Determination of chlorogenic acid,polyphenols and antioxidants in green coffee by thin‐layer chromatography,effect‐directed analysis and dot blot – comparison to HPLC and spectrophotometry methods

The great prevalence of thin‐layer chromatography over high‐performance liquid chromatography is connected with the possibility of analyzing many samples in parallel. Therefore, the method is often used in screening and/or effect directed analysis to compare composition and chemical/biological properties of many samples in one run. It was already proved, that high performance thin‐layer chromatography, in many cases, can replace high‐performance liquid chromatography for quantitative analysis. The main aim of the paper is to show that simple thin‐layer chromatography can also be used as a quantitative or at least as a semi‐quantitative method, even when it concerns effect directed analysis e.g. direct bioautography. Chlorogenic acid content was measured in four methanol extracts of various green coffees and in one extract of black coffee using thin‐layer chromatography with ultraviolet detection and thin‐layer chromatography with effect directed detection. High‐performance liquid chromatography was used as a reference method. Additionally, total contents of polyphenols and antioxidants were estimated using thin‐layer chromatography or dot‐blot on chromatography plates. These results were compared to spectrophotometric methods. It was proved that thin‐layer chromatography can be used as a quantitative (using densitometry) or semi‐quantitative method (using other detection methods including effect directed detection) as well as for estimating total antioxidants or polyphenols content.     

Comparison of various easy-to-use procedures for extraction of phenols from apricot fruits

Phenols are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention due to their potential antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer. The objective of this study was to investigate a suitable method for determination of protocatechuic acid, 4-aminobenzoic acid, chlorogenic acid, caffeic acid, vanillin, p-coumaric acid, rutin, ferulic acid, quercetin, resveratrol and quercitrin from apricot samples. A high-performance liquid chromatograph with electrochemical and UV detectors was used. The method was optimized in respect to both the separation selectivity of individual phenolic compounds and the maximum sensitivity with the electrochemical detection. The lowest limits of detection (3 S/N) using UV detection were estimated for ferulic acid (3 μM), quercitrin (4 μM) and quercetin (4 μM). Using electrochemical detection values of 27 nM, 40 nM and 37 nM were achieved for ferulic acid, quercitrin and quercetin, respectively. It follows from the acquired results that the coulometric detection under a universal potential of 600 mV is more suitable and sensitive for polyphenols determination than UV detection at a universal wavelength of 260 nm. Subsequently, we tested the influence of solvent composition, vortexing and sonication on separation efficiency. Our results showed that a combination of water, acetone and methanol in 20:20:60 ratio was the most effective for p-aminobenzoic acid, chlorgenic acid, caffeic acid, protocatechuic acid, ferulic acid, rutin, resveratrol and quercetin, in comparison with other solvents. On the other hand, vortexing at 4 °C produced the highest yield. Moreover, we tested the contents of individual polyphenols in the apricot cultivars Mamaria, Mold and LE-1075. The major phenolic compounds were chlorgenic acid and rutin. Chlorgenic acid was found in amounts of 2,302 mg/100 g in cultivar LE-1075, 546 mg/100 g in cultivar Mamaria and 129 mg/100 g in cultivar Mold. Generally, the cultivar LE-1075 produced the highest polyphenol content values, contrary to Mold, which compared to cultivar LE-1075 was quite poor from the point of view of the phenolics content.     

Polyphenol Content and Antioxidant Activity of Merlot and Shiraz Wine

The total polyphenol content, antocyanins, proantoacyanidins, and some phenols were determined in fifteen red wine samples of the same vintage from single-blended Merlot or Shiraz cultivars. The principal goal of this study was to use these compounds for wine identification and to evaluate the correlation with antioxidant activity. High-performance liquid chromatography (HPLC) was used to determine catechin, epicatechin, rutin, quercetin, malvidin, peonidin, petunidin, cyanidin, delphinidin, pelargonidin, trans-resveratrol, gallic acid, chlorogenic acid, and p-coumaric acid. Spectrophotometric methods were employed to determine chromatic profile, total polyphenol content, antocyanins, proanthocyanidins, and antioxidant activity. There were some differences in the concentrations of phenolics for Shiraz and Merlot wines of 2009 vintage, including total polyphenols, antocyanidins, and proanthocyanidins. The analysis revealed a significantly higher antioxidant activity of Merlot than Shiraz and a strong correlation between antioxidant capacity and total polyphenol concentration.     

