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- Analytical methodologies for the determination of biogenic amines in wines: an overview of the recent trendsPublication . Miranda, Andreia; Leça, João M.; Pereira, Vanda; Marques, José CarlosBiogenic amines are naturally present in grapes or can occur during the vinification and aging processes, essentially due to the microorganism’s activity. When present in wines in high amount, biogenic amines may cause not only organoleptic defects but also adverse effects in sensitive human individuals, namely due to the toxicity of histamine, tyramine and putrescine. Even though there are no legal limits for the concentration of biogenic amines in wines, some European countries only recommend maximum limits for histamine. In this sense, biogenic amines in wines have been widely studied. The determination of amines in wines is commonly achieved by liquid chromatography, using derivatization reagents in order to promote its separation and detection. In alternative, other promising methodologies have been developed using capillary electrophoresis or biosensors, revealing lower costs and faster results, without needing a derivatization step. Nowadays, it is still a challenge to develop faster and inexpensive techniques or methodologies to apply in the wine industry. Thus, this review will be focused on the studies published in the last decade that involves the determination of biogenic amines in wines, highlighting the novelty, improvement and optimization of the analytical methods. The sample preparation procedures (such as derivatization reagents), the analytical methodologies and the new trends being followed by the wine industry are also described and discussed.
- Impact of indigenous non-saccharomyces yeasts isolated from Madeira Island vineyards on the formation of ethyl carbamate in the aging of fortified winesPublication . Leça, João Micael; Pereira, Vanda; Miranda, Andreia; Vilchez, José Luis; Malfeito-Ferreira, Manuel; Marques, José CarlosThe impact of selected non-Saccharomyces yeasts on the occurrence of ethyl carbamate (EC) was evaluated. Hanseniaspora uvarum, Starmerella bacillaris, Pichia terricola, Pichia fermentans and Pichia kluyveri isolated from Madeira Island vineyards were inoculated in Tinta Negra musts. Urea, citrulline (Cit) and arginine (Arg) were quantified when the density of musts attained the levels to obtain sweet (1052 ± 5 g/L) and dry (1022 ± 4 g/L) Madeira wines. The urea concentration varied between 1.3 and 5.3 mg/L, Cit from 10.6 to 15.1 mg/L and Arg between 687 and 959 mg/L. P. terricola and S. bacillaris generated lower levels of urea (<2.5 mg/L), Cit (<11.0 mg/L) and Arg (<845.6 mg/L). The five resulting fortified wines, individually fermented by the selected non-Saccharomyces yeast, were exposed to laboratory-accelerated aging at 70 °C for 1 month. From the studied yeasts, P. terricola and S. bacillaris revealed a lower potential to form EC (<100 µg/L); therefore, both yeasts can be a useful tool for its mitigation in wines.
- A sensitive method for the rapid determination of underivatized ethyl carbamate in fortified wine by liquid chromatography-electrospray tandem mass spectrometryPublication . Leça, João M.; Pereira, Vanda; Pereira, Ana C.; Marques, José C.This work presents the optimization of a miniaturized liquid-liquid extraction (mLLE) followed by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (RP-HPLC-MS/MS) for the determination of ethyl carbamate (EC) in fortified wine, without using derivatizing agents. The mLLE was optimized by an experimental design. Thus, 15 mL of wine and 8 mL of ethyl acetate were used for the extraction. After concentration, each extract was injected into the HPLC MS/MS equipment and the characteristic secondary ion transition of EC (m/z = 90.10 → 62.05) was used for the quan tification purposes. The proposed method presented a good lin earity (R2 = 0.9999) and a high sensitivity with low limits of detection (LOD) and quantification (LOQ), 0.17 and 0.52 μg L−1 , respectively. The precision (repeatability and reproducibility) never exceeded 8% of variation, and the recoveries varied between 93 and 114%. The applicability of the method was checked through the analysis of 24 fortified wines, with EC values ranging between 23 ± 1 and 194 ± 5 μg L−1 . All chromatograms revealed good peak resolutions. This new method is efficient for the simple, fast, and reliable determination of EC in fortified wines, providing great sensitivity without using derivatizing agents or large volumes of organic solvents.
