Total Phenols and Flavonoids in Germinated Barley Using Different Solvents.

Sprouts are a source of secondary metabolites as phenolic compounds. Germination and the use of solvents can affect their content. The aim of this work was to identify the total content of phenols and flavonoids in ungerminated and germinated (3, 5, and 7 days) Esmeralda and Perla barley. Different solvents (water, 50 % acetone, 80 % methanol, 80 % ethanol) were used to recover total phenols and flavonoids. The 7-day germination proved to be ideal for total phenol and flavonoid obtention from Esmeralda barley and the highest total phenol and flavonoid content in Perla variety was observed at 5 and 7 days of germination, respectively. Methanol and ethanol (80 %) yielded the highest extraction percentage of total phenols; 50 % acetone recovered the highest flavonoid concentrations in Esmeralda barley and 80 % methanol in Perla barley. The highest total phenol concentration was obtained from Perla samples at 13.60 mg GAE/g, and the highest total flavonoids were observed in Esmeralda barley at 1.73 mg QE/g. A high correlation was found between the concentration of phenols (0.995) and total flavonoids (0.780) with the radicle size in the Esmeralda samples.

ACE-Inhibitory Activity of Whey Proteins Fractions Derived of Fermentation by Lacticaseibacillus rhamnosus GG and Streptococcus thermophilus SY-102.

Many studies have reported the benefits of probiotic microorganisms and the production of angiotensin-converting enzyme (ACE) inhibitors. Determining the proteolytic and ACE inhibition capacities during whey fermentation was the goal of the study. Lacticaseibacillus rhamnosus GG, Streptococcus thermophilus SY-102, and both bacteria together were initially inoculated into whey, reaching an initial concentration of 108 CFU per milliliter in each fermentation system. Through the use of TNBS, SDS-PAGE, and SEC-HPLC methods, the proteolytic profile was examined. An in vitro investigation was performed to test the ACE inhibition capacity. With S. thermophilus, the logarithmic phase of microbial development was shorter than with L. rhamnosus (6 and 12 h, respectively). The logarithmic phase in the co-culture fermentation, however, was extended to 24 h. There were no significant differences in pH between the fermentations. However, the co-culture had a greater concentration of protein hydrolysis (453 ± 0.06 µg/mL), as indicated by the amount of free amino groups. Similarly, this fermentation produced more low molecular weight peptides. The higher inhibition activity, which increased at the conclusion of the fermentation with the co-culture and reached 53.42%, was influenced by the higher peptide synthesis. These findings highlighted the significance of creating useful co-culture products.

Removal of heavy metal ions with magnetic carbon prepared from corncob biomass.

Four novel magnetic-activated carbons (MACs) were prepared, characterised, and used as adsorbents to remove heavy metal ions from wastewater samples. The MACs prepared, are advanced adsorbents for the removal of Hg(II), Cr(III), Cd(II), and Pb(II). The nature of the acid, amount, composition of the MACs, and the remotion time were evaluated in aqueous solutions. The ions removal percentages obtained, under the best conditions, were 93% for Hg(II) and higher than 99% for Pb(II), Cr(III), and Cd(II) (100 mg L-1, initial concentration in solution), with 100 mg of the MAC-3 in HNO3 3 mM. The capacity of the best adsorbent, MAC-3, for removing heavy metals ions Hg(II), Cr(III), Cd(II), and Pb(II) was studied using Langmuir and Freundlich adsorption isotherms under the best condition. The maximum adsorption capacities of Hg(II), Cr(III), Cd(II), and Pb(II) were found to be 10.72, 11.51, 11.49 and 11.49 mg g-1, the values of constants of Freundlich models were 17.98, 26.83, 9.18, and 7.18 mg g-1 respectively. For Hg(II) and Pb(II) the correlation factor (R2) was better for Freundlich model, while Cr(III) and Cd(II) showed better R2 with Langmuir model. Finally, the treatment for the elimination of heavy metal ions was carried out, with wastewater samples of industrial and domestic origin, and used for crop irrigation. The samples were collected in Irrigation District 003, Hidalgo, Mexico. The MAC-3 removes heavy metal ions from the wastewater matrix above 99%.

Nephroprotective Activity of Papaloquelite (Porophyllum ruderale) in Thioacetamide-Induced Injury Model.