Comprehensive Evaluation of Amino Acids and Polyphenols in 69 Varieties of Green Cabbage (Brassica oleracea L. var. capitata L.) Based on Multivariate Statistical Analysis

The biological activities of the primary metabolites and secondary metabolites of 69 green cabbage varieties were tested. The LC-MS detection method was used to determine the content of 19 free amino acids (lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, arginine, asparagine, glycine, proline, tyrosine, glutamine, alanine, aspartic acid, serine, and glutamate). The content of 10 polyphenols (chlorogenic acid, gallic acid, 4-coumaric acid, ferulic acid, gentisic acid, cymarin, erucic acid, benzoic acid, rutin, and kaempferol) was determined by the HPLC detection method. Considering the complexity of the data obtained, variance analysis, diversity analysis, correlation analysis, hierarchical cluster analysis (HCA), and principal component analysis (PCA) were used to process and correlate amino acid or polyphenol data, respectively. The results showed that there were significant differences between the different amino acids and polyphenols of the 69 cabbage varieties. The most abundant amino acids and polyphenols were Glu and rutin, respectively. Both amino acids and polyphenols had a high genetic diversity, and multiple groups of significant or extremely significant correlations. The 69 cabbage varieties were divided into two groups, according to 19 amino acid indexes, by PCA. Among them, seven varieties with high amino acid content all fell into the fourth quadrant. The HCA of amino acids also supports this view. Based on 10 polyphenols, the 69 cabbage varieties were divided into two groups by HCA. Based on 29 indexes of amino acids and polyphenols, 69 cabbage varieties were evaluated and ranked by PCA. Therefore, in this study, cabbage varieties were classified in accordance with the level of amino acids and polyphenols, which provided a theoretical basis for the genetic improvement of nutritional quality in cabbage.     

Metabolite Differences of Polyphenols in Different Litchi Cultivars (Litchi chinensis Sonn.) Based on Extensive Targeted Metabonomics

Litchi is an important fruit cultivated in tropical and subtropical areas with high nutritious and delicious flavor and the pulp is the main part of the fruit consumed. Previous studies found that litchi had high total phenol content and antioxidant activity, but most of them focused on the identification of single or a few phenolic components with a low throughput test, and the metabolic differences of cultivars are still unknown to a some extent. In this study we used widely targeted metabolome based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) to analyze the polyphenol metabolites of five different genotypes of mature litchi fruit. A total of 126 polyphenol metabolites in eight categories were identified to reveal the composition and differences of polyphenol; 15 common differential metabolites and 20 specific differential metabolites to each cultivar were found for the first time. The results infer that flavonoids, flavonols, hydroxycinnamoyls and catechins are the main polyphenol metabolites of litchi pulp. Cluster analysis showed that there were three groups of polyphenols from high to low; early maturing Feizhixiao is a kind of high polyphenol content cultivars, especially in catechins, anthocyanins, flavonols, quinic acids and hydroxycinnamoyls. The polyphenols in the flesh of mature litchi are rich, and there are significant differences among cultivars; there was a level of correlation between the contents of phenolics and the maturity of litchi cultivars; the content of phenolics in early maturing litchi cultivars appeared higher than those of mid- to late-maturing cultivars. This experiment will provide significant reference information for cultivation, breeding, processing and consumption.     