- Unveiling the evolution of Madeira Wine key metabolites: a three-year follow-up studyPublication . Pereira, Vanda; Leça, João M.; Freitas, Ana I.; Pereira, Ana C.; Pontes, Marisela; Albuquerque, Francisco; Marques, José C.Madeira wine (MW) encompasses an unusual oxidative ageing process that makes it distinc tive. Several metabolites have been related to its quality and safety, such as 5-hydroxymethylfurfural (HMF), furfural, sotolon, and ethyl carbamate (EC). These compounds were quantified over a three year period to assess their formation rate according to the ageing procedure used: canteiro vs. estufagem. Estufagem, which includes thermal processing of young MWs, promoted greater HMF, furfural, and sotolon accumulation, especially in sweet wines, in which sotolon contributed significantly to aroma (odour active values up to 17.5). Tinta Negra revealed a higher predisposition to form EC while Malvasia and Sercial were less prone to its formation. The formation of furfural, HMF, and EC strongly correlated with the ageing time. Sotolon had a strong correlation with the ageing time in canteiro (r = 0.79) and a moderate correlation in estufagem (r = 0.65). In both ageing procedures, sotolon, furfural, and HMF formation trends strongly correlated with each other (r = 0.74–0.90). In turn, EC also correlated with all furans (r = 0.51–0.85). Yellow tones (b*) correlated with these metabolites only when wines undergo estufagem. This study provides valuable insights to improve MW quality and safety management procedures.
- Definitive screening designs and latent variable modelling for the optimization of solid phase microextraction (SPME): case study-quantification of volatile fatty acids in winesPublication . Pereira, Ana C.; Reis, Marco S.; Leça, João M.; Rodrigues, Pedro M.; Marques, José C.In the present study, we apply the recently proposed Definitive Screening Designs (DSD) to optimize HS-SPME extraction in order to analyze volatile fatty acids (VFA) present in wine samples. This is the first attempt to apply this new class of designs to one of the most well-known and widely applied extraction techniques. The latent structure of the responses is also explored for defining the optimal extraction conditions. DSD is a new screening design with the potential to significantly reduce the number of experiments required to estimate the model pa rameters and to establish the optimum operation conditions. Therefore, there is an obvious interest in assessing the benefits of DSD in practice. In this work, this design framework is applied to the simultaneous optimization of seven extraction parameters (responses). Both qualitative and quantitative extraction parameters are considered, in order to test the flexibility of DSD designs: a two-level qualitative variable, the fiber coating, and six quanti tative variables, namely the pre-incubation time, the extraction time and temperature, the headspace/sample volume, the effect of agitation during extraction and the influence of the ethanol content (sample dilution). Optimization of analytes' chromatographic responses was carried out both individually (response by response) and altogether, by modelling the responses in the latent variable space (i.e., explicitly considering their under lying correlation structure). In the end, a consensus analysis of all perspectives was considered in the definition of the overall optimal extraction conditions for the quantification of VFA in fortified wines. The solution found was to use a DVB/Car/PDMS fiber, 10 mL of samples in 20 mL vial, 40 min of extraction at 40 C. The analysis also revealed that the factors incubation time, agitation and sample dilution do not play a significant role in explaining the variability of extraction parameters. Therefore, they were set to the most convenient levels. The methodology followed was thoroughly validated and the following figures of merit were obtained: good linearity (R2 > 0.999, for all compounds), high sensitivity (LOD and LOQ are close or below the values found in literature), recoveries of approximately 100% and suitable precision (repeatability and reproducibility lower than 7.21% and 8.61%, respectively). Finally, the optimized methodology was tested in practice. Several wine samples were analyzed and the odor activity value calculated to facilitate the identification of their importance as odor active compounds in different aged fortified wines. This work demonstrates the benefits of using DSD and latent variable modelling for the optimization of analytical techniques, contributing to the implementation of rigorous, systematic and more efficient optimization protocols.
