Search results for: HPLC method

Authors: Mohamed Hefnawy, Abdulrahman Al-Majed, Aymen Al-Suwailem

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Enantioseparation of drugs with multiple stereogenic centers is challenging. This study objectives to evaluate the efficiency of different mobilized and/or immobilized polysaccharide-based chiral stationary phases to separate enantiomers of some drugs containing multiple stereogenic centers namely indenolol, nadolol, labetalol. The critical mobile phase variables (composition of organic solvents, acid/base ratios) were carefully studied to compare the retention time and elution order of all isomers. Different chromatographic parameters such as capacity factor (k), selectivity (α) and resolution (Rs) were calculated. Experimental conditions and the possible chiral recognition mechanisms have been discussed.

Keywords: HPLC, polysaccharide columns, enantio-resolution, indenolol, nadolol, labetalol

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Authors: Kira Rysakova, Vitaly Novikov

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An improved method of obtaining enzymatic collagen hydrolysate from the tissues of marine hydrobionts is proposed, which allows to obtain hydrolysate without pre-isolation of pure collagen. The method can be used to isolate enzymatic collagen hydrolysate from the waste of industrial processing of Red King crab and non-traditional objects - marine holothurias. Comparative analysis of collagen hydrolysates has shown the possibility of their use in a number of nutrient media, but this requires additional optimization of their composition and biological tests on wide sets of test strains of microorganisms.

Keywords: collagen hydrolysate, marine hydrobionts, red king crab, marine holothurias, enzymes, exclusive HPLC

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Authors: Khaled Khaladi, Rafika Bibi, Hind Mokrane, Boubekeur Nadjemi

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Sorghum (Sorghum bicolor (L.) Moench) is an important cereal crop grown in the semi-arid tropics of Africa and Asia due to its drought tolerance. Sorghum grain has protein content varying from 6 to 18%, with an average of 11%, Sorghum proteins can be broadly classified into prolamin and non-prolamin proteins. Kafirins, the major storage proteins, are classified as prolamins, and as such, they contain high levels of proline and glutamine and are soluble in non-polar solvents such as aqueous alcohols. Kafirins account for 77 to 82% of the protein in the endosperm, whereas non-prolamin proteins (namely, albumins, globulins, and glutelins) make up about 30% of the proteins. To optimize the extraction of sorghum proteins, several variables were examined: detergent type and concentration, reducing agent type and concentration, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC.

Keywords: sorghum, protein extraction, detergent, food science

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Authors: Chandrima Das, Sirshendu Chatterjee

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Tea (Camellia sinensis) is known as nature's low calorie wonder drink. Since ancient times hot consumptions of tea is very much popular. We have observed that many heat sensitive secondary metabolites which get destroyed on heating, moreover by people, who are permanently live at higher altitude or the members of high altitude expedition team, are deprived of various tea brewing facilities like electricity, fuel, etc. and the hence cold decoction of tea might be a good alternative. In this backdrop present study aims at the analysis of antioxidants like polyphenols, flavonoids and free radical scavenging activity as well as the l-theanine concentration of different types of cold brewed teas like black, green, white and oolong and compared with its hot decoction. Further, we also analysed in details about the bioactive components by using HPLC followed by green synthesis of nanoparticles. The study highlighted that the difference between the concentration of antioxidant in cold and hot brewed tea is insignificant and hence intake of cold decoction will be beneficial to health.

Keywords: antioxidants, flavanoid, polyphenols, HPLC, nanoparticles

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Authors: Renata Adriana Labanca, Gabriela Rezende Costa, Nilton de Oliveira Couto e Silva, José Marcos Gontijo Mandarino, Rodrigo Santos Leite, Nilson César Castanheira Guimarães, Roberto Gonçalves Junqueira

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The objective of this study was to evaluate the differences in composition between six brands of conventional soybean and six genetically modified cultivars (GM), all of them from Minas Gerais State, Brazil. We focused on the isoflavones profile and mineral content questioning the substantial equivalence between conventional and GM organisms. The statement of compliance label for conventional grains was verified for the presence of genetic modified genes by real time polymerase chain reaction (PCR). We did not detect the presence of the 35S promoter in commercial samples, indicating the absence of transgene insertion. For mineral analysis, we used the method of inductively coupled plasma-optical emission spectrometry (ICP-OES). Isoflavones quantification was performed by high performance liquid chromatography (HPLC). The results showed no statistical difference between the conventional and transgenic soybean groups concerning isoflavone content and mineral composition. The concentration of potassium, the main mineral component of soy, was the highest in conventional soybeans compared to that in GM soy, while GM samples presented the highest concentrations of iron.

