Optimizacija i validacija tečno-hromatografske metode za određivanje flavanona i njihovih derivata u farmaceutskim formulacijama i hrani

U ovom radu razvijene su LC-MS/MS i HPLC-UV/DAD metode za istovremeno određivanje bioflavonoida: rutina, kvercetina, hesperidina, hesperetina,kaempferola, apigenina, luteolina, katehina i epikatehina u nekim uzorcima hrane (kora pomorandže, crveni luk, med, karfiol, brokoli i prokelj). Takođe, razvijene su i HPLC-DAD i spektrofotometrijske metode za određivanje moksifloksacina (analoga flavonoida) i njegovih srodnih jedinjenja i degradacionih proizvoda u farmaceutskim formulacijama i humanoj krvnoj plazmi. Procenjen je matrični efekat prilikom određivanja rutina, kvercetina, hesperetina, hesperidina i kaempferola u uzorcima kore pomorandže, crvenog luka i meda. Uticaj matriksa procenjen je korišćenjem metode post-ekstrakcije pri čemu se dobijene negativne vrednosti u opsegu od -44 do -0,5 %, što ukazuje na jonsku supresiju. Matrični efekat jako zavisi od koncentracije ispitivanih bioflavonoida. Jonska supresija se može objasniti prisustvom koeluiranih fenolnih kiselina, koje zbog svojih kiselinsko-baznih i hidrofilnih karakteristika utiču na jonizaciju bioflavonoida u gasnoj plazmi. Ekstrakti uzoraka hrane su dobijeni primenom različitih načina ekstrakcije (ekstrakcija po Soksletu, ultrazvučna ektrakcija, ekstrakcija maceracijom i ekstrakcija ključalom vodom). Za prečišćavanje i prekoncentrisavanje dobijenih ekstrakata analita korišćena je ekstrakcija na čvrstoj fazi primenom Supelco kertridža.In the present work the LC-MS/MS and HPLC-UV/DAD methods with solid phase extraction for simultaneous determination of bioflavonoids rutin, quercetin, hesperidin,hesperetin, kaempferol, apigenin, luteolin, catehin and epicatehin in some food samples (red onion, orange peel, honey, cauliflower, broccoli and Brussel sprouts) were developed. Also, HPLC-UV/DAD and spectrophotometric methods for determination moxifloxacin (flavonoids analog) and their related substances and degradation products in pharmaceutical forms and human plasma were developed. The matrix effect accompanying determination rutin, quercetin, hesperidin, hesperetin and kaempferol in orange peel, red onion and honey was quantified. The matrix effect evaluated using postextraction addition method was found to be negative in the range -44-0,5 %, indicating ionization suppression and strongly depended on bioflavonoid concentration. Ion suppresion was explained taking into account the co-elution of phenolic acids, in terms of their acid-base and hydrophilic properties, which affected ionisation of bioflavonoids in gas phase. Extracts of food samples were obtained by different methods of extraction. Purification and preconcentration of extracts, on the solid phase using the Supelco cartridges was performed

Spectrophotometric zinc(II) based determination of quercetin in pharmaceutical formulations

Flavonoids are a group of polyphenolic compounds widely present in the herbal world representing an important part of human diet. Quercetin, which is a flavanol, makes 70% of total daily intake of flavonoids. Because of the characteristic chemical structure, quercetin has the ability of complexing metals and antioxidative ability. Using equimolar solution variation method it was determined that quercetin makes a complex with zinc(II)-ion in acidic environment (pH 5.25), in stoichiometric relation quercetin:zinc(II)-ion = 2:1, with absorption maximum on λmax=363 nm. The ability of quercetin to make complex compounds with zinc(II)-ion was used to develop simple, precise and accurate assay to determine the content of quercetin in various samples of heterogeneous composition. The proposed indirect spectrophotometric method can selectively determine quercetin in concentrations ranging from 0.1 to 6.0 mg/L. LOD and LOQ were derived from the calibration curve and estimated as 0.03 mg/L and 0.1 mg/L respectively. Developed method is reproductive and accurate, as indicated by high value of correlation coefficient R=0.99996 and low value of SD=0.00122. Method was successfully used to determine quercetin content in dietary supplement tablets. Dietary supplements, proscribed for therapeutic and/or profilactic pruposes, usualy content quercetin combined with other flavonoids and ascorbic acid. Therefore, it was necessary to test the selectivity of proposed method. The reliability of the method was checked out by newly developed RP-HPLC/UV method for capsules with direct determination of quercetin after separation. The good agreement between the two methods indicates the applicability of the proposed spectrophotometric method for quercetin determination in dietary supplement tablets with high reproducibility, and enables direct and simple determination without its prior extraction from samples. In addition, the antioxidative ability of quecetin and zinc(II)-quercetin complex was determined using oxidation-reduction standardized methods DPPH and FRAP. The same samples were tested for antimicrobial activity against seven laboratory control strains of bacteria and one yeast. As a result of those tests, there are no obstacles to combine quercetin and zinc in the same formulation

Zinc complex-based determination of rutin in dietary supplements

The aim of this study was to develop and validate a simple, rapid, sensitive and low-cost method for determination of rutin in tablets. The proposed spectrophotometric method is based on the formation of the Zn2+-rutin complex in methanol 70% (v/v) at pH 8.52, and detection at lambda(max)= 410 nm. The concentration range over which the response was linear was 0.3-12.2 mu g ml(-1). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.21 mu g ml(-1) and 0.63 mu g ml(-1), respectively. The proposed method was successfully applied to the determination of rutin in herbal dietary supplements. The reliability of the method was checked by comparison with results obtained by the established RP-HPLC/UV method. The proposed method fulfills all aimed requirements

Spectrofluorimetric and HPLC Determination of Morin in Human Serum

Morin is a flavonol antioxidant. In ethanol-water mixtures (70 wt% of ethanol) it reacts with Al(3+) to give Al(Morin)(2) in the pH range 3-6. The conditional stability constant of this complex at 298 K was found to be log beta(2) = 16.96 +/- 0.02 at pH 4.40. The complex shows strong fluorescence emission at 500 nm upon excitation at 410 nm. The fluorescence intensity is pH dependent with maximum emission at pH 4.40. Since the complexation reaction enhances the fluorescence of morin, this property was used for the determination of morin in human serum. A linear dependence of the intensity of fluorescence of the complex on the concentration of morin was obtained in morin concentration range from 1.5-30.5 ng mL(-1), relative standard error of measurements was 1.4%. The LOD was 0.02 ng mL(-1) while LOQ was 1.0 ng mL(-1). Serum concentration of morin was also determined using HPLC as a reference method. A C-18 Hypersil Gold AQ column was used with acetonitrile-0.1% v/v phosphoric acid (30:70% v/v) as the mobile phase at 1.0 mL min(-1) flow rate and UV detection at 250 nm. Acceptable relative standard errors (less than 5%) between determinations obtained by the two methods indicate that the fluorescence method is reliable

Spectrofluorimetric determination of quercetin in pharmaceutical dosage forms

A simple, accurate and precise method based on the fluorescence properties of the aluminum(III)-quercetin complex, for the determination of quercetin, has been developed and validated. The complex has strong emission at pH 3.30, lambda(em) = 480 nm, with lambda(ex) = 420 nm. The linearity range of quercetin determination was 1.5-60.5 ng ml(-1) with LOD 0.09 ng ml(-1) and LOQ 0.27 ng ml(-1). Recovery values in the range of 99.9-100.2% indicate a good accuracy of the method. The established method was applied for the determination of quercetin in capsules, with a recovery value of 98.3%, standard deviation of 0.22% and coefficient of variation of 0.09%. The reliability of the method was checked by the newly developed RP-HPLC/UV method for capsules with the direct determination of quercetin after separation. The good agreement between the two methods indicates the applicability of the proposed spectrofluorimetric method for quercetin determination in pharmaceutical dosage forms, with high reproducibility, and enables the direct and simple determination without its prior extraction from samples. The proposed spectrofluorimetric method has much better sensitivity and LOD and LOQ values that are about 1000 times lower than data reported in the literature

Allelopathic potential of selected woody species growing on fly-ash deposits

The objective of this study was to determine the allelopathic potential of Robinia pseudoacacia L., Ailanthus altissima (Mill.) Swingle and Amorpha fruticosa L. that grow on the fly-ash deposits at the “Nikola Tesla – A” thermoelectric power plant in Obrenovac. The chemical characteristics of fly ash, such as pH, electrical conductivity (EC), content of carbon (C) and nitrogen (N), contents of available phosphorus (P2O5) and potassium (K2O), the contents of total and available Fe, Cu, Mn, Ni and Zn as well as of phenolic acids (3,5 dihydroxybenzoic acid (3,5-DHBA) and ferulic acid) and flavonoids (rutin and quercetin) were analyzed in control fly ash (bare zones without vegetation cover) and plant rhizospheric fly ash. In order to determine the allelopathic activity of phenolic compounds in fly ash, modified soil sandwich allelopathic biotests were performed, and Trifolium pratense L. (red clover) was used as the indicator species. A. fruticosa showed the highest allelopathic activity, followed by A. altissima whereas R. pseudoacacia showed the lowest allelopathic potential. Negative correlation was noted between radicle and hypocotyl growth inhibition of red clover and the pH of fly ash. Positive correlations were found between radicle growth inhibition and the content of C, P2O5, total concentrations of Cu, available concentrations of Mn and Ni, the contents of ferulic acid, 3,5-DHBA, and rutin. Our results indicate that A. fruticosa and A. altissima increased the content of phenolics in fly ash, which can act as allelochemicals leading to radicle growth inhibition of red clover in the pioneer plant community on fly-ash deposits. These woody species that colonized fly-ash deposits can initiate the beginning of pedogenetic processes altering the ecosystem processes at degraded site

Green One-Pot Synthesis of Coumarin-Hydroxybenzohydrazide Hybrids and Their Antioxidant Potency

Compounds from the plant world that possess antioxidant abilities are of special importance for the food and pharmaceutical industry. Coumarins are a large, widely distributed group of natural compounds, usually found in plants, often with good antioxidant capacity. The coumarin-hydroxybenzohydrazide derivatives were synthesized using a green, one-pot protocol. This procedure includes the use of an environmentally benign mixture (vinegar and ethanol) as a catalyst and solvent, as well as very easy isolation of the desired products. The obtained compounds were structurally characterized by IR and NMR spectroscopy. The purity of all compounds was determined by HPLC and by elemental microanalysis. In addition, these compounds were evaluated for their in vitro antioxidant activity. Mechanisms of antioxidative activity were theoretically investigated by the density functional theory approach and the calculated values of various thermodynamic parameters, such as bond dissociation enthalpy, proton affinity, frontier molecular orbitals, and ionization potential. In silico calculations indicated that hydrogen atom transfer and sequential proton loss–electron transfer reaction mechanisms are probable, in non-polar and polar solvents respectively. Additionally, it was found that the single- electron transfer followed by proton transfer was not an operative mechanism in either solvent. The conducted tests indicate the excellent antioxidant activity, as well as the low potential toxicity, of the investigated compounds, which makes them good candidates for potential use in food chemistry

Optimizacija i validacija tečno-hromatografske metode za određivanje flavanona i njihovih derivata u farmaceutskim formulacijama i hrani

U ovom radu razvijene su LC-MS/MS i HPLC-UV/DAD metode za istovremeno određivanje bioflavonoida: rutina, kvercetina, hesperidina, hesperetina,kaempferola, apigenina, luteolina, katehina i epikatehina u nekim uzorcima hrane (kora pomorandže, crveni luk, med, karfiol, brokoli i prokelj). Takođe, razvijene su i HPLC-DAD i spektrofotometrijske metode za određivanje moksifloksacina (analoga flavonoida) i njegovih srodnih jedinjenja i degradacionih proizvoda u farmaceutskim formulacijama i humanoj krvnoj plazmi. Procenjen je matrični efekat prilikom određivanja rutina, kvercetina, hesperetina, hesperidina i kaempferola u uzorcima kore pomorandže, crvenog luka i meda. Uticaj matriksa procenjen je korišćenjem metode post-ekstrakcije pri čemu se dobijene negativne vrednosti u opsegu od -44 do -0,5 %, što ukazuje na jonsku supresiju. Matrični efekat jako zavisi od koncentracije ispitivanih bioflavonoida. Jonska supresija se može objasniti prisustvom koeluiranih fenolnih kiselina, koje zbog svojih kiselinsko-baznih i hidrofilnih karakteristika utiču na jonizaciju bioflavonoida u gasnoj plazmi. Ekstrakti uzoraka hrane su dobijeni primenom različitih načina ekstrakcije (ekstrakcija po Soksletu, ultrazvučna ektrakcija, ekstrakcija maceracijom i ekstrakcija ključalom vodom). Za prečišćavanje i prekoncentrisavanje dobijenih ekstrakata analita korišćena je ekstrakcija na čvrstoj fazi primenom Supelco kertridža.In the present work the LC-MS/MS and HPLC-UV/DAD methods with solid phase extraction for simultaneous determination of bioflavonoids rutin, quercetin, hesperidin,hesperetin, kaempferol, apigenin, luteolin, catehin and epicatehin in some food samples (red onion, orange peel, honey, cauliflower, broccoli and Brussel sprouts) were developed. Also, HPLC-UV/DAD and spectrophotometric methods for determination moxifloxacin (flavonoids analog) and their related substances and degradation products in pharmaceutical forms and human plasma were developed. The matrix effect accompanying determination rutin, quercetin, hesperidin, hesperetin and kaempferol in orange peel, red onion and honey was quantified. The matrix effect evaluated using postextraction addition method was found to be negative in the range -44-0,5 %, indicating ionization suppression and strongly depended on bioflavonoid concentration. Ion suppresion was explained taking into account the co-elution of phenolic acids, in terms of their acid-base and hydrophilic properties, which affected ionisation of bioflavonoids in gas phase. Extracts of food samples were obtained by different methods of extraction. Purification and preconcentration of extracts, on the solid phase using the Supelco cartridges was performed

Seed Priming Improves Biochemical and Physiological Performance of Wheat Seedlings under Low-Temperature Conditions

Wheat is a widely cultivated cereal throughout the world and stress caused by low temperatures significantly affects all stages of wheat development. Seed priming is an effective method to produce stress-resistant plants. This work was carried out to determine whether different priming methods (hormo-, halo-, osmo-, and hydropriming) can increase the resistance of wheat to low-temperature conditions (10 °C). The effect of priming on growth, as well as the biochemical and physiological performance of wheat seedlings were monitored. In general, priming had a significant stimulatory effect on the monitored characteristics. Hormo- and halopriming had a positive effect on the growth, vigor index, and total soluble protein content of wheat seedlings. Additionally, hormopriming reduced the malondialdehyde (MDA) content in wheat seedlings compared to unprimed seeds. A dominant effect on antioxidant enzymes (superoxide-dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and pyrogallol peroxidase) was recorded after seed priming with KNO3. The effectiveness of priming was also confirmed through the increased content of phenolic compounds (including flavonoids), and total antioxidant activity. The HPLC analysis showed increased content of chlorogenic acid, catechin, 4-hydroxy benzoic acid, sinapic acid, rutin, naringin, and quercetin in primed wheat seedlings compared to unprimed grown seedlings under low-temperature conditions with the best effects achieved by hormo- and hydropriming. It is concluded that seed priming can be regarded as a promising approach for increasing the resistance of wheat seedlings to low-temperature stress

Chemometric-Assisted Optimization of RP-HPLC Method for Determination of Some Bioflavonoids in Brassica oleracea Species and Their Antioxidative Activity

In the present work, the rapid RP-HPLC method with UV (DAD) detection for simultaneous quantification of bioflavonoids: quercetin, apigenin, catechin, epicatechin, kaempferol, and luteolin in Brassica oleracea species samples (cauliflower, broccoli, and Brussels sprouts) was developed with the aid of LC-Simulator (ACD LabsA (R) suite) software. A series of extracts obtained with different extraction method were evaluated for antioxidant activity. The optimal conditions for separation and quantification were established after nine scouting runs entered to LC-Simulator software. The optimized separation was achieved on Hypersil GOLD aQ column with isocratic elution and mobile phase composition A:2 % acetic acid in water and B:acetonitrile in 91:9 (v/v %) ratio. The R (s) values were in the range from 2.6 to 8.00, indicating good selectivity of the method. The obtained results generally show good agreement with published data. Low detection limits (0.02-0.055 mu g/mL) were obtained with acceptable recoveries (90-109 %). Total time of analysis was less than 11 min; therefore, the proposed method represents significant improvement over existing methods. Extracts from Brassica vegetables, obtained using different extraction procedures, were studied for their radical scavenging effects. Scavenging of DPPH showed different kinetics at the beginning of the assay period and after 15 min from the initialization of reaction. Different kinetics suggested the presence of polymerized and/or less active antioxidants with different scavenging mechanisms for particular polyphenolic compounds