Evaluation of the Chemiluminometric Method for Determination of Polyphenols in Wine

The Pholasin chemiluminometric method for determination of polyphenols in wine was evaluated by comparison with liquid chromatography with mass-spectrometric detection. The results were also compared with the conventional Singleton-Rossi spectrophotometric method for determination of total polyphenols.

A total of 141 wine samples were examined using all three methods and the results indicated a good agreement between the chemiluminometric and the Singleton-Rossi methods; however, the chromatographic data correlated reasonably well with the chemiluminometric data only for gallic acid, but less well or not at all with other determined phenolic antioxidants: trans-resveratrol, (+)-catechin, (?)-epicatechin, quercetin, vanillic-, caffeic-, caftaric-, p-coumaric-, 3,4-dihydroxybenzoic-, ellagic-, sinapic-, ferullic-, and ellagic acid.     

Separation of Phenols from the Leaves of Toona Sinensis (Meliaceae) by Capillary Electrophoresis

A micellar electrokinetic capillary chromatography (MEKC) method, using UV detection, was developed for the determination of polyphenols in Toona sinensis (Meliaceae); the procedure involved precipitation of polyphenols from the leaves of T. sinensis using methanol. The structures can be established with fifteen compounds including methyl gallate, gallic acid, kaempferol, quercitin, quercitrin, rutin, kaempferol‐glucoside, catechin, epicatechin, stearic acid, palmitic acid, β‐sitosterol, stigmasterol, β‐sitosteryl‐glucoside, and stigmasteryl‐glucoside by spectroscopic analysis. However, there has been no investigation to quantitate the polyphenols that form T. sinensis. Thus, seven polyphenols of T. sinensis with UV absorbance, catechin (C), epicatechin (EC), methyl gallate (MG), rutin (R), gallic acid (G), quercitrin (Q), and kaempferol (K) were separated within 10 min with a 40 cm uncoated fused‐silica column, with the RSD < 3% (migration times), voltage at 15 kV using this method. On‐column detection was carried out at 254 nm. The detection limit of this method for all analytes ranged from 19.5 to 0.02 μM (RSD < 3.1%). The method provided a rapid and sensitive identification of polyphenols of interest in T. sinensis and is suitable for biological activity studies.     

Fractionation of red wine polyphenols by solid-phase extraction and liquid chromatography

A systematic method for separation of aged red wine polyphenols into various distinct fractions using combined techniques of solid-phase extraction and liquid chromatography was proposed. The aged red wine polyphenols were separated into various distinct fractions including phenolic acid fraction, monomer flavanol fraction, oligomer procyanidin fraction, anthocyanin and its pyruvic acid derivative fraction, free or non-colored proanthocyanidin fraction, fraction of direct condensation products between anthocyanins and proanthocyanidins and fraction of other pigmented complexes. The phenolic composition of each fraction was verified by HPLC with diode array detection (HPLC-DAD), thiolysis, vanillin assay, HPLC coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) and multi-stage MS fragment analysis. For the first time, anthocyanins and their pyruvic derivatives were separated from other phenolic compounds, while free or non-pigmented polymer proanthocyanidins from other pigmented complexes. The fractionation method would be of particular interest in further studying the detailed composition of polymeric polyphenols in red wine.     

Liquid Chromatographic Analysis of Phenolic Compounds in Organically and Conventionally Grown Varieties of Sour Cherries

Liquid chromatographic analysis of the polyphenols in sour cherries was achieved on bi-functional RP columns with a gradient prepared from methanol and aqueous formic acid. For quantitative analysis the compounds were detected at 320 nm. The method was validated for linearity, precision, and recovery. Chlorogenic acid, neochlorogenic acid, and rutin were detected in sour cherry cultivars. Amounts of neochlorogenic acid were in the range 471–590 μg g⁻¹. For the three sour cherry cultivars investigated in this study there was no significant difference between the chlorogenic acid and rutin content of cherries produced by conventional and organic farming. There were, however, significant differences between the amounts of these compounds in cherries harvested in different years.