Acute kidney injury and impaired kidney function is associated with reduced survival and increased morbidity. Porophyllum ruderale is an edible plant endemic to Mexico used in Mexican traditional medicine. The aim of this study was to evaluate the nephroprotective effect of a hydroalcoholic extract (MeOH:water 70:30, v/v) from the aerial parts of P. ruderale (HEPr). Firstly, in vitro the antioxidant and anti-inflammatory activity of HEPr was determined; after the in vivo nephroprotective activity of HEPr was evaluated using a thioacetamide-induced injury model in rats. HEPr showed a slight effect on LPS-NO production in macrophages (15% INO at 40 µg/mL) and high antioxidant activity in the ferric reducing antioxidant power (FRAP) test, followed by the activity on DPPH and ABTS radicals test (69.04, 63.06 and 32.96% of inhibition, respectively). In addition, values of kidney injury biomarkers in urine (urobilinogen, hemoglobin, bilirubin, ketones, glucose, protein, pH, nitrites, leukocytes, specific gravity, and the microalbumin/creatinine) and serum (creatinine, urea, and urea nitrogen) of rats treated with HEPr were maintained in normal ranges. Finally, 5-O-caffeoylquinic, 4-O-caffeoylquinic and ferulic acids; as well as 3-O-quercetin glucoside and 3-O-kaempferol glucoside were identified by HPLC as major components of HEPr. In conclusion, Porophyllum ruderale constitutes a source of compounds for the treatment of acute kidney injury.

Influence of keeping the calyces during fermentation on physicochemical and sensory properties of Hibiscus sabdariffa wines.

Hibiscus (Hibiscus sabdariffa) wines are usually made through the fermentation of its calyces extracts. However, the exhausted calyces could still contain a considerable amount of extractable bioactive compounds. This work analyzed the effect of keeping the hibiscus calyces during fermentation on the physicochemical and sensory characteristics and acceptance of its wines. Hibiscus wines Filtered-Ch and Filtered-C were generated by fermentation of filtered musts of China and Colima varieties, respectively, while Unfiltered-Ch and Unfiltered-C were obtained by fermentation of musts from calyces that were kept for 120 days. Unfiltered-C and Unfiltered-Ch wines had higher contents of total monomeric anthocyanins (32.5 mg/L in Unfiltered-C and 48.5 mg/L in Unfiltered-Ch), condensed tannins (around 200 mgCE/L), total phenolic compounds (800 mgGAE/L), and antioxidant activity (8.4-8.8 mMTE/L), as well as a higher concentration of tartaric, citric and malic acids (0.26, 0.32 and 2.25 g/L, respectively) than Filtered-C and Filtered-Ch wines (P < 0.05). Moreover, Unfiltered-C and Unfiltered-Ch wines had darker red colors, evidenced by lower lightness, chroma and hue values, than those observed in Filtered-C and Filtered-Ch wines (P < 0.01). Furthermore, Unfiltered-Ch and Unfiltered-C wines were more appreciated, having a more intense taste and smell according to the descriptions obtained from consumers by applying the Check-All-That-Apply technique. Sensory attributes observed in hibiscus wines were statistically correlated with the physicochemical characteristics. Keeping hibiscus calyces during fermentation allows for the production of wines with acceptable sensory characteristics and a higher concentration of bioactive compounds than producing wines from filtered musts.

Novel bioactive peptides sequences released by in vitro digestion of proteins isolated from Amaranthus hypochondriacus.

Amaranth is a protein-rich seed, highly consumed in Mexico and reported as a potential source of bio-peptides with physiological function. Therefore, the objective of this study was to evaluate and identify bioactive peptide sequences from amaranth protein fractions released by pepsin and pancreatin in vitro digestion. Multifunctional bioactivities of peptides were measured through antihypertensive, antithrombotic and antioxidant assays. Peptide sequences were identified by analysis with a Nano-LC-ESI-MS/MS. With these results, it was demonstrated that the in vitro enzymatic digestion mechanism allows the release of multifunctional bioactive peptide fractions from amaranth proteins.

Cereals as a Source of Bioactive Compounds with Anti-Hypertensive Activity and Their Intake in Times of COVID-19.