- New insights into ethyl carbamate occurrence in fortified winesPublication . Leça, João M.; Pereira, Vanda; Miranda, Andreia; Vilchez, José Luis; Marques, José C.The occurrence of ethyl carbamate (EC) in fortified wines was studied testing different model wines under accelerated ageing (45 °C for 4 months and 70 °C for 1 month), to investigate the possibility of arginine (Arg) being a direct precursor of EC in fortified wines, comparing it with known major precursors, urea and citrulline (Cit). Wine main sugars were appraised as possible catalysts, as previously pointed out. Model wines showed that: Arg can induce the EC formation under accelerated ageing, even without being metabolised by microorganisms; although much less reactive, Arg can play a significant role since high residual levels can occur in young fortified wines; glucose (Glc) and fructose (Fru) suppressed the EC formation from urea and Cit pathways, in about 11–26%. Moreover, Madeira wine samples were investigated in order to appraise the eventual contribution of the alcoholic fortification. The results revealed that this step can promote a decrease of the amount of these EC precursors up to 46%. Despite preliminary, additional information about the EC formation in fortified wines was obrained namely for designing new mitigation strategies, which can pass through the reduction of residual Arg.
- Evaluation of fucoxanthin contents in seaweed biomass by vortex-assisted solid-liquid microextraction using high-performance liquid chromatography with photodiode array detectionPublication . Nunes, N.; Leça, João M.; Pereira, Ana C.; Pereira, Vanda; Ferraz, S.; Barreto, Maria Carmo; Marques, José C.; Carvalho, M.A.A. Pinheiro deFucoxanthin is considered an important marine bioactive compound with biological properties with promising effects, namely on health. A simple and efficient analytical methodology is proposed for its quantification in seaweed biomass by using vortex-assisted solid-liquid microextraction (VASLME) followed by reversed phase high-performance liquid chromatography (RP-HPLC) photodiode array detection (PDA) analysis. This microextraction uses reduced quantities of sample (25 mg) and solvent (300 μL of ethanol) to efficiently extract this high-valued xanthophyll, in a vortex time of 15 min. These extraction parameters were optimized performing a Central Composite Design (CCD) analysis, running 32 individual experiments. In turn, the method validation was assessed. The linearity of the method was confirmed (R2 = 0.99998) in a concentration range from 12 to 3600 μg·g−1 dw. Also, good sensitivity and accuracy results were observed through the LOD (3.33 μg·g−1 ), LOQ (10.09 μg·g−1 ) and recovery (varied from 95 to 97%) assessments. Good precision was also verified, with intra-day variation within 2.0–3.3%, and inter-day within 1.0–3.8%. Matrix effect was also evaluated and an acceptable variation of 3.4% was found. The method applicability was confirmed by the analysis of 22 seaweed biomass samples and fucoxanthin content was found to vary from about 10 to 853 μg·g−1 dw. This method demonstrated a good performance and can be successfully implemented for a rapid, reliable and accurate screening of fucoxanthin in seaweed biomass.
- Evolution of ethyl carbamate during Madeira wine ageing by GC-MS: a new methodologyPublication . Leça, João Micael da Silva; Marques, José Carlos Antunes; Pereira, Vanda Nulita GomesRecently, ethyl carbamate (EC) was reclassified by the International Agency for Research on Cancer (IARC) as "probably carcinogenic to humans" and occurs mainly in fermented beverages. Nowadays many countries have set limit values for EC in alcoholic beverages. In this sense and taking into account the low concentrations found in alcoholic beverages, the scientific community has shown interest for the development of new analytical methods, whereby its simplification plays an important role in the EC control and prevention. Firstly, a simple, rapid and sensitive methodology was developed for the EC quantification in fortified wines by microextraction by packed sorbent (MEPS) with gas chromatography coupled with a mass spectrometer detector (GC-MS). This method showed good linearity (R2 = 0.999) and sensitivity (LOD = 1.5 μg/L). The accuracy of the method was assessed by means of repeatability and reproducibility (RSD < 7%). Moreover, a good recovery has been demonstrated (97 – 106%) as well as its applicability (16 fortified wines). Thus, the developed methodology has proven to be an excellent approach for routine quantification of EC in fortified wines. The EC evolution was also evaluated during a year and half of Madeira wine ageing submitted to two traditional ageing methods, estufagem and canteiro, in order to evaluate the formation kinetic. The results revealed that estufagem process increased the formation kinetic and promoted a linear increase of the EC concentration (R2 ≥ 0.977), proportionally to the ageing time (4 months). However, when the wines are firstly submitted to estufagem and then undergo canteiro ageing, the EC values remain almost constant during the following 14 months. The results suggest that estufagem does not seem to be the critical factor in the EC formation, but instead the amount of precursors in the medium.
