Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta 4-hydrazinobenzoic acid as a derivatizing agent for aldehyde analysis by HPLC-UV and CE-DAD Lucas F. de Limaa,1, Pedro F. Brandãob,1, Tiago A. Donegattia, Rui M. Ramosb, Luís Moreira Gonçalvesb,c, Arnaldo A. Cardosod, Elisabete A. Pereiraa,⁎, José A. Rodriguesb a Federal University of São Carlos, Sorocaba, SP, Brazil b REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal c Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo (USP), São Paulo, SP, Brazil dUniversidade Estadual Paulista (UNESP), Instituto de Química, Araraquara, SP, Brazil A R T I C L E I N F O Keywords: Carbonyl compounds Derivatization Sample preparation Secondary ketamine Schiff base Volatile and semi-volatile extraction A B S T R A C T Aldehydes are relevant analytes in a wide range of samples, in particular, food and beverages but also body fluids. Hydrazines can undergo nucleophilic addition with aldehydes or ketones giving origin to hydrazones (a group of stable imines) that can be suitably used in the identification of aldehydes. Herein, 4-hydrazinobenzoic acid (HBA) was, for the first time, used as the derivatizing agent in analytical methodologies using liquid chromatography aiming the determination of low-molecular aldehydes. The derivatization reaction was si- multaneously performed along with the extraction process, using gas-diffusion microextraction (GDME), which resulted in a clean extract containing the HBA-aldehyde derivates. The corresponding formed imines were de- termined by both high-performance liquid chromatography (LC) with UV spectrophotometric detection (HPLC- UV) and capillary electrophoresis with diode array detection (CE-DAD). HBA showed to be a rather advanta- geous derivatization reagent due to its stability, relatively high solubility in water and other solvents, high selectivity and sensibility, reduced impurities, simple preparation steps and applicability to different separation and/or different detection techniques. Limits of detections (LODs) of the optimized methodologies (in terms of time and pH among other experimental variables) were all below 0.5mg L−1, using both instrumental techni- ques. Furthermore, for the first time, the HBA-aldehyde derivatives were analyzed by LC with mass spectrometry (LC-MS), demonstrating the possibility of identification by MS of each compound. The developed methodologies were also successfully applied in the analysis of formaldehyde and acetaldehyde in several alcoholic beverages. This was also the first time GDME was combined with CE, showing that it can be a valuable sample preparation tool for electrophoresis, in particular by eliminating the interference of ions and inorganic constituents present in the samples. 1. Introduction Aldehydes are a group of chemical compounds of the utmost im- portance in food and environmental science [1,2]. In alcoholic bev- erages, for example, aldehydes are commonly formed during the fer- mentation process. Due to the addition of flavors such as nut, fat, fruit or grass to the final product, their presence can have an important role on the flavor characteristics of these beverages. But, as aldehydes can greatly improve food quality, they can also generate unpleasant flavors, product deterioration and even cause health hazards [1,3–7]. En- dogenous aldehydes are generated during oxidative stress and are associated with many pathogenic processes [8]. Not surprisingly, there are many analytical methodologies reported in literature based in a separation by gas-chromatography (GC) [8], liquid chromatography (LC) [3,9–12], and capillary electrophoresis (CE) for low molecular weight aldehydes on various matrices [2,13–17]. Derivatization procedures are a clever way to improve the sus- ceptibility of analytes to be detected and quantified by many analytical instruments. It consists on the reaction between the aimed analytes with a derivatizing reagent producing derivates. Particularly, in chro- matographic techniques, derivatization not only may lead to detectable compounds but can also improve their resolution and their symmetry; https://doi.org/10.1016/j.talanta.2018.04.091 Received 8 March 2018; Received in revised form 27 April 2018; Accepted 28 April 2018 ⁎ Correspondence to: Universidade Federal de São Carlos -UFSCAR, Departamento de Física, Química e Matemática, Rodovia João Leme dos Santos, Km 110, SP 264, CEP 18052-780 Sorocaba, SP, Brazil. 