Cereals have phytochemical compounds that can diminish the incidence of chronic diseases such as hypertension. The angiotensin-converting enzyme 2 (ACE2) participates in the modulation of blood pressure and is the principal receptor of the virus SARS-CoV-2. The inhibitors of the angiotensin-converting enzyme (ACE) and the block receptors of angiotensin II regulate the expression of ACE2; thus, they could be useful in the treatment of patients infected with SARS-CoV-2. The inferior peptides from 1 to 3 kDa and the hydrophobic amino acids are the best candidates to inhibit ACE, and these compounds are present in rice, corn, wheat, oats, sorghum, and barley. In addition, the vitamins C and E, phenolic acids, and flavonoids present in cereals show a reduction in the oxidative stress involved in the pathogenesis of hypertension. The influence of ACE on hypertension and COVID-19 has turned into a primary point of control and treatment from the nutritional perspective. The objective of this work was to describe the inhibitory effect of the angiotensin-converting enzyme that the bioactive compounds present in cereals possess in order to lower blood pressure and how their consumption could be associated with reducing the virulence of COVID-19.

Human milk oligosaccharides as bioactive compounds in infant formula: recent advances and trends in synthetic methods.

Human milk oligosaccharides (HMO) have attracted great interest in recent years due to their role in boosting infants and adults health. According to several in vitro, in vivo and clinical studies, gastrointestinal and immune physiological systems benefit the most from HMO intake. Other organ systems, such as the respiratory, central nervous, circulatory, locomotor, and urinary systems have also been found to be affected by the HMO consumption in the recent decade. Due to their positive impact on human health, the incorporation of HMO into the infant formula or other functional foods has become highly desirable. Currently, their large-scale production is limited to 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) that are obtained through fermentation and added to the infant formula as fortifiers. Fewer advances have been made for other HMO to reach the industrial scale synthesis. The present paper summarizes the latest research on HMO in terms of their health benefits and synthetic methodologies, with the overall aim to establish the current status and trends in both fields.

Impact of the Gut Microbiota Balance on the Health-Disease Relationship: The Importance of Consuming Probiotics and Prebiotics.

Gut microbiota is a group of microorganisms that are deposited throughout the entire gastrointestinal tract. Currently, thanks to genomic tools, studies of gut microbiota have pointed towards the understanding of the metabolism of important bacteria that are not cultivable and their relationship with human homeostasis. Alterations in the composition of gut microbiota could explain, at least in part, some epidemics, such as diabetes and obesity. Likewise, dysbiosis has been associated with gastrointestinal disorders, neurodegenerative diseases, and even cancer. That is why several studies have recently been focused on the direct relationship that these types of conditions have with the specific composition of gut microbiota, as in the case of the microbiota-intestine-brain axis. In the same way, the control of microbiota is related to the diet. Therefore, this review highlights the importance of gut microbiota, from its composition to its relationship with the human health-disease condition, as well as emphasizes the effect of probiotic and prebiotic consumption on the balance of its composition.

Biogenic amines levels in food processing: Efforts for their control in foodstuffs.

Fermented and/or protein-rich foods, the most widely consumed worldwide, are the most susceptible to the presence of high levels of biogenic amines (BAs). Many reviews have focused on BAs toxicity and presence in foods; however, technological strategies such as evaluation of physical parameters, the addition of natural or synthetic compounds or the use of specific starter cultures of BAs reduction, and quick detection methods have been scarcely approached. In current research, there has been a focus on fast detection of BAs through colorimetric methods that allow these compounds to be quickly and easily identified by consumers. To reduce BAs presence in food, several alternatives have been developed and investigated with the aim of preventing negative effects caused by their intake, which can be applied before, during, or after processing. Food safety is one of the most important concerns of consumer and sanitary authorities. Therefore, detecting toxins such as BAs in food has become a priority for research. Recent reports that focus on the development of rapid detection methods of BAs are reviewed in this analysis. These methods have been successfully applied to food matrices with little to no sample pretreatment. Several alternatives for BAs reduction in food was also summarized. These findings will help the food industry to improve its processes for developing safe food.

Pomegranate as a Potential Alternative of Pain Management: A Review.

