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A fast and environment-friendly MEPS PEP /UHPLC-PDA methodology to assess 3-hydroxy-4,5-dimethyl-2(5H)-furanone in fortified wines

Publication . Freitas, Jorge; Perestrelo, Rosa; Cassaca, Rubina; Castillo, Mariangie; Santos, Mariana; Pereira, Jorge; Câmara, José S.

Sotolon is widely associated with the quality of fortified aged wines, and has also been linked to premature oxidative aging (premox). Here we developed a single, fast and environmental-friendly microextraction by packed sorbent ultra-high pressure liquid chromatography analysis (MEPS/UHPLC-PDA) for sotolon quantification in different wines. The best extraction conditions (loading three times 250μL samples through the MEPSPEP sorbent and elution with 100μL of 50% MeOH) were combined with a fast UHPLC separation (5min separation using acidified 10% MeOH isocratic flow in a CORTECS C18 column) to allow unparalleled minimum sample and solvents volumes usage. The validated methodology showed good linearity (r(2)>0.993) and precision (<5.6%); high recovery (>81%) and detection limits (0.45-2.51μg/L) far below sotolon odor threshold for any type of wine. The methodology was successfully applied to selected white table and Madeira wines, encompassing therefore a wide range of alcohol and sugar contents. Furthermore, as far we may know, this is the first time a single methodology can be used to assess both wine aging or premox according to the type of wine.

2017Journal article

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A fast and innovative microextraction technique, μSPEed, followed by ultrahigh performance liquid chromatography for the analysis of phenolic compounds in teas

Publication . Porto-Figueira, Priscilla; Figueira, José A.; Pereira, Jorge A. M.; Câmara, José S.

The objective of this study was to evaluate the efficiency of a promising solid phase microextraction technique, μSPEed, in the analysis of selected phenolic compounds from teas by ultrahigh performance liquid chromatography with photodiode array detection (μSPEed/UHPLC-PDA). The innovative μSPEed configuration uses 3-μm sorbent particles tightly packed in a disposable needle equipped with a pressure-driven valve to withdraw samples in a single direction. The system was operated by the electronic pipette eVol® and different parameters influencing the extraction efficiency, as the nature of sorbent, pH, loading and elution conditions, and solvents were optimized. The best extracting conditions were obtained by loading twice 100μL of tea samples through the PS/DVB-RP sorbent and eluting with 50μL of acidified MeOH 95%. The following chromatographic separation was carried out in an Acquity C18 BEH capillary column using a gradient of 0.1% FA and acetonitrile. The optimized μSPEed/UHPLC-PDA methodology is selective and specific and was properly validated for 8 phenolic compounds widely reported in different teas. Overall, an excellent analytical performance was obtained in the 0.2-20μg/L linear dynamic range (LDR), with very low limits of detection (LODs) and quantification (LOQs), ranging between 3.5-16.8ng/mL and 10.6-50.6ng/mL, respectively, high recoveries (89.3-103.3%), good precision (RSD<5%) and negligible matrix effect. The methodology was used to assess the target polyphenols concentration in several tea samples. Rutin and quercetin-3-glucoside were the most abundant phenolics in all tea samples analysed and, with exception of naringenin and cinnamic acid, which are present in high amounts in the investigated citric teas, remain phenolic compounds are present in trace levels.

2015Journal article

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Ultrasound-assisted liquid-liquid extraction followed by ultrahigh pressure liquid chromatography for the quantification of major carotenoids in tomato

Publication . Figueira, José A.; Pereira, Jorge A.M.; Porto-Figueira, Priscilla; Câmara, José S.

Lycopene and β-carotene, the main carotenoids present in different tomatoes varieties (gordal, cherry, roma and campari) of Solanum lycopersicum L. (Solanaceae), were investigated using ultrasound-assisted liquid-liquid extraction (LLUSAE) followed by ultra-high pressure liquid chromatography with PDA detection (UHPLC-PDA) analysis. Experimental parameters influencing the LLUSAE were optimized using an univariate design, resulting in a 30-min ACN/MeOH extraction by sonication of a lyophilized sample, followed by PSA/C18/MgSO4 clean-up and fast centrifugation before UPLC analysis. Using this greener methodology, high recoveries (above 97%), good linearity (r2 > 0.98) and improved sensitivity, with limits of detection and quantification of 24.0 and 80.0 ng/mL for lycopene and 3.0 and 9.9 ng/mL for β-carotene, respectively, were obtained. This sensitivity is about five times better than previously reported in literature, making LLUSAE/UHPLC-PDA a promising strategy for lycopene and β-carotene quantification in tomato and eventually in other matrices. The carotenoids studied, lycopene and β-carotene, were found at highest concentrations in the gordal tomato variety, followed by cherry, roma and campari (727.1, 342.2, 267.2 and 218.2 μg/g and 80.4, 44.0, 45.7 and 44.0 μg/g for lycopene and β-carotene, respectively). Additionally, an exponential increase of both carotenoids occurs during ripening and mainly in the skin and locular cavity of the gordal variety. These results provide further evidences of the potential of tomatoes as an interesting source of lycopene and β-carotene.

