M.M. Delgado-Zamarreño

Simultaneous extraction of tocotrienols and tocopherols from cereals using pressurized liquid extraction prior to LC determination.

Tocopherols and tocotrienols have been simultaneously determined in food samples using a rapid and simple analytical method including pressurized liquid extraction (PLE) and LC with electrochemical detection. Separation was carried out on a Phenomenex Synergi 4 microm Hydro-RP 80A column, using a solution of 2.5 mM acetic acid/sodium acetate in methanol/water (99:1, v/v) as mobile phase at a flow rate of 1.0 mL/min. Column temperature was maintained at 30 degrees C. Detection was performed by coulometric detection at 500 mV except for (beta+gamma)-tocotrienol, in wheat and rye samples, which was at +350 mV. A palm oil containing a relatively large amount of gamma-tocotrienol and lower concentrations of alpha- and delta-tocotrienols and alpha- and gamma-tocopherols was used to provide reference retention times for the tocotrienols. Analyte quantification was performed using the external standard method. The calibration equations of tocopherols were used to quantify both tocopherols and their corresponding tocotrienols. The extraction recoveries obtained using the optimized PLE conditions were in the 80-114% range, with RSDs lower than 15%. The method was successfully applied to the determination of tocotrienols and tocopherols in cereal (wheat, rye, barley, maize and oat) and palm oil samples.

A modified QuEChERS method as sample treatment before the determination of isoflavones in foods by ultra-performance liquid chromatography-triple quadrupole mass spectrometry.

This paper reports the development of an analytical method for the determination of isoflavones in legumes using LC-MS/MS. A modified approach of the QuEChERS methodology was used to extract the analytes from the food samples. The proposed method includes a two-step extraction process and allows the determination of isoflavones in pulses without the need of a clean-up step. Use of this methodology for the extraction of natural occurring substances provides advantages such as simplicity and ease of use, especially taking into account the complexity of food matrices. The method was applied successfully for the determination of eight isoflavones, including aglycones and glucosides, in legumes of Spanish origin (chickpeas, lentils and beans from the region of Castilla y León). The target compounds were the glucosides daidzin, glycitin and genistin, and the aglycones daidzein, glycitein, genistein, formononetin, and biochanin A. The detection limits were in the 0.7 μg L(-1) to 1.5 μg L(-1) range for formononetin and glycitin respectively. Recoveries ranged from 72% to 119%, and standard deviations lower than 25% were obtained for the inter-day precision. The method described is precise, selective and not time-consuming.

Analysis of isoflavones in soy drink by capillary zone electrophoresis coupled with electrospray ionization mass spectrometry

Capillary zone electrophoresis coupled with electrospray ionization mass spectrometry (CZE-ESI-MS) has been applied for the first time for the separation and quantification of isoflavones in soy products. The proposed method was successfully applied to the determination of seven isoflavones, including aglycones and glucosides, in soy drink. The target compounds were the glucosides daidzin and genistin, and the aglycones daidzein, genistein, formononetin, biochanin A and glycitein. During CE separation in positive mode, the analytes were present as anions, and MS detection was carried out in ESI positive-ion mode. To prevent the frequent drops in current and to improve the resolution in the separation of analytes in anionic form, a programmed nebulizing gas pressure (PNP) was applied along the analysis. Extraction of isoflavones from soy drinks was carried out by liquid-liquid extraction using ethanol. The proposed extraction procedure is simple, efficient, and affords reproducible results. Quantification of the isoflavones in soy drinks using the external standard method did not produce good results; therefore, both internal standard and standard addition quantification methods were used, obtaining significantly similar results. The detection limits found were lower than 3.2 μg L(-1).

Off-line coupling of pressurized liquid extraction and LC/ED for the determination of retinyl acetate and tocopherols in infant formulas.