The use of complementary medicine has recently increased in an attempt to find effective alternative therapies that reduce the adverse effects of drugs. Punica granatum L. (pomegranate) has been used in traditional medicine for different kinds of pain. This review aims to explore the scientific evidence about the antinociceptive effect of pomegranate. A selection of original scientific articles that accomplished the inclusion criteria was carried out. It was found that different parts of pomegranate showed an antinociceptive effect; this effect can be due mainly by the presence of polyphenols, flavonoids, or fatty acids. It is suggested in the literature that the mechanisms of action may be related to the activation of the L-arginine / NO pathway, members of the TRP superfamily (TRPA1 or TRPV1) and the opioid system. The implications for the field are to know the mechanisms of action by which this effect is generated and thus be able to create alternative treatments for specific types of pain, which help alleviate it and reduce the adverse effects produced by drugs. The results propose that pomegranate and secondary metabolites could be considered in the treatment of inflammatory, nociceptive, and neuropathic pain.

Lactobacillus pentosus ABHEAU-05: An in vitro digestion resistant lactic acid bacterium isolated from a traditional fermented Mexican beverage.

The health benefits attributed to probiotics generate interest in the search of competent strains adapted to several ecological niches, especially those related to traditional beverages and foods of each country. Pineapple tepache, a traditional Mexican fermented beverage, was used for the isolation of lactic acid bacteria with probiotic potential, one of which withstood the in vitro tests. The isolated strain AB-05, which exhibited the tested probiotic functional properties, was designated as Lactobacillus pentosus ABHEAU-05. The sequence was registered in GenBank under access code MK587617. This study is the first report of a lactic acid bacterium with in vitro digestion resistance isolated from pineapple tepache. The survival of L. pentosus ABHEAU-05 in a symbiotic medium was proven using fermented milk enriched with inulin. The in vitro digestion-resistant probiotic activity of lactobacilli was measured through analysis of pH and proteolysis. Results showed that L. pentosus grew properly in fermented milk; therefore, this microorganism could be used in the manufacture of this kind of products. The concentration of L. pentosus reached up to 8.5logCFU/ml after 40h of fermentation. In addition, the production of peptides and the decrease in pH indicated the vigorous and active metabolic state of the lactic acid bacterium tested. The activity and the concentration of this microorganism were maintained during refrigeration. The results of this research conclude that L. plantarum ABHEAU-05 is an in vitro digestion-resistant microorganism that can be used as a starter culture for the production of functional foods of dairy origin.

Development of a dispersive liquid-liquid microextraction coupled with magnetic dispersive microsolid phase extraction for determination of triazines in wastewater samples.

The combination of two microextraction techniques (dispersive liquid-liquid microextraction [DLLME] and magnetic dispersive microsolid phase extraction [MDMSPE]) was developed and reported for atrazine and simazine preconcentration from wastewater samples. The proposal methodology involved the use of magnetite supports functionalized with different alkyl or phenyl groups. The magnetic adsorbents were synthesized by the solvothermal method assisted by microwave, characterized, and used in the sample preconcentration of atrazine and simazine. The method validation included parameters such as the wastewater matrix effect, repeatability, and recovery. The analyte separation and quantification were performed by high-performance liquid chromatography with ultraviolet detection (HPLC-DAD). Parameters, such as the polarity and mass of magnetic solids and pH, were evaluated to provide better extraction performance. The highest recoveries (> 95%) were obtained with 50 mg of the phenyl group support (CS2) at pH 5, using 5 mL of the sample and carbon tetrachloride and methanol, as extraction and dispersive solvents, respectively. The lowest limits of detection (LOD) achieved were 13.16 and 13.86 ng L-1, and the limits of quantification (LOQ) were 43.89 and 46.19 ng L-1 for simazine and atrazine, respectively, with repeatability (expressed as %RSD) below 5% in all cases. The developed method is simple, easy, and low cost for the analysis of two herbicides potentially dangerous for environmental and human health. Graphical abstract.

Physicochemical characterization of an arabinoxylan-rich fraction from brewers' spent grain and its application as a release matrix for caffeine.