1 These authors contributed equally for this work. E-mail address: ealves@ufscar.br (E.A. Pereira). Talanta 187 (2018) 113–119 Available online 30 April 2018 0039-9140/ © 2018 Published by Elsevier B.V. T http://www.sciencedirect.com/science/journal/00399140 https://www.elsevier.com/locate/talanta https://doi.org/10.1016/j.talanta.2018.04.091 https://doi.org/10.1016/j.talanta.2018.04.091 mailto:ealves@ufscar.br https://doi.org/10.1016/j.talanta.2018.04.091 http://crossmark.crossref.org/dialog/?doi=10.1016/j.talanta.2018.04.091&domain=pdf derivates can be more stable, both chemically and thermally, thus a suitable analyte separation can be greatly improved [2,3,18–24]. One example of this is the group of compounds called hydrazines, that are a well-known group of derivatization agents for the detection and de- termination of carbonyl compounds in various samples [25]. There are other derivatizing agents for aldehydes in literature [24,26,27], in- cluding 2-aminoethanethiol, 2-diphenylacetyl-1,3-indandione-1-hy- drazone, cyclohexane-1,3-dione, dansylhydrazine, 2,4-dini- trophenylhydrazine (DNPH) [3,28–30], 3-methylbenzothiazolin-2-one hydrazone (MBTH) [31], 4-(2-((4-bromophenethyl)dimethylammonio) ethoxy)benzenaminium dibromide (4-APEBA) [32], 2,4,6-tri- chlorophenylhydrazine [38], O-2,3,4,5,6-(pentafluorobenzyl) hydro- xylamine hydrochloride (PFBO) [8,33], and benzoylhydrazine [2], all with their inherent advantages and disadvantages. As an example, DNPH, that is perhaps the most popular reagent for carbonyl com- pounds, also presents some disadvantages: large amount of impurities are found in the reagent, which may require additional purification steps prior to its use; furthermore, interferences with ozone and ni- trogen dioxide [34,35] have been reported, as well as low solubility in water and limited applicability in CE [36]. To overcome this limited applicability to CE, the use of one other hydrazine, 4-hydrazinobenzoic acid (HBA) as a derivatizing agent for aldehydes is suggested. It was initially developed for the CE analysis of aldehydes present in air samples [13]. Considering the great potential of HBA, it was intended to apply it to more complex samples and observe if it was functional with LC. The derivatizing reaction is schematized in Fig. 1, the terminal primary amine in HBA reacts with the carbonyl group forming an imide. Food and biological samples are very complex matrices with many different compounds present. A sample preparation step seems ideal not only to prolong the life span of the chromatographic columns and de- tectors but also to actually make a suitable detection possible [19]. In this work, gas-diffusion microextraction (GDME) was applied, a tech- nique that merges microextration with gas-diffusion (hence ideal for volatile and semi-volatile compounds [37]) that is fully enhanced when a derivatizing reagent is used accordingly [38–40]. The GDME extrac- tion device consists of a small hollow PTFE cylinder with a membrane at its bottom. Membrane type and characteristics can be adjusted ac- cording to the specific conditions in order to increase selectivity and sensitivity. Although GDME had been used before for aldehydes [38,41,42], it has never been associated with HBA and never before associated with CE (although indeed other gas-diffusion techniques, particularly in flow systems, have been associated with CE [43–45]). When compared with other separation methods, like HPLC, CE has the advantages of a shorter time of analysis and lower sample consumption [46]. On the other hand, it has limited sensitivity and, when used with samples like beverages, can have the interference of ions and inorganic constituents [46]. These two issues can be solved using GDME, since not only it is possible to obtain enrichment factors (‘pre-concentration’) when applying derivatization but also only volatile and semi-volatile compounds are extracted (‘clean-up’). 