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- A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in winesPublication . Silva, Catarina L.; Gonçalves, João L.; Câmara, José S.A new approach based on microextraction by packed sorbent (MEPS) and reversed-phase high-throughput ultra high pressure liquid chromatography (UHPLC) method that uses a gradient elution and diode array detection to quantitate three biologically active flavonols in wines, myricetin, quercetin, and kaempferol, is described. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (selectivity, linearity, sensitivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters such as the type of sorbent material (C2, C8, C18, SIL, and C8/SCX), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, on the MEPS performance. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). Under optimized conditions, excellent linearity View the MathML source(Rvalues2>0.9963), limits of detection of 0.006 μg mL−1 (quercetin) to 0.013 μg mL−1 (myricetin) and precision within 0.5–3.1% were observed for the target flavonols. The average recoveries of myricetin, quercetin and kaempferol for real samples were 83.0–97.7% with relative standard deviation (RSD, %) lower than 1.6%. The results obtained showed that the most abundant flavonol in the analyzed samples was myricetin (5.8 ± 3.7 μg mL−1). Quercetin (0.97 ± 0.41 μg mL−1) and kaempferol (0.66 ± 0.24 μg mL−1) were found in a lower concentration. The optimized MEPSC8 method was compared with a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis HLB) were used as reference. MEPSC8 approach offers an attractive alternative for analysis of flavonols in wines, providing a number of advantages including highest extraction efficiency (from 85.9 ± 0.9% to 92.1 ± 0.5%) in the shortest extraction time with low solvent consumption, fast sample throughput, more environmentally friendly and easy to perform.
- An attractive, sensitive and high-throughput strategy based on microextraction by packed sorbent followed by UHPLC-PDA analysis for quantification of hydroxybenzoic and hydroxycinnamic acids in winesPublication . Gonçalves, João; Silva, Catarina L.; Castilho, Paula C.; Câmara, José S.An attractive, fast, low-consumption and very promising high-throughput strategy based on microextraction by packed sorbent (MEPS), and reversed phase ultra-high pressure liquid chromatography (UHPLC), was developed and validated for the determination and quantification of hydroxybenzoic (gallic acid, protocatechuic acid, gentisic acid, vanillic acid, syringic acid), and hydroxycinnamic acids (p-coumaric acid, m-coumaric acid, o-coumaric acid, ferrulic acid and cinnamic acid) in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL and C8/SCX), number of extraction cycles (extract–discard), sample volume and elution volume were tested and optimized. The optimal MEPS extraction conditions were obtained using C8 phase as sorbent and 50 μL of the sample in five extraction cycles. The analytes were separated on a new Trifunctional High Strength Silica analytical column (HSS T3; 100% silica particle), specially designed for polar compounds, using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). This new stationary phase, proved to be an excellent alternative to conventional C18 columns for the determination of benzoic and cinnamic acids derivatives in wines. The analytical method was fully validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), extraction yield, accuracy and inter/intra-day precision, using a synthetic wine spiked with hydroxybenzoic and hydroxycinnamic acids, and demonstrated good linearity with r2‐values > 0.998 within the established concentration range (0.2–40 μg mL− 1). Under the optimized conditions, LOD of 0.01–0.2 μg mL− 1, LOQ of 0.03–0.7 μg mL− 1, and precision, expressed as the relative standard deviation (RSD %), within 0.3–6% were observed. The proposed method was also applied to the analysis of hydroxybenzoic and hydroxycinnamic acids in red and white wines and recoveries of 80–100% and 77–99%, respectively, were obtained. The new approach offers an attractive alternative for analysis of phenolic acids from wine samples, providing a number of advantages including decreased sample preparation, faster sample throughput and easy to perform as compared to traditional methodologies. Moreover it could potentially be extended to other extraction media.