporphyrins can be achieved using MS/MS. These results can also be useful for the interpretation of the mass spectra of other nitroalkenyl substituted compounds.

Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids

&NA; Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole‐tandem mass spectrometry (HPLC–MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3 &mgr;m, 100 × 2.1 mm) at 45 °C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35 mL min−1. Total run time was 9 min, with retention time of 2.8 and 4.1 min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI‐) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500 ng mL−1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7 ng mL−1 in sample extracts, respectively. The method was found to be specific, accurate (96.0–106.0% of nominal values) and precise (RSD < 2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24 h at room temperature (20 °C) or in the autosampler, and after three freeze‐thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs. Graphical abstract Figure. No caption available. HighlightsQuantification of tenofovir (TFV) and efavirenz (EFV) was attained for 4–500 ng mL−1.Validated LC–MS/MS method for vaginal tissue, lavage and blood plasma was developed.Matrix effects were observed for EFV in tissue (+17%) and plasma (−50%) extracts.LOD and LOQ values were ≤0.4 and ≤0.7 ng mL−1 in sample extracts.PK profile after intravaginal administration showed low systemic exposure.

Evaluation of the photooxidation of galactosyl‐ and lactosylceramide by electrospray ionization mass spectrometry

RATIONALE Glycosphingolipids are important lipid molecules namely as constituents of the plasma membrane organized in lipid rafts, in signal transduction, and cell-cell communication. Although many human diseases are associated with oxidative stress and lipid oxidation, a link between oxidative stress and modification of glycosphingolipids has never been addressed. METHODS In this study, the structural changes caused by UVA-induced photooxidation of galactosyl- (GalCer) and lactosylceramide (LacCer) molecular species were studied by electrospray ionization mass spectrometry (ESI-MS and MS/MS), using a quadrupole time-of-flight (QTOF) mass spectrometer and high-performance liquid chromatography/tandem mass spectrometry with a C5 stationary phase (C5 HPLC/MS/MS) using a linear ion trap. RESULTS ESI-MS spectra of GalCer and LacCer after photooxidation showed new ions with a mass shift of +32 Da when compared with the ions of the non-modified glycosphingolipids. These new species were assigned as hydroperoxyl derivatives, confirmed by HPLC/MS/MS and through FOX 2 assay. In the ESI-MS and LC/MS of lactosylceramide a new ion with lower m/z value, assigned as glucosylceramide (GlcCer) + 32 Da, was also detected and proposed to be formed due to oxidative cleavage of lactosyl moieties. ESI-MS/MS of the oxidized species allowed us to infer the presence of isomeric hydroperoxyl derivatives, with the hydroperoxyl moiety either linked to the sphingosine backbone or in the unsaturated acyl chain. Oxidation in the sugar moieties was observed in the case of LacCer, suggesting an oxidation via radical reactive oxygen species that can induce the oxidative cleavage of the lactosyl moiety. CONCLUSIONS This study shows that glycosphingolipids are prone to oxidation and the identified mass spectrometry fingerprint of oxidized galactosyl- and lactosylceramide species will support their future identification in lipidomic studies of biological samples under oxidative conditions.

Tandem mass spectrometry based investigation of cinnamylideneacetophenone derivatives: valuable tool for the differentiation of positional isomers

Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24 derivatives were systematically studied by tandem mass spectrometry (MS(2)) with electrospray ionization (ESI), in positive ion mode. The protonated molecules, [M + H](+), formed under ESI conditions were induced to dissociate and the fragmentation patterns were studied. The information collected provided important structural information on the type of substituents present and constitute an important database concerning this family of compounds. Overall, it was found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI-MS(2) fragmentation considerably. These results indicate that ESI-MS(2) is a useful technique for distinguishing positional isomers of these cinnamylideneacetophenone derivatives.

Do cinnamylideneacetophenones have antioxidant properties and a protective effect toward the oxidation of phosphatidylcholines

Cinnamylideneacetophenones (CA) are an important group of α,β,γ,δ-diunsaturated ketones that have been widely used in a variety of synthetic transformations. Biological studies concerning these compounds are scarce and refer mainly to antiviral and antibacterial evaluations. Curcumin (CR), a natural polyphenol, is a yellow pigment extracted from the plant Curcuma longa, which is one of the major spices used in the Indian culinary. It has been reported that CR has cancer chemopreventive properties in a range of animal models of chemical carcinogenesis, along with antioxidative and anti-inflammatory properties. Inspired by the biological activity shown by CR and their structural resemblance with CA, it was considered to study the ability of the latter molecules to inhibit lipid oxidation induced by the hydroxyl radical (Fenton reaction) by electrospray ionization (ESI) mass spectrometry (MS) using phosphatidylcholine (PC) liposomes as a model of cell membrane. Compound 4, holding a methylated hydroxy group in the position R(2), and CR showed similar effects in inhibiting lipid peroxidation. In the presence of 7, the extension of oxidation was higher than the one verified in all other compounds. Other methodologies, namely DPPH radical scavenging and oxygen radical absorption capacity (ORAC) assays, were performed to complement and clarify the results attained by oxidation of PC monitored by ESI-MS and to evaluate the antioxidant profile of compounds. For both assays, compound 7 showed to be rather efficient due to its specific structure. This derivative can form a quite stable allylic radical by abstraction of a hydrogen atom which accounts for these results.

Characterisation of (E)‐2‐styrylchromones by electrospray ionisation mass spectrometry: singular gas‐phase formation of benzoxanthenones

RATIONALE The electrospray ionisation (ESI) mass spectra (ESI-MS(n) , n = 2 and 3) of six (E)-2-styrylchromones containing electron-donating and electron-withdrawing groups were studied for the first time. The potential application of these compounds as drug candidates brings a need for the development of an analytical method to ensure a detailed knowledge of their structures. METHODS ESI-MS, CID-MS(2) and MS(3) spectra of all the studied compounds were acquired using a LXQ linear ion trap mass spectrometer with an electrospray source, operating in the positive ion mode. ESI conditions were: nitrogen 8.00 sheath gas flow rate (arb), spray voltage 5 kV, heated capillary temperature 275 °C, capillary voltage 10.99 V and tube lens voltage 75.01 V. CID-MS(2) and MS(3) experiments were performed on mass-selected precursor ions using standard isolation and excitation procedures (activation q value of 0.25, activation time of 30 ms). RESULTS A comparable pattern of product ions was observed for all the studied compounds. Only the product ion ((1,2)(3,4)) A(+) is common to all the studied compounds. Common to compounds 1 (R(2) = H), 1a (R(2) = OMe) and 1c (R(2) = Cl) are the product ions formed by loss of H(2)O, CO, C(6)H(5)OH, and the product ions ((1,2)(2,3))B(+) and ((1,2)(3,4))B(+). Among all compounds, 2-SC 1b (R(2) = NO(2)) shows an obvious differentiation showing the lowest number of product ions under CID-MS(2) conditions. CONCLUSIONS The analysed compounds revealed a comparable pattern of product ions formed for each compound only different in what concerns the relative abundance of these formed product ions. A rather interesting and unexpected loss of 2 Da was observed for some compounds. The structure of a benzoxanthenone was proposed for this product ion based on evidence obtained by synthesised benzoxanthenone as a model compound.