     

Relationships between the Content of Phenolic Compounds and the Antioxidant Activity of Polish Honey Varieties as a Tool for Botanical Discrimination

The study compared the content of eight phenolic acids and four flavonoids and the antioxidant activity of six Polish varietal honeys. An attempt was also made to determine the correlations between the antioxidant parameters of the honeys and their polyphenol profile using principal component analysis. Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (ABTS) and reduction capacity (FRAP) were determined spectrophotometrically, and the phenolic compounds were determined using high-performance liquid chromatography (HPLC). The buckwheat honeys showed the strongest antioxidant activity, most likely because they had the highest concentrations of total phenols, total flavonoids, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, vanillic acid and chrysin. The principal component analysis (PCA) of the data showed significant relationships between the botanic origin of the honey, the total content of phenolic compounds and flavonoids and the antioxidant activity of the six Polish varietal honeys. The strongest, significant correlations were shown for parameters of antioxidant activity and TPC, TFC, p-hydroxybenzoic acid, caffeic acid and p-coumaric acid. Analysis of four principal components (explaining 86.9% of the total variance), as a classification tool, confirmed the distinctiveness of the Polish honeys in terms of their antioxidant activity and content of phenolic compounds.     

Characterization and Identification of Bioactive Polyphenols in the Trapa bispinosa Roxb. Pericarp Extract

In this study, we present the isolation and characterization of the structure of six gallotannins (1–6), three ellagitannins (7–9), a neolignan glucoside (10), and three related polyphenolic compounds (gallic acid, 11 and 12) from Trapa bispinosa Roxb. pericarp extract (TBE). Among the isolates, the structure of compound 10 possessing a previously unclear absolute configuration was unambiguously determined through nuclear magnetic resonance and circular dichroism analyses. The α-glucosidase activity and glycation inhibitory effects of the isolates were evaluated. Decarboxylated rugosin A (8) showed an α-glucosidase inhibitory activity, while hydrolyzable tannins revealed stronger antiglycation activity than that of the positive control. Furthermore, the identification and quantification of the TBE polyphenols were investigated by high-performance liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry analysis, indicating the predominance of gallic acid, ellagic acid, and galloyl glucoses showing marked antiglycation properties. These findings suggest that there is a potential food industry application of polyphenols in TBE as a functional food with antidiabetic and antiglycation activities.     

Effect of radiation process on antinutrients and HCl extractability of calcium,phosphorus and iron during processing and storage

Whole and dehulled flours of millet cultivars Ashana and Dembi were stored for 30 and 60 days before and after radiation and/or cooking. Phytic acid and polyphenols contents were assayed for all treatments. The results revealed that the storage period was found to have no effect on phytate and polyphenols contents. Moreover, dehulling of the grains reduced more than 50% of phytate and polyphenols of both cultivars. Cooking of the raw whole and dehulled flour significantly (P≤0.05) reduced phytate and polyphenols contents for both cultivars. Radiation process alone had no effect on phytate and polyphenols contents but when followed by cooking significantly (P≤0.05) reduced the level of such antinutrients for the whole and dehulled flour of both cultivars. Dehulling alone significantly (P≤0.05) decreased Ca and P content but slightly decreased Fe content. Radiation alone or in combination with cooking was found to have slight effect on minerals content of the whole and dehulled raw flour for both cultivars. Cooking alone or in combination with radiation of whole or dehulled raw flour significantly (P≤0.05) improved the extractable Ca but had no significant (P≤0.05) effect on extractable P and Fe for both cultivars.     

An optimized method to extract poplar leaf proteins for two‐dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High‐quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE‐42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.     