The brewers' spent grain is a by-product generated during brewery process and is a potential source for arabinoxylans (AX) extraction. In the present work, the extraction and characterization of an arabinoxylan-rich fraction from brewers' spent grain (BSG-AX) were performed, and BSG-AX was evaluated as release matrix for caffeine. The BSG-AX showed an AX content of 72% (w/w), a ferulic acid content of 3.52 µg/mg BSG-AX, an Ara/Xyl ratio of 0.89, an intrinsic viscosity of 41.18 mL g-1, and a molecular weight of 43.80 kDa. The studied BSG-AX showed a good antioxidant capacity compared with other polysaccharide gums and was estimated by DPPH (114.41 µM Trolox equivalent/g BSG-AX) and FRAP (49.01 µmol Fe2+/g BSG-AX) assays. The partial specific volume (0.63 cm3 g-1), loss on drying (10.68%), swelling (10.87%), solubility (80.93%) and electrostatic interactions (by zeta potential, -3.44 to -9.17 mV) were determined and used to evaluate the application of the BSG-AX as release matrix. A film containing the BSG-AX, glycerol (as plasticizer) and caffeine (target drug) was prepared as release matrix. Glycerol promoted an increase in the extensibility and the surface smoothness of the BSG-AX-caffeine film. The drug was released (≈98%) in about 7 h. These results are promising to concern the design and use of BSG-AX based biofilms for the controlled release of bioactive compounds.

Bioactive compounds in aqueous extracts of lemon balm (Melissa officinalis) cultivated in Mexico

Lemon balm (Melissa officinalis) is a plant in the family of Lamiaceae. In Mexican traditional medicine it is used to alleviate gastrointestinal and hepatic problems. Studies carried out mainly in ethanolic and methanolic extracts, have revealed the presence of diverse compounds to which those medicinal properties are attributed. The objective of this research work was to extract in aqueous solution the antioxidants present in lemon balm and identify them through HPLC-MS. A Box-Behnken design was applied to determine the physical conditions of antioxidant extraction, where the analyzed variables were time, temperature and sample quantity. The antioxidant activity was determined through methodologies of DPPH*, FRAP and total phenolics. The aqueous solution with the highest antioxidant activity was analyzed through HPLC-MS. The results showed that the interaction temperature-time has a positive influence on the liberation of antioxidants. The best condition for a conventional extraction of antioxidants was 90°C, 15 min and 2 g of sample. Higher correlations were observed at r2>0.6 between determined antioxidant activity by DPPH* (or FRAP) vs Total phenolics; this would indicate that such activity could be attributed to phenolic compounds whose presence was confirmed through an analysis by HPLC-MS(AU)

El toronjil (Melissa officinalis) es una planta de la familia Lamiaceae. En la medicina tradicional mexicana es utilizado para aliviar problemas gastrointestinales y hepáticos. Algunos estudios realizados con extractos etanólicos y metanólicos de dicha planta, han revelado la presencia de diversos compuestos a los que se les atribuye sus propiedades medicinales. El objetivo de esta investigación fue extraer en solución acuosa los antioxidantes presentes en el toronjil e identificarlos a través de HPLC-MS. Para ello, se aplicó un diseño de experimentos Box-Behnken a fin de determinar las condiciones físicas de extracción de antioxidantes; las variables analizadas fueron tiempo, temperatura y cantidad de muestra. La actividad antioxidante fue determinada a través de las metodologías de DPPH*, FRAP y fenoles totales. El extracto acuoso con la mayor actividad antioxidante fue analizado mediante HPLCMS. Los resultados mostraron que la interacción tiempotemperatura tuvo una influencia positiva en la liberación de antioxidantes. La mejor condición para la extracción de antioxidantes presentes en el toronjil fue 90°C, 15 min y 2 g de muestra. Correlaciones superiores a r2>0.6 fueron determinadas entre la actividad antioxidante medida por DPPH* (o FRAP) vs Fenoles totales; esto podría indicar que la actividad antioxidante encontrada podría atribuirse a compuestos de tipo fenólico cuya presencia fue confirmada por el análisis en HPLC-MS(AU)

Effect of thermoultrasound on aflatoxin M1 levels, physicochemical and microbiological properties of milk during storage.