2. Materials and methods 2.1. Chemicals and samples All reagents were of analytical grade and, except when mentioned otherwise, were used without further purification. All aqueous solutions were prepared using ultrapure water with resistivity not lower than 18.2MΩ cm at 298 K (Direct-Q 3 UV, Millipore, Bedford, USA). Formaldehyde (For, methanal), acetaldehyde (Ace, ethanal), propio- naldehyde (Pro, propanal), furfural (Fur, furan-2-carbaldehyde), acro- lein (Acr, propenal), benzaldehyde (Ben), butyraldehyde (But, butanal), sodium dodecyl sulfate (SDS), sodium tetraborate (STB) and 4-hy- drazinobenzoic acid (HBA) were obtained from Sigma-Aldrich (St. Louis, USA). Methanol, acetonitrile and hydrochloric acid were ob- tained from Merck (Darmstadt, Germany). Stock standard solutions (1000mg L−1) of each aldehyde were prepared in methanol, except For which was prepared in water. Daily work standard solutions (100mg L−1) of each aldehyde were prepared by dilution of the stock solutions in methanol/water (1:1 in v/v). For the CE-DAD experiments, the stock derivatizing solution, 10 mL of HBA, concentration of 1000mg L−1, was prepared daily in methanol/water (1:1 in v/v). This solution was protected from light. Working HBA so- lutions were prepared by diluting the stock solution in methanol/water (1:1 in v/v). For the HPLC-UV experiments, the stock derivatization solution, 10mL of HBA, concentration 2500mg L−1, was daily pre- pared in HCl 0.1 mol L−1. This solution was kept in the dark, protected from light. Samples (wine, cachaça and the other liquors) were purchased in local supermarkets. 2.2. HPLC-UV The HPLC system (Thermo Electron Corp., USA) was composed of a low-pressure gradient quaternary pump with an autosampler (200-vial capacity sample) and a DAD detector (Finnigan Surveyor Plus). Chromatographic separations of the aldehydes-HBA derivatives were performed with a Phenomenex Gemini C18 (250×4.60mm, 5 µm of particle size), the eluents and the gradient profile were optimized. Initial conditions consisted of acetonitrile (27%) and formic acid, 0.1%, in water (73%) (v/v); the gradient began with 27% of acetonitrile and increased to 43% in the following 25min; an increase to 51% in the next 7min was performed, then returned to the initial conditions in 7min; an additional 5min step was used for conditioning, before the next injection. The flow rate was 1.0 mLmin−1, the injection volume was 20 μL, and the UV detection was performed at 320 nm. All se- parations were made at room temperature (approximately 20 °C). 2.3. CE-DAD CE was performed using a capillary electrophoresis system, model G7100A from Agilent Technologies (Palo Alto, USA). Separations were performed using a fused silica capillary (PolymicroTechnologies, Phoenix, USA) 58 cm of total length, 50 cm of effective length, 75 µm i.d. x 375 µm o.d., equipped with a diode array detector set at 290 nm and 320 nm, the temperature control device was set at 27 °C. The samples were injected using hydrodynamic mode (25 mBar) during 10 s. The instrument was operated under positive polarity at + 25 kV. Every day, before the first analysis, the capillary was conditioned by flushing with sodium hydroxide, 1 mol L−1 solution, during 5min, followed by a 5min flush of purified water and 20min flush of running electrolyte. In between samples injections, the capillary was rinsed by a 3min flush with electrolyte solution (sodium tetraborate, 40mmol L−1, 85%, and acetonitrile, 15%), which was prepared daily. Fig. 1. Scheme of the derivatizing re- action, reaction between HBA and the different aldehydes forming an imine and a water molecule. L.F. de Lima et al. Talanta 