An analytical methodology including pressurized liquid extraction (PLE) as sample treatment to isolate retinyl acetate and tocopherols from infant formulas has been developed. The milk extracts were kept at -18 degrees C for 30min and after filtration could be injected directly into the chromatographic system. Thus, a rapid and simple routine control method of these products is possible. The parameters affecting both the extraction process and the liquid chromatography (LC) system were optimized. PLE was performed using one cycle of extraction during a static time of 5min. Methanol was chosen as the extraction solvent for a temperature of 50 degrees C. Chromatographic separation was accomplished using a RP-18 column; the mobile phase used was methanol-water (94:6, v/v) containing 2.5mM acetic acid/sodium acetate buffer. Electrochemical detection in amperometric mode with a glassy carbon electrode at +1100mV was applied. The proposed methodology was successfully used for the determination of retinyl acetate, delta-tocopherol, (beta+gamma)-tocopherol and alpha-tocopherol in different infant formulas. The analytes were evaluated in the same chemical form present in the samples. Recoveries were between 92 and 106%. A certified reference material of milk powder was also analyzed.

Determination of tocopherols and sitosterols in seeds and nuts by QuEChERS-liquid chromatography.

In the present work a simple, reliable and affordable sample treatment method for the simultaneous analysis of tocopherols and free phytosterols in nuts was developed. Analyte extraction was carried out using the QuEChERS methodology and analyte separation and detection were accomplished using HPLC-DAD. The use of this methodology for the extraction of natural occurring substances provides advantages such as speed, simplicity and ease of use. The parameters evaluated for the validation of the method developed included the linearity of the calibration plots, the detection and quantification limits, repeatability, reproducibility and recovery. The proposed method was successfully applied to the analysis of tocopherols and free phytosterols in samples of almonds, cashew nuts, hazelnuts, peanuts, tiger nuts, sun flower seeds and pistachios.

Comparative Study of the Methodology Used in the Extraction of Isoflavones from Legumes Applying a Modified QuEChERS Approach

INTRODUCTION Isoflavones are phytochemicals of great interest because of their association with a large variety of positive effects on human health. The major sources of isoflavones in the diet are plants of the Leguminosae family, especially soybeans, although many other legumes more widely consumed in the Mediterranean diet have also been reported to contain these compounds. In previous work we extracted isoflavones from samples using a modified QuEChERS (Quick Easy Cheap Effective Rugged Safe) methodology. OBJECTIVE To compare different methods for placing the sample and the solvent in contact to optimise the extraction of isoflavones from legumes (chickpeas, lentils and white beans) using a modified QuEChERS methodology. METHOD Five different approaches to sample agitation were tested: vortex agitation, thermostatted stirring agitation and thermostatted tray shaking, and a thermostatted ultrasound bath and an ultrasound probe. To evaluate the different methodologies a modified QuEChERS approach was used as the extraction method. The separation and quantification of isoflavones was carried out using liquid chromatography-triple quadrupole/mass spectrometry (LC-MS/MS). RESULTS The best methods were found upon using a thermostatted shaking tray for the extraction of chickpeas and white beans and the ultrasound probe for lentil samples. These methods were chosen based on the highest amount of analytes obtained as well as the best recovery values. CONCLUSION Determination of isoflavones in foods may be affected by the different methods used to place the sample and the solvent in contact in the extraction step. The main advantages of the proposed extraction procedures are their simplicity, speed, reliability and low cost.

Classification of Lentils, Chickpeas and Beans Based on Their Isoflavone Content

Food legumes (Fabaceae) form an important part of the human diet; besides, several Fabaceae species are acknowledged for their high levels of bioactive compounds, among which are isoflavones, being recognised for their varied types of biological activity. The aim of this work was to classify different varieties of three types of legumes (chickpeas, lentils and beans) according to their isoflavone contents. The analysis of isoflavones was carried out using high-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (HPLC-MS/MS). To extract the analytes, a modified QuEChERS approach was used. The chromatographic peak areas obtained, after scaling in Pareto mode, were used to build statistical models. Both supervised and unsupervised techniques were applied for the classification of the different types of pulses analysed in the study: principal component analysis (PCA), hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLS-DA). The statistical models were validated by internal validation, obtaining satisfactory results for the different matrices. PCA models allowed the differentiation between subspecies, but not subspecies, varieties or ecotypes. The results provided by HCA and PLS-DA revealed that the different species and subspecies of beans and the different varieties and subvarieties of lentils can be distinguished, and even the different ecotypes of the same variety in the case of chickpeas. This study revealed that it was possible to differentiate among species, subspecies, varieties and even ecotypes of different types of legumes based on their isoflavone content.