Ana M. Ares

Hydrophilic interaction liquid chromatography in food analysis

The use of hydrophilic interaction liquid chromatography (HILIC) in food analysis in the last decade is reviewed. The HILIC mechanism is briefly discussed, but main emphasis is put on the use of HILIC for separation of different food matrices. The food matrices are divided into food of animal origin and related products, vegetables, fruits and related compounds, and other food-related matrices. A list on important applications is provided for each category including experimental conditions and a brief summary of the results. The 100 references included will provide the reader a comprehensive overview and insight into HILIC applications to food analysis.

Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds.

Broccoli (Brassica oleracea L. var. Italica) contains substantial amount of health-promoting compounds such as vitamins, glucosinolates, phenolic compounds, and dietary essential minerals; thus, it benefits health beyond providing just basic nutrition, and consumption of broccoli has been increasing over the years. This review gives an overview on the extraction and separation techniques, as well as the biological activity of some of the above mentioned compounds which have been published in the period January 2008 to January 2013. The work has been distributed according to the different families of health promoting compounds discussing the extraction procedures and the analytical techniques employed for their characterization. Finally, information about the different biological activities of these compounds has been also provided.

Optimized extraction, separation and quantification of twelve intact glucosinolates in broccoli leaves

A new method has been developed and validated to determine twelve intact glucosinolates (glucoiberin, GIB; glucoraphanin, GRA; glucoerucin GER; gluconapin, GNA; glucotropaeolin, GTL; glucobrassicin, GBC; gluconasturtiin, GST; glucoalyssin, ALY; 4-hydroxyglucobrassicin, 4-OH; 4-metoxyglucobrassicin, 4ME; neoglucobrassicin, NEO; sinigrin, SIN) in broccoli leaves using liquid chromatography (LC) coupled to diode array (DAD) and electrospray ionization mass spectrometry (ESI-MS) detection. An extraction procedure has also been proposed and optimized by means of statistical analysis (the Box-Behnken design and analysis of variance); this is based on the deactivation of myrosinase using a microwave and heated water. Low limits of detection and quantification were obtained, ranging from 10 to 72 μg/g with DAD and 0.01 to 0.23 μg/g with ESI-MS, and the resulting recovery values ranged from 87% to 106% in all cases. Finally, glucosinolates were analyzed in broccoli leaf samples from six different cultivars (Ramoso calabrese Parthenon, Marathon, Nubia, Naxos and Viola).

Hydrophilic interaction chromatography in drug analysis

AbstractHydrophilic interaction chromatography (HILIC) is an increasingly popular alternative to conventional HPLC for drug analysis. It offers increased selectivity and sensitivity, and improved efficiency when quantifying drugs and related compounds in complex matrices such as biological and environmental samples, pharmaceutical formulations, food, and animal feed. In this review we summarize HILIC methods recently developed for drug analysis (2006–2011). In addition, a list of important applications is provided, including experimental conditions and a brief summary of results. The references provide a comprehensive overview of current HILIC applications in drug analysis.

Liquid chromatography coupled to ion trap-tandem mass spectrometry to evaluate juvenile hormone III levels in bee hemolymph from Nosema spp. infected colonies

It has been described a fast, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure juvenile hormone III (JH III), which was used to study of the effects of Nosema spp. infection on JH III levels in bee hemolymph. Honey bee hemolymph was extracted by centrifugation and mixed with a solution of phenylthiourea in methanol. This mixture was then centrifuged and the supernatant removed and evaporated to dryness. The residue was reconstituted in methanol containing the internal standard (methoprene) and injected onto an LC-MS/MS (ion-trap) system coupled to electrospray ionization (ESI) in positive mode. Chromatography was performed on a Synergi Hydro-RP column (4 μm, 30 mm × 4.60 mm i.d.) using a mobile phase of 20 mM ammonium formate and methanol in binary gradient elution mode. The method was fully validated and it was found to be selective, linear from 15 to 14,562 pg/μL, precise and accurate, with %RSD values below 5%. The limits of detection and quantification were: LOD, 6 pg/μL; LOQ, 15 pg/μL. Finally, the proposed LC-MS/MS method was used to analyze JH III levels in the hemolymph of worker honey bees (Apis mellifera iberiensis) experimentally infected with different Nosema spp. (Nosema apis, Spanish and Dutch Nosema ceranae strains). The highest concentrations of JH III were detected in hemolymph from bees infected with Spanish N. ceranae.

Simultaneous determination of tryptophan, kynurenine, kynurenic and xanthurenic acids in honey by liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection.

A liquid chromatography method using diode array-fluorescence detection and atmospheric pressure chemical ionization mass spectrometry (LC-DAD-FLD and LC-APCI-MS/MS) was developed to quantify the levels of tryptophan (TRP), kynurenine (KYN), kynurenic (KYNA) and xanthurenic (XA) acids in honey. This procedure involved isolating the compounds of interest via solid-phase extraction (SPE) with mixed-mode polymeric cartridges. Chromatographic separation of the analytes was performed in isocratic mode on a Synergi 4μ Hydro-RP 80 Å (150×4.60 mm i.d.) analytical column at 30 °C. The mobile phase of 20mM ammonium formate (pH 4) and methanol was passed at a flow rate of 0.5 mL/min. In replicate sets of spiked honey samples, the average analyte recoveries ranged from 60 to 98% for TRP, 55 to 120% for KYN, 65 to 106.5 for KYNA and 56 to 114% for XA. Detection limits ranged from 4 to 36 μg/kg for LC-DAD-FLD to 0.2 and 1.0 μg/kg for LC-APCI-MS/MS. A strong matrix effect was found when MS/MS was employed, necessitating calibration using the standard addition method on matrix-matched standards for each honey type. The method was used to quantify each of the compounds of interest in 17 honey samples of distinct botanical origins.