The aim of this research was to determine the physicochemical properties, microbial counts and aflatoxin M1 (AFM1) levels of thermoultrasonicated, pasteurized and untreated milk (control) at days 1, 7 and 14 of storage. Thermoultrasound treatment was performed at a rate of 20 kHz for 10 or 15 min and 95% amplitude on homogenized and non-homogenized milk samples. Results showed that most physicochemical parameters were within the Mexican norms established for milk. Ultrasound treatment for 15 min reduced solids precipitation (p < 0.05) in unhomogenized milk during storage as compared to the pasteurized milk. All samples complied with aerobic mesophilic counts limits set by the Mexican norm except the control and the homogenized milk sample which was thermoultrasonicated for 10 min. Enterobacteriaceae counts of pasteurized and 15 min-thermoultrasound homogenized milks complied with the norm. The lowest levels of AFM1 were found in the 10 min-thermoultrasound unhomogenized milk (0.15 ±â€¯0.05 pg AFM⁠1E/mL) one day after storage. Thermoultrasound did not affect the color of samples but homogenized milk treated for 10 min exhibited less total color difference. A high phenolic content was found in thermoultrasound and pasteurized milks on day 1. Thermoultrasound could be an alternative to milk pasteurization that preserves the physicochemical and microbiological quality of milk while reducing AFM1 levels.

Antithrombotic Activity of Milk Protein Hydrolysates by Lactic Acid Bacteria Isolated from Commercial Fermented Milks

ABSTRACT In the last decade, thrombosis has been one of the pathologies with high incidence creating great concern in Health Institutes all around the world. Considering this, the aim of this research was to determine the antithrombotic activity of peptides released during lactic fermentation. Reconstituted skim milk powder was fermented by Lactobacillus casei Shirota and Lactobacillus johnsonii LA1 isolated from commercial fermented milks. The hydrolysis degree and proteolytic profile were analyzed by trinitrobenzenesulfonic acid spectrophotometry method and by peptide polyacrylamide electrophoresis gel separation. The milk fermented by Lactobacillus casei Shirota exhibited a higher concentration of free amino groups than that fermented by Lactobacillus johnsonii LA1. Additionally, in both fermentation systems peptides with molecular weights lower than 1.4 kDa were observed. The highest inhibition of thrombin (31.67±2.35%) was observed in milk fermented by Lactobacillus johnsonii LA1 at 10 hours of fermentation. Finally, no relationship was found between the antithrombotic capacity and the proteolytic profile.

Optimization of Physical Conditions for the Aqueous Extraction of Antioxidant Compounds from Ginger (Zingiber officinale) Applying a Box-Behnken Design.

Since ancient times, ginger (Zingiber officinale) has been widely used for culinary and medicinal purposes. This rhizome possesses several chemical constituents; most of them present antioxidant capacity due mainly to the presence of phenolic compounds. Thus, the physical conditions for the optimal extraction of antioxidant components of ginger were investigated by applying a Box-Behnken experimental design. Extracts of ginger were prepared using water as solvent in a conventional solid-liquid extraction. The analyzed variables were time (5, 15 and 25 min), temperature (20, 55 and 90 °C) and sample concentration (2, 6 and 10 %). The antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl method and a modified ferric reducing antioxidant power assay while total phenolics were measured by Folin & Ciocalteu's method. The suggested experimental design allowed the acquisition of aqueous extracts of ginger with diverse antioxidant activity (100-555 mg Trolox/100 g, 147-1237 mg Fe2+/100 g and 50-332 mg gallic acid/100 g). Temperature was the determining factor in the extraction of components with antioxidant activity, regardless of time and sample quantity. The optimal physical conditions that allowed the highest antioxidant activity were: 90 °C, 15 min and 2 % of the sample. The correlation value between the antioxidant activity by ferric reducing antioxidant power assay and the content of total phenolics was R2 = 0.83. The experimental design applied allowed the determination of the physical conditions under which ginger aqueous extracts liberate compounds with antioxidant activity. Most of them are of the phenolic type as it was demonstrated through the correlation established between different methods used to measure antioxidant capacity.

Microextraction method of medium and long chain fatty acids from milk.

The aim of this research was to develop a method for microextraction and quantification of long and medium chain fatty acids from milk. The proposed method was carried out on three steps: (1) lipids extraction from 10 µL of whole or skimmed milk containing omega-3 fatty acids (FAs) during 12, 24, 48, 72 and 96 h, in 2 mL chloroform: methanol mixture (2:1); (2) acid methylation of FAs; and (3) quantification of FAs methyl esters by gas chromatography. Using this method, the percentage recovery of FAs was higher (67% for whole and 85% for skimmed milk) compared with AOAC method 905.02 recovery (49% and 83%, respectively). Good reproducibility and repeatability (<3%) were obtained. The method developed can be useful for researching and in routine quality control. Additionally, it is simpler, faster and cheaper than the reference procedure since it requires minimum sample and solvents volume as well as fewer steps.