- Optimal design of experiments applied to headspace solid phase microextraction for the quantification of vicinal diketones in beer through gas chromatography-mass spectrometric detectionPublication . Leça, João M.; Pereira, Ana C.; Vieira, Ana C.; Reis, Marco S.; Marques, José C.Vicinal diketones, namely diacetyl (DC) and pentanedione (PN), are compounds naturally found in beer that play a key role in the definition of its aroma. In lager beer, they are responsible for off-flavors (buttery flavor) and therefore their presence and quantification is of paramount importance to beer producers. Aiming at developing an accurate quantitative monitoring scheme to follow these off-flavor compounds during beer production and in the final product, the head space solid-phase microextraction (HS-SPME) analytical procedure was tuned through experiments planned in an optimal way and the final settings were fully validated. Optimal design of experiments (O-DOE) is a computational, statistically oriented approach for designing experiences that are most informative according to a well-defined criterion. This methodology was applied for HS-SPME optimization, leading to the following optimal extraction conditions for the quantification of VDK: use a CAR/PDMS fiber, 5 ml of samples in 20 ml vial, 5 min of pre-incubation time followed by 25 min of extraction at 30 C, with agitation. The validation of the final analytical methodology was performed using a matrix-matched calibration, in order to mini mize matrix effects. The following key features were obtained: linearity (R2 > 0.999, both for diacetyl and 2,3-pentanedione), high sensitivity (LOD of 0.92 mg L 1 and 2.80 mg L 1 , and LOQ of 3.30 mg L 1 and 10.01 mg L 1 , for diacetyl and 2,3-pentanedione, respectively), recoveries of approximately 100% and suitable precision (repeatability and reproducibility lower than 3% and 7.5%, respectively). The applicability of the methodology was fully confirmed through an independent analysis of several beer samples, with analyte concentrations ranging from 4 to 200 g L 1
- Multiresponse and multiobjective latent variable optimization of modern analytical instrumentation for the quantification of chemically related families of compounds: case study—Solid‐phase microextraction (SPME) applied to the quantification of analytes with impact on wine aromaPublication . Reis, Marco S.; Pereira, Ana C.; Leça, João M.; Rodrigues, Pedro M.; Marques, José C.The optimized operation of modern analytical instrumentation is a critical but complex task. It involves the simultaneous consideration of a large number of factors, both qualitative and quantitative, where multiple responses should be quantified and several goals need to be adequately pondered, such as global quantification performance, selectivity, and cost. Furthermore, the problem is highly case specific, depending on the type of instrument, target analytes, and media where they are dispersed. Therefore, an optimization procedure should be conducted frequently, which implies that it should be efficient (requiring a low number of experiments), as simple as possible (from experi mental design to data analysis) and informative (interpretable and conclusive). The success of this task is fundamental for achieving the scientific goals and to justify, in the long run, the high economic investments made and significant costs of operation. In this article, we present a systematic optimization proce dure for the prevalent class of situations where multiple responses are available regarding a family of chemical compounds (instead of a single analyte). This class of problems conducts to responses exhibiting mutual correlations, for which, furthermore, several goals need to be simultaneously considered. Our approach explores the latent variable structure of the responses created by the chemical affinities of the compounds under analysis and the orthogonality of the interpretable extracted components to conduct their simultaneous opti mization with respect to different analysis goals. The proposed methodology was applied to a real case study involving the quantification of a family of analytes with impact on wine aroma.