Antifungal activity of mango peel and seed extracts against clinically pathogenic and food spoilage yeasts

The antioxidant and antifungal (antiyeast) properties of mango (Mangifera indica) peel and seed by-products were investigated. Nine extracts were obtained using three cultivars and two extraction methods. Significant differences between cultivars and extraction methods were detected in their bioactive compounds and antioxidant activity. The antifungal property was determined using agar diffusion and broth micro-dilution assays against 18 yeast species of the genera Candida, Dekkera, Hanseniaspora, Lodderomyces, Metschnikowia, Pichia, Schizosaccharomyces, Saccharomycodes and Zygosaccharomyces. All mango extracts showed antifungal activity. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) values were lower for seed than for peel extracts. MICs and MFCs ranged from values <0.1 to 5 and 5 to >30 mgGAE/mL, respectively. The multivariate analysis showed a relationship between antifungal activity, the capacity to inhibit lipid peroxidation and total phenol content. These properties were associated with high levels of proanthocyanidins, gallates and gallotannins in the extracts.     

Identification and Quantification of Lignans,Carboxylic Acids and Sugars in the Leaves of Forsythia Species and Cultivars

Lignans, carboxylic acids and sugars were determined by gas chromatography (GC) with mass selective detection, the lignans also by high-performance liquid chromatography with UV detection in the leaf extracts of four species and four cultivars of Forsythia plant. Three methods were used to optimize the extraction of the main lignan constituents (arctiin, arctigenin), which possess significant pharmaceutical effects. Supercritical fluid extraction (SFE) proved to be the most efficient method compared to sonication and refluxation. The total lignan content of the leaves varied from 31.6 (F. ovata ‘Tetragold’) to 113.8 mg g⁻¹ (F. ovata ‘Robusta’).

     

Sea Buckthorn Leaf Powders: The Impact of Cultivar and Drying Mode on Antioxidant,Phytochemical, and Chromatic Profile of Valuable Resource

Sea buckthorn (Hippophae rhamnoides L. (HR)) leaf powders are the underutilized, promising resource of valuable compounds. Genotype and processing methods are key factors in the preparation of homogenous, stable, and quantified ingredients. The aim of this study was to evaluate the phenolic, triterpenic, antioxidant profiles, carotenoid and chlorophyll content, and chromatic characteristics of convection-dried and freeze-dried HR leaf powders obtained from ten different female cultivars, namely ‘Avgustinka’, ‘Botaniceskaja Liubitelskaja’, ‘Botaniceskaja’, ‘Hibrid Percika’, ‘Julia’, ‘Nivelena’, ‘Otradnaja’, ‘Podarok Sadu’, ‘Trofimovskaja’, and ‘Vorobjovskaja’. The chromatic characteristics were determined using the CIELAB scale. The phytochemical profiles were determined using HPLC-PDA (high performance liquid chromatography with photodiode array detector) analysis; spectrophotometric assays and antioxidant activities were investigated using ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and FRAP (ferric ion reducing antioxidant power) assays. The sea buckthorn leaf powders had a yellowish-green appearance. The drying mode had a significant impact on the total antioxidant activity, chlorophyll content, and chromatic characteristics of the samples; the freeze-dried samples were superior in antioxidant activity, chlorophyll, carotenoid content, and chromatic profile, compared to convection-dried leaf powder samples. The determined triterpenic and phenolic profiles strongly depend on the cultivar, and the drying technique had no impact on qualitative and quantitative composition. Catechin, epigallocatechin, procyanidin B3, ursolic acid, α-amyrin, and β-sitosterol could be used as quantitative markers in the phenolic and triterpenic profiles. The cultivars ‘Avgustinka’, ‘Nivelena’, and ‘Botaniceskaja’ were superior to other tested cultivars, with the phytochemical composition and antioxidant activity.     

Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential

Presented work summarizes the data about polyphenolic profiles in various plant parts (leaves, shoots, berries) of sea buckthorn (Hippophaë rhamnoides L.) during the annual growth cycle. A reversed-phase high performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) was optimized for determination of catechin, epicatechin, gallic acid, p-coumaric acid, caffeic acid, ferulic acid, rutin (quercetin 3-rutinoside) and quercitrin (quercetin 3-rhamnoside). The content of these polyphenolic compounds was monitored in extracts of sea buckthorn plant samples from April to October. The total antioxidant activity was determined using scavenging of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) cation radical (ABTS^·+) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^·). The total content of polyphenols was estimated by conventional spectrophotometric method using Folin-Ciocalteu reagent. The monitoring of temporal changes of selected polyphenolic compounds by RP-HPLC showed that catechin, epicatechin and gallic acid were the most abundant analytes in annual green shoots and leaves, and their content varied significantly during the studied period.      

Innovative Combination of Dispersive Solid Phase Extraction Followed by NIR-Detection and Multivariate Data Analysis for Prediction of Total Polyphenolic Content

Recently polyphenols attracted great interest in the field of food and nutrition as well as in the pharmaceutical and cosmetics industries due to their health benefits through antioxidative behavior in the human body. However, because of the high number of compounds characterized as phenols and their structural diversity, quantification of polyphenols turns out to be a highly complex task. Although, a wide variety of analytical methods are used for the determination of total polyphenolic content, they are all found to be lacking in a variety of different tasks, such as their limits of detection and quantification, repeatability, accuracy and specificity. For this reason, a novel approach combining the advantages of solid phase purification, near infrared analysis and multivariate data analysis was investigated for the prediction of total polyphenolic content, suitable for a wide range of sample matrices. Dispersive solid phase extraction was performed and optimized using polyvinylpyrrolidone as sorbent, known to selectively bind polyphenols. Near-infrared detection of adsorbed polyphenols was carried out subsequently. Furthermore, the method was in-house validated, examining selectivity, repeatability and accuracy, working range, as well as multivariate limit of detection and limit of quantification, comparing it with two routinely used methods—namely, Folin–Ciocalteu photometric assay and Löwenthal titration. The novel established method was applied for the prediction of total polyphenolic content in tea and wine samples.     

The Effect of Brewing Process Parameters on Antioxidant Activity and Caffeine Content in Infusions of Roasted and Unroasted Arabica Coffee Beans Originated from Different Countries

Coffee is one of the most often consumed beverages almost all over the world. The multiplicity of beans, as well as the methods and parameters used to brew, encourages the optimization of the brewing process. The study aimed to analyze the effect of roasting beans, the brewing technique, and its parameters (time and water temperature) on antioxidant activity (determined using several in vitro methods), total polyphenols, flavonoids, and caffeine content. The infusions of unroasted and roasted Arabica beans from Brazil, Colombia, India, Peru, and Rwanda were analyzed. In general, infusions prepared from roasted beans had higher antioxidant activity and the content of above-mentioned compounds. The hot brew method was used to obtain infusions with a higher antioxidant activity, while the cold brew with higher caffeine content. The phenolic compound content in infusions prepared using both techniques depended on the roasting process. Moreover, the bean’s origin, roasting process, and brewing technique had a significant effect on the tested properties, in contrary to brewing time and water temperature (below and above 90 °C), which had less impact. The results confirm the importance of coffee brewing optimization.     

Determination of sorbic acid in margarine and butter by high-performance liquid chromatography with fluorescence detection

A procedure is reported for the separation and determination of sorbic acid, as a derivative of 4-bromoethyl-6,7-dimethoxycoumarin, by reversed-phase high-performance liquid chromatography with fluorescence detection using enanthic acid as an internal standard. Sorbic acid, separated from samples of commercial margarine and butter by steam distillation, was evaluated using the proposed procedure and by UV absorption and visible spectrophotometric methods (AOAC). The preparation of the calibration graph and the determination of sorbic acid with the visible spectrophotometric method was improved. The sorbic acid content determined using UV and visible spectrophotometric methods was higher than that obtained with the reversed-phase performance liquid chromatographic method owing to the presence of interfering substances in the samples. The range of recovery and the precision of the proposed method and the reference methods are also reported.