187 (2018) 113–119 114 2.4. LC-MS The separation in the LC-MS application was performed with a Phenomenex Gemini C18 column (150× 4.6mm, 3 µm of particle size) and a guard column with the same characteristics was used at room temperature, with a flow rate of 0.5mLmin−1 with an injection volume of 25 μL. The gradient and eluents used are shown in Table S1 in the Supporting information. A quadrupole ion-trap mass spectrometer (Finnigan LCQ Deca XP Plus) equipped with an electrospray ionization (ESI) source in the positive ion mode was used under the following conditions: capillary temperature, 325 °C; source voltage, 5.0 kV; ca- pillary voltage, 3.0 V; sheath gas (N2) flow at 60 arbitrary units and auxiliary gas (N2) flow at 22 arbitrary units. The mass detection was performed in the range m/z 0–1000. Xcalibur software version 1.4 (Thermo Electron Corp.) was used for data acquisition and processing. 2.5. Derivatization and extraction procedure The initial studies of the derivatizing reaction were performed without any extraction (direct reaction). For the HPLC-UV analysis: 125 μL of each working aldehyde standard solution were placed in a 5mL volumetric flask and the volume was completed with HBA (312.5 mg L−1), the mixed solution was allowed to react at room tem- perature, protected from the light for about 60min. For the CE-DAD analysis: 50 μL of each working aldehyde standard solution were placed in a 5mL volumetric flask and the volume was completed with HBA (300mg L−1), the mixed solution was allowed to react at room tem- perature protected from the light for about 60min. Extraction was performed with a system of GDME [3,30,39] with appropriate minor modifications. Concerning the extraction conditions: a) HPLC-UV: 10mL of the standard solution or sample (donor solution) were placed in the thermostatized flask; 500 μL of HBA (312.5mg L−1) was used as the acceptor solution, the time of extraction was 15min; b) CE-DAD: 15mL of the standard solution or sample (donor solution) were placed in the thermostatized flask; 500 μL of HBA (300mg L−1) was used as the acceptor solution, the time of extraction was 20min. In both cases: the membrane used was PTFE with 0.5 µm pore size (Mitex, Millipore), and the temperature was maintained at 50 °C using a water bath, the extracts were left to react protected from light for about 40min. 3. Results and discussion 3.1. Optimization of the separation conditions Initial conditions for the HPLC separation of aldehydes-HBA deri- vatives were based in the literature which described the analysis of HBA pharmaceutical formulations [47]. Several gradient programs were evaluated. Fig. S1 in the Supporting information presents the separation of HBA-aldehydes with an initial gradient tested. There were some problems separating HBA-Pro and HBA-Acr that could be solved with a slower increase in the acetonitrile percentage. The optimized separation of HBA derivatives using the final gradient profile is shown in Fig. 2. According to literature [37] different parameters such as the ac- ceptor solution's volume, whether the GDME device is immersed or suspended in the headspace, temperature and time of extraction, among others, can affect the extraction's efficiency. The acceptor solution's volume (derivatization solution, HBA 200mg L−1) was studied on the extraction efficiency simultaneously comparing both immersion and headspace modes (Fig. 3). The volume of the HBA solution placed in- side of the extracting probe varied in the range from 300 to 1000 μL (experiments HM 300, HM 500, HM 700 and HM 1000 in Fig. 3), keeping the time and temperature of extraction constant (20min and 50 °C, respectively). With increasing HBA solution volume, a decrease in the analytical signal for all aldehydes was observed. A volume of 500 μL was selected for the following experiments for practical aspects, the handling of the extract was experimentally simpler. This volume was compared in the immersion and headspace mode experiments (HM 500 and IM 