2017Journal article

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Quantification of δ-, γ- and α-Tocopherol in tomatoes using an improved liquid-dispersive solid-phase extraction combined with ultrahigh pressure liquid chromatography

Publication . Figueira, José A.; Pereira, Jorge A. M.; Câmara, José S.

Tomato (Solanum lycopersicum L.) consumption has been correlated with a lower incidence of cardiovascular diseases and cancer. This protective effect has been ascribed to different bioactive compounds present in this fruit. Therefore, to gain insights on the potential of S. lycopersicum L. as bioactive food, a fast and sensitive methodology, based on liquid–liquid extraction (LLE), dispersive solid phase extraction (dSPE) followed by ultrahigh pressure liquid chromatography (UHPLC-FLR) analysis, was developed and validated to quantify δ-, γ- and α-tocopherol in tomatoes. Upon the optimization of different parameters, a fast extraction and separation, and simultaneously, increased resolution and sensitivity was attained. The methodology was validated, retrieving better analytical performance than most methods reported so far. This included good linearity, (r 2 > 0.99) and precision (<6.4%), high recoveries (>79.5%) and improved limits of detection and quantification (LODs of 2.15, 5.52 and 1.67 ng/mL and LOQs of 7.18, 18.40 and 5.58 ng/mL, for δ- γ- and α-tocopherol, respectively). These limits are about 1000 times lower than those reported in literature. Furthermore, as far we are aware, this is the first time δ-tocopherol presence in tomato is fully characterized and quantified. The methodology was applied to different tomato varieties, ripening stages and fruit sections, revealing high levels of δ-tocopherol that increase along fruit ripening, while the α-tocopherol follows the inverse trend. Moreover, δ-tocopherol is almost fully concentrated in the seeds and skin of ripe tomato. Finally, ORAC and DPPH assays revealed that the selected tocopherols contribute to approximately half of tomato total antioxidant capacity.

2017Journal article

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A new and fast methodology to assess oxidative damage in cardiovascular diseases risk development through eVol-MEPS–UHPLC analysis of four urinary biomarkers

Publication . Mendes, Berta; Silva, Pedro; Mendonça, Isabel; Pereira, Jorge; Câmara, José S.

In this work, a new, fast and reliable methodology using a digitally controlled microextraction by packed sorbent (eVol(®)-MEPS) followed by ultra-high pressure liquid chromatography (UHPLC) analysis with photodiodes (PDA) detection, was developed to establish the urinary profile levels of four putative oxidative stress biomarkers (OSBs) in healthy subjects and patients evidencing cardiovascular diseases (CVDs). This data was used to verify the suitability of the selected OSBs (uric acid-UAc, malondialdehyde-MDA, 5-(hydroxymethyl)uracil-5-HMUra and 8-hydroxy-2'-deoxyguanosine-8-oxodG) as potential biomarkers of CVDs progression. Important parameters affecting the efficiency of the extraction process were optimized, particularly stationary phase selection, pH influence, sample volume, number of extraction cycles and washing and elution volumes. The experimental conditions that allowed the best extraction efficiency, expressed in terms of total area of the target analytes and data reproducibility, includes a 10 times dilution and pH adjustment of the urine samples to 6.0, followed by a gradient elution through the C8 adsorbent with 5 times 50 µL of 0.01% formic acid and 3×50 µL of 20% methanol in 0.01% formic acid. The chromatographic separation of the target analytes was performed with a HSS T3 column (100 mm × 2.1 mm, 1.7 µm in particle size) using 0.01% formic acid 20% methanol at 250 µL min(-1). The methodology was validated in terms of selectivity, linearity, instrumental limit of detection (LOD), method limit of quantification (LOQ), matrix effect, accuracy and precision (intra-and inter-day). Good results were obtained in terms of selectivity and linearity (r(2)>0.9906), as well as the LOD and LOQ, whose values were low, ranging from 0.00005 to 0.72 µg mL(-1) and 0.00023 to 2.31 µg mL(-1), respectively. The recovery results (91.1-123.0%), intra-day (1.0-8.3%), inter-day precision (4.6-6.3%) and the matrix effect (60.1-110.3%) of eVol(®)-MEPS/UHPLC-PDA method were also very satisfactory. Finally, the application of the methodology to the determination of target biomarkers in normal subjects and CVDs patients' revealed that the DNA adducts 5-HMUra and 8-oxodG levels are much more abundant in CVDs patients while no statistic differences were obtain for MDA and UAc. This result points to the importance of 5-HMUra and 8-oxodG as biomarkers of CVDs risk progression and further epidemiological studies are needed to explore the importance of this correlation.

2013Journal article

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