Development and validation of a liquid chromatography with mass spectrometry method to determine resveratrol and piceid isomers in beeswax

This paper represents the first report of a liquid chromatography coupled to electrospray ionization mass spectrometry method for simultaneously analyzing resveratrol and piceid isomers (cis and trans) in beeswax. An efficient extraction procedure has been proposed (average analyte recoveries were between 89 and 95%); this involved a solid-liquid extraction using a mixture of ethanol and water (80:20, v/v) and a concentration step in a rotary evaporator. The separation of all the compounds was achieved using a C18 column and a mobile phase composed of ammonium formate 0.03 M in water and acetonitrile in gradient elution mode at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification ranged from 1.0 to 1.7 and 3.5 to 5.5 μg/kg, respectively. Finally, the proposed method was applied to analyze beeswax samples collected from experimental and organic apiaries.

Fast determination of intact glucosinolates in broccoli leaf by pressurized liquid extraction and ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

In this study, we investigate for the first time the efficiency of an environmentally sustainable extraction technique (pressurized liquid extraction, PLE) in conjunction with a fast separation technique (ultra-high performance liquid chromatography, UHPLC) coupled to a selective mass spectrometry (MS) detector (quadrupole time-of-flight, qTOF) to extract, separate and quantify fifteen intact-glucosinolates (GLSs) in broccoli leaves. Firstly, we have developed and optimized by means of an experimental design an efficient extraction procedure based on PLE (using ethanol/water as a solvent), giving complete extraction within 15min; meanwhile, the average analyte recoveries were between 85% and 96% in all cases. Chromatography was performed on a UHPLC BEH Shield RP18 1.7μm 110Å (2.1×100mm) analytical column with a mobile phase composed by formic acid in water (0.5%, v/v) and formic acid in acetonitrile (0.5%, v/v) in gradient elution mode at 0.3mL/min, resulted in baseline-separated peaks and a run time of 13min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, precision, and trueness; meanwhile a study of the matrix effect was also performed. A good selectivity, low LODs and LOQs, ranging from 2 to 26μg/g, wide linear ranges from LOQ to 2500μg/g, and satisfactory precision and trueness with relative standard deviation and relative error values lower than or equal to 9%, were obtained for the studied GLSs. Finally, the proposed method was successfully applied to the analysis of intact-GLSs in fifteen broccoli leaf samples from three different cultivars (Parthenon, Nubia, and Naxos). Nine intact-GLSs were detected in all the varieties, although in different concentrations, which ranged between 14 and 1136μg/g, depending on the broccoli cultivar. In addition, the highest total content of GLSs was found in broccoli leaf samples from Parthenon cultivar, being the Naxos cultivar the poorest in GLS content. This study demonstrates the efficiency of PLE as an environmentally sustainable alternative to extract intact-GLS from broccoli leaves, and that UHPLC-qTOF-MS allowed a rapid, selective and sensitive determination of intact-GLSs in this matrix.

Extraction and determination of bioactive compounds from bee pollen

HighlightsBee pollen is a good source of bioactive compounds.The composition of bee pollen depends strongly on plant source.Solvent extraction has been the most used sample treatment.UV–vis and titration have been mainly employed for determining the total content.LC (RP or NP) has been the technique of choice when determining individual content. ABSTRACT Since ancient times bee pollen has been considered a good source of bioactive substances and energy. Taking into account the current demand for healthy and natural foods, it is not surprising that bee pollen has been attracting commercial interest in recent years, making it one of the most widely consumed food supplements. It has been extensively reported that bee pollen contains several health‐promoting compounds, such as proteins, amino acids, lipids, phenolic compounds, vitamins or minerals. Thus, this study aims to give an overview of the extraction and determination techniques of several of the above‐mentioned compounds which have been published in the last few years (2011‐2017). The design of the study is in accordance with the different families of bioactive compounds, and the extraction procedures together with the analytical techniques employed and their determination are discussed. A list of some of the most relevant applications is provided for each category, including a brief summary of the experimental conditions. The references included will provide the reader with a comprehensive overview of and insight into the analysis of bioactive compounds from bee pollen.

Determination of flubendiamide in honey at trace levels by using solid phase extraction and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

In this study, a new method has been developed to determine flubendiamide in honey using liquid chromatography coupled to a selective mass spectrometry detector (quadrupole-time-of-flight). An efficient sample treatment involving a solid phase extraction with a C18 sorbent was proposed (average analyte recoveries were between 94 and 104%). Chromatographic analysis (9min) was performed on a C18 column (Gemini C18, 50×2.0mm, 3µm, 110Å). The mobile phase consisted of water and acetonitrile, with a flow rate of 0.5mL/min in gradient elution mode. The method was fully validated in terms of selectivity, limits of detection and quantification, matrix effect, linearity, trueness and precision. Low limits of detection and quantification were obtained, ranging from 0.1 to 0.2µg/kg and 0.4 to 0.6µg/kg, respectively. The method was applied to analyze flubendiamide in honey from different botanic origins (multifloral, rosemary and heather).