500 in Fig. 3), the immersion presented an increase on the analytical signal, mainly for For, Fur and Ben, probably due to lower volatility than the other aldehydes. The effect of temperature on extraction was evaluated in the range from 40° to 60 °C (Fig. 4 - A). The For analytical signal increased with increasing temperature, however, no significant variations on peak area were observed for Ace, Pro, Fur and Ben, though it was observed a decrease in Acr. Another parameter evaluated was the extraction time, it was tested in a range from 10 to 30min (Fig. 4 - B). It was observed that the analytical signal increases with increasing extraction time for Ace, Pro and Acr, however, a loss of signal was observed for For, Fur and Ben for a time larger than 30min. Consequently, the optimized temperature and time were set to 50 °C and 20min, respectively. The extraction using the immersion mode was evaluated by com- paring the extraction system with and without stirring (Fig. S2 in the Supporting information). The stirring of the sample caused a small in- crease in the analytical signal for Ace, Pro and Acr, no significant variations for Fur and Ben and slight decrease to For, probably due to competition for the derivatizing agent. Since the temperature and time of extraction had been evaluated and optimized in HPLC-UV experiments, the same conditions were used for the CE-DAD analysis. Initial results with CE showed that poor re- solution occurred between peaks of aldehydes evaluated with electro- lyte containing only STB (Fig. 5 - A). The effect of addition of methanol and acetonitrile on the separation was evaluated using each organic solvent in the range of 5–15%, with constant electrolyte concentration, injection, applied voltage and temperature (40mmol L−1 STB pH 9.45, 10 s x 25 mBar, + 25 kV and 27 °C). It was found that 15% acetonitrile improved resolution of all peaks (Fig. 5 – B and C). When studying the influence of the reaction medium solvent, it was observed that me- thanol showed better performance (improved signal analytical for Ace, Pro, Fur and Ben) than acetonitrile. Studies with binary mixtures of Fig. 2. Chromatograms of the optimized separation of the aldehydes-HBA de- rivates. Inlay: schematics of the gradient used in each case. All aldehydes had concentration of 10mg L−1, with the exception of For that was 2.5 mg L−1. Peaks identification: 1 - HBA-For (4-(2-methylenehydrazineyl)benzoic acid), 2 - HBA-Ace (4-(2-ethylidenehydrazineyl)benzoic acid), 3 - HBA-Pro (4-(2-propy- lidenehydrazineyl)benzoic acid), 4 - HBA-Acr (4-(2-allylidenehydrazineyl)ben- zoic acid), 5 – HBA-Fur (4-(2-(furan-2-ylmethylene)hydrazineyl)benzoic acid), and 6 - Hba-Ben (4-(2-benzylidenehydrazineyl)benzoic acid). Gradient elution: starting with 80:20 (% of buffer of phosphoric acid, 20mmol L−1, pH 2.5; and % of acetonitrile), gradually changing to 70:30 during 15min, then gradually changing to 65:35 during 8min, another gradual change to 40:60 during 7min, stable gradient for 10min, gradually changing to 80:20 during 5min, finishing with a stable gradient for 5 min. L.F. de Lima et al. Talanta 187 (2018) 113–119 115 1:1 v/v purified water:methanol and 1:1 v/v water:acetonitrile with different times of reaction can be found in the Supporting Information (Fig. S3). The derivatization reaction between HBA and the aldehydes was conducted under three pH values conditions: 2.5, 5.1 and 7.0. The pH of the derivatization solution was adjusted with hydrochloric acid and sodium hydroxide. The pH value of 5.1 was not adjusted (dissolution of reagent and binary mixture). Derivatization time was about 60min [13]. When the pH was increased from 5.1 to 7.0, no further im- provements in the analytical signal for Ace and Pro were observed, however it was observed a decrease in the analytical signal for For, Fur and Ben (Fig. 6). The derivatization reagent without any pH adjustment was selected as the optimum condition. This result is explained by the reaction mechanism schematized in