Keywords: glycine max, genetically modified organism, bioactive compounds, ICP-OES, HPLC

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Authors: Lila Boulekbache-Makhlouf, Kahina Djaoud, Myriam Tazarourte, Samir Hadjal, Khodir Madani

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In Algeria, important quantities of secondary date variety (Phoenix dactylifera L.) are generated in each campaign; their chemical composition is similar to that of commercial dates. The present work aims to valorize this common date variety (Degla-Beida) which is often poorly exploited. In this context, we tried to prepare syrup from the secondary date variety and to evaluate the effect of conventional extraction (CE) or water bath extraction (WBE) and alternative extraction (microwaves assisted extraction (MAE), and ultrasounds assisted extraction (UAE)) on its total sugar content (TSC), using response surface methodology (RSM). Then, the analysis of individual sugars was performed by high-performance liquid chromatography (HPLC). Maximum predicted TSC recoveries under the optimized conditions for MAE, UAE and CE were 233.248 ± 3.594 g/l, 202.889 ± 5.797 g/l, and 233.535 ± 5.412 g/l, respectively, which were close to the experimental values: 233.796 ± 1.898 g/l; 202.037 ± 3.401 g/l and 234.380 ± 2.425 g/l. HPLC analysis revealed high similarity in the sugar composition of date juices obtained by MAE (60.11% sucrose, 16.64% glucose and 23.25% fructose) and CE (50.78% sucrose, 20.67% glucose and 28.55% fructose), although a large difference was detected for that obtained by UAE (0.00% sucrose, 46.94% glucose and 53.06% fructose). Microwave-assisted extraction was the best method for the preparation of date syrup with an optimal recovery of total sugar content. However, ultrasound-assisted extraction was the best one for the preparation of date syrup with high content of reducing sugars.

Keywords: dates, extraction, RSM, sugars, syrup

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Authors: Zafar Iqbal, Sana Irshad Khan

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Organic extract from newly isolated plant growth promoting fungus (PGPF) Penicillium sp EU0013 was subjected to bioassays including anti fungal (disc diffusion) cytotoxicity (brine shrimp lethality), herbicidal (Lemna minor) and nematicidal activities. Metabolite profile of the extract was also assessed using HPLC analysis with the aim to identify bioactive natural products in the extract as new drug candidate(s). The extract showed anti fungal potential against tested fungal pathogens. Growth of the Wilt pathogen Fusarium oxyosproum was inhibited up to 63% when compared to negative reference. Activity against brine shrimps was weak and mortality up to 10% was observed at concentration of 200 µg. mL-1. The extract exhibited no toxicity against Lemna minor frond at 200 µg. mL-1. Nematicidal activity was observed very potent against root knot nematode and LC50 value was calculated as 52.5 ug. mL-1 using probit analysis. Methodically assessment of metabolites profile by HPLC showed the presence of kojic acid (Rt 1.4 min) and aflatoxin B1 (Rt 5.9 min) in the mycellial extract as compared with standards. The major unidentified metabolite was eluted at Rt 8.6 along with other minor peaks. The observed high toxicity against root knot nematode was attributed to the unidentified compounds that make fungal extract worthy of further exploration for isolation and structural characterization studies for development of future commercial nematicidal compound(s).

Keywords: penicillium, nematicidal activity, metabolites, HPLC

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Authors: Carol Yue-En Ong, Angelina Hui-Min Tan, Quan Liu, Paul Chi-Lui Ho

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A public general hospital in Singapore has recently implemented an automated unit-dose machine in their inpatient dispensary, Swisslog, with the objective of reducing human error and improving patient safety. However, a concern in stability arises as tablets are removed from their original packaging (bottled loose tablets/capsules) and are repackaged into individual, clear plastic wrappers as unit doses in the system. Drugs that are light-sensitive and hygroscopic would be more susceptible to degradation as the wrapper does not offer full protection. Hence, this study was carried out to study the stability of haloperidol tablets and phenytoin capsules that are light-sensitive and hygroscopic respectively. Validated HPLC-UV assays were first established for quantification of these two compounds. The medications involved were put in the Swisslog and sampled every week for one month. The collected data was analysed and showed no degradation over time. This study also explored an alternative approach for drug stability determination-Raman spectroscopy. The advantage of Raman spectroscopy is its high time efficiency and non-destructive nature. The results suggest that drug degradation can indeed be detected using Raman microscopy, but further research is needed to establish this approach for quantification or qualification of compounds. NanoRam®, a portable Raman spectrocope was also used alongside Raman microscopy but was unsuccessful in detecting degradation in this study.