Fig. 6. The reaction between a primary amine and an aldehyde to form an imine follows this alkyli- mino-de-oxo-bisubstitution mechanism: 1) nucleophilic attack from the amine's nitrogen to the aldehyde's carbon; 2) proton transfer originating an hemianal (also known as carbinolamine); 3) protonation of the hy- droxyl (in this case the Lewis’ acid is an hydronium ion); 4) elimination of water; 5) deprotonation. Step 1 and step 4 are the rate-determining steps depending on the pH: a) for a pH below 4, step 1 limits the re- action because too much acid will protonate the unprotected amine (i.e. rate-determining addition); b) for a pH higher than 6, step 4 is limited because the elimination of water is not favored (i.e. rate-determining dehydration) [48]. As a result, theoretically the optimum pH is around 5 [48], this is precisely what was obtained experimentally. Imines are in general rather unstable, one of the exceptions are hydrazones, the electronegativity of the extra nitrogen participates in the delocalization of the imine's double bond, delocalization decreases the small positive Fig. 3. Extraction optimization concerning the acceptor solution's volume, and the immersed mode (IM) vs. headspace mode (HM). Extraction conditions: temperature of 50 °C, extraction time of 20min, aldehydes concentra- tions of 10mg L−1. (HM 300) Headspace mode, HBA vo- lume of 300 μL; (HM 500) Headspace mode, HBA volume of 500 μL; (HM 700) Headspace mode, HBA volume of 700 μL; (HM 1000) Headspace mode, HBA volume of 1000 μL; (IM 500) Immersion mode, HBA volume of 500 μL. On the right side there is a scheme depicting what are the headspace and immersion modes. Fig. 4. Extraction optimization studies, in both a mixture of aldehydes with concentrations of 2.5 mg L−1 of each aldehyde, immersion mode, HBA volume of 500 μL, without shaking: (A) Effect of temperature on extraction procedure, extraction time of 20min; (B) Effect of time in the extraction, temperature of 50 °C. Fig. 5. Electropherograms of the separation of the different aldehyde-HBA derivatives, at a concentration of 10mg L−1, injection of 10 s at 25 mBar, po- tential of + 25 kV, temperature of 27 °C, wavelength of 290 nm. Peak identi- fication: R - excess of HBA, 1 – HBA-For, 2 – HBA-Ace, 3 – HBA-Acr, 4 – HBA- Pro, 5 – HBA-Fur and 6 – HBA-Ben. (A) electrolyte: 40mmol L−1 STB, pH 9.45; (B) 40mmol L−1 STB, pH 9.45+15% methanol; (C) 40mmol L−1 STB, pH 9.45+ 15% acetonitrile. L.F. de Lima et al. Talanta 187 (2018) 113–119 116 charge on the imine's carbon making it less susceptible to nucleophilic attack [48]. In order to provide an extra dimension of information to the already rich data of HPLC-UV and CE-DAD, HPLC-DAD-MS/MS studies were also performed. The combination between the retention time and the MS spectral information of the several aldehyde derivatives present from standards enabled highly reliable identification and confirmation of the compounds. The most relevant data is summarized in Fig. 7. It seems that all derivates were mostly fragmented in the carboxylic group releasing a water molecule, thus the most intense fragment is 18 Da short from the root compound. The N-N covalent bond also seems to be a ‘weak’ interaction easily broken. 3.2. Analytical parameters and sample analysis The method performance parameters were obtained from several calibration curves, for both GDME-HPLC-UV and GDME-CE-DAD, using the analytical procedures previously described, results are summarized in Table 1. The obtained results showed that linearity for all aldehydes was suitable with all coefficients of determination (r2) above 0.99. The linear ranges using GDME-CE-DAD were up to 15mg L−1 for all com- pounds, and using GDME-HPLC-UV were up to 10mg L−1 for all com- pounds. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as three and ten times the standard deviation of the intercept divided by the slope, respectively, and were all below 0.5 mg L−1 (Table 1). Apart from other aldehydes or ketones, it is not expected that other compounds can be simultaneously extracted and react with HBA, thus no selectivity tests were performed. The practical applicability of the developed methodologies was tested in the analysis of For and Ace using spiking techniques in dif- ferent kind of alcoholic beverages. The obtained results are shown in Table 2 (Fig. S4 in the Supporting information shows an example of a chromatogram obtained in the analysis one of the samples by GDME- HPLC-UV, and Fig. S5 in the Supporting information shows an example of an electropherogram obtained in the analysis one of the samples by Fig. 6. Effect of the pH in the derivatization reaction, measurements were performed with CE-DAD (injection of 10 s at 25 mBar, potential of +25 kV, temperature of 27 °C, wavelength of 290 nm, electrolyte: 40mmol L−1 STB, pH 9.45+ 15% acetonitrile), the different aldehyde-HBA derivatives were at the concentration of 10mg L−1. Below the reaction mechanism: 1) nucleophilic attack; 2) proton transfer; 3) protonation of the hydroxyl; 4) elimination of water; 5) deprotonation. Fig. 7. Overview of the MS fragmentation. Main fragments are listed for each aldehyde-HBA derivative, other fragments are listed in order of magnitude. Table 1 Analytical parameters. Values of the slope and intercept are expressed along with the confidence interval at the significance level of 0.05. For GDME-CE- DAD n=5 and for GDME-HPLC-UV n=5. All measurements were performed in duplicate or triplicate. GDME-HPLC-UV GDME-CE-DAD LOD (mg L−1) Ace 0.15 0.45 Pro 0.17 0.35 Acr 0.08 – Fur 0.21 0.35 Ben 0.15 0.39 For 0.005 0.36 But 0.07 – LOQ (mg L−1) Ace 0.50 1.49 Pro 0.57 1.18 Acr 0.25 – Fur 0.69 1.16 Ben 0.50 1.32 For 0.017 1.21 But 0.23 – r2 Ace 0.996 0.999 Pro 0.995 0.999 Acr 0.999 – Fur 0.993 0.999 Ben 0.996 0.999 For 0.999 0.999 But 0.997 – L.F. de Lima et al. Talanta 187 (2018) 113–119 117 GDME-CE-DAD). No pre-treatment of the samples was applied and the obtained values were in accordance with the ones commonly found in similar products [30]. It was chosen to analyze only these two com- pounds since the concentration of other aldehydes was expected to be too low and might require longer extraction times. 4. Conclusions A new derivatizing agent was successfully applied in a novel methodology aiming the determination of low molecular weight alde- hydes. The derivatization with HBA was simple, quick, and only one derivative is formed from each different aldehyde. The formed deri- vates were analyzed by HPLC-UV and CE-DAD, being further studied by LC-MS. GDME is ideal to be used in complex samples since it allows simple and simultaneous clean-up and pre-concentration steps. The developed methodologies showed satisfactory results concerning line- arity and precision. Acknowledgements TAD wishes to acknowledge to Programa de Pós-Graduação em Biotecnologia e Monitoralmento (PPGBMA) UFSCar, Campus Sorocaba and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of Brazil for the fellowship. EAP also would like to acknowl- edge Fundação de Amparo à Pesquisa do Estado de São Paulo for fi- nancial support (FAPESP 11/23286-1). This work received financial support from Fundação para a Ciência e a Tecnologia/Ministro da Ciência, Tecnologia e Ensino Superior (FCT/MCTES) through national funds and co-financed by (CAPES/FCT 99999.008406/2014-06) and Fundo Europeu de Desenvolvimento Regional (FEDER), under the Partnership Agreement PT2020 through projects UID/QUI/50006/ 2013 and POCI/01/0145/FEDER/007265, which includes a student- ship to PFB and RMR. LMG further wishes to acknowledge FCT (SFRH/ BPD/76544/2011 and FCT/13277/4/8/2015/S) Conflicts of interest There are no conflicts of interest to declare. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2018.04.091. References [1] J. Schultheiss, D. Jensen, R. Galensa, Determination of aldehydes in food by high- performance liquid chromatography with biosensor coupling and micromembrane suppressors, J. Chromatogr. 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