Keywords: drug stability, haloperidol, HPLC, phenytoin, raman spectroscopy, Swisslog

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Authors: Fatih Özogul, Sezen Özçeli̇k, Yesim Özogul

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Lactic, acetic, succinic, propionic, formic and butyric acid production by lactic acid bacteria (LAB) were monitored in M17 broth (the control) and some fish (squid, shrimp, octopus, and eel) infusion broth by using HPLC method. There were significant differences in terms of lactic, acetic, succinic, propionic, formic and butyric acid production (p < 0.005) among bacterial strains. Acetic acid production was the lowest by LAB while succinic acid followed by propionic acid was synthesized at the highest levels. Lactic acid production ranged from 0 to 938 mg/L by all LAB strains in different infusion broth. The highest acetic acid production was found by Lb. acidophilus and Lb. delbrueckii subsp. lactic in octopus and shrimp infusion broth, with values of 872 and 674 mg/L, respectively while formic acid formation ranged from 1747 mg/L by Lb. acidophilus in octopus infusion broth to 69 mg/L by Lb. delbrueckii subsp. lactis in shrimp infusion broth. Propionic acid and butyric acid productions by St. thermophilus were 9852 and 3999 mg/L in shrimp infusion broth while Leu. mes. subsp. cremoris synthesized 312 and 9 mg/L of those organic acid in European squid infusion broth, respectively. Apparently, LAB strains had a great capability to generate succinic acid followed by propionic and butyric acid. In addition, other organic acid production differed significantly depending on bacterial strains and growth medium.

Keywords: Lactic acid bacteria , organic acid, HPLC analysis, growth medium

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Authors: Hee-Jae Suh, Mi-Na Hong, Min-Ji Kim, Yeon-Seong Jeong, Ok-Hwan Lee, Jae-Wook Shin, Hyang-Sook Chun, Chan Lee

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Brown HT is a bis-azo dye which is permitted in EU as a food colorant. So far, many studies have focused on HPLC using diode array detection (DAD) analysis for detection of this food colorant with different columns and mobile phases. Even though these methods make it possible to detect Brown HT, low recovery, reproducibility, and linearity are still the major limitations for the application in foods. The purpose of this study was to compare various methods for the analysis of Brown HT and to develop an improved analytical methods including pretreatment. Among tested analysis methods, best resolution of Brown HT was observed when the following solvent was applied as a eluent; solvent A of mobile phase was 0.575g NH4H2PO4, and 0.7g Na2HPO4 in 500mL water added with 500mL methanol. The pH was adjusted using phosphoric acid to pH 6.9 and solvent B was methanol. Major peak for Brown HT appeared at the end of separation, 13.4min after injection. This method exhibited relatively high recovery and reproducibility compared with other methods. LOD (0.284 ppm), LOQ (0.861 ppm), resolution (6.143), and selectivity (1.3) of this method were better than those of ammonium acetate solution method which was most frequently used. Precision and accuracy were verified through inter-day test and intra-day test. Various methods for sample pretreatments were developed for different foods and relatively high recovery over 80% was observed in all case. This method exhibited high resolution and reproducibility of Brown HT compared with other previously reported official methods from FSA and, EU regulation.

Keywords: analytic method, Brown HT, food colorants, pretreatment method

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Authors: Semuel Unwakoly, Reinner Puppela, Maresthy Rumalean, Healthy Kainama

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Fish is a kind of food that contains many nutritions, one of those is the long chain of unsaturated fatty acids as omega-3 and omega-6 fatty acids and essential amino acid in enough amount for the necessity of our body. Like pelagic fish that found in the sea of Maluku. This research was done to identify fatty acids and amino acids composition in Moonfish (M. maculata) using transesterification reaction steps and Gas Chromatograph-Mass Spectrophotometer (GC-MS) and High-Performance Liquid Chromatography (HPLC). The result showed that fatty acids composition in Moonfish (M. maculata) contained tridecanoic acid (2.84%); palmitoleic acid (2.65%); palmitic acid (35.24%); oleic acid (6.2%); stearic acid (14.20%); and 5,8,11,14-eicosatetraenoic acid (1.29%) and 12 amino acids composition that consist of 7 essential amino acids, were leucine, isoleucine, valine, phenylalanine, methionine, lysine, and histidine, and also 5 non-essential amino acid, were tyrosine, glycine, alanine, glutamic acid, and arginine.Thus, these fishes can be used by the people to complete the necessity of essential fatty acid and amino acid.

Keywords: Moonfish (M. maculata), fatty acid, amino acid, GC-MS, HPLC

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Authors: Thi Huong Vu, Vijay Jayasena, Zhongxiang Fang, Gary Dykes

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D-pinitol (3-O-methyl ether of D-chiro-inosito) has been known to have health benefits for diabetic patients. Carob kibble has received attention due to the presence of high value D-pinitol and polyphenol antioxidants. D-pinitol was concentrated from carob kibble using submerged fermentation with Saccharomyces cerevisiae. Total carbohydrates and D-pinitol were determined by the phenol-sulphuric acid method and HPLC, respectively. The content of D-pinitol increased from approximately 43 to 70 mg/g dry weight after fermentation. The yeast consumed over 70% of total carbohydrates in carob kibble without any negative effect on D-pinitol content. A range of substrate medium pH’s from 5.0 – 7.0 had no significant effect on the removal of carbohydrates and D-pinitol. This method may provide a practical solution for production of D-pinitol from carob in a cost effective manner.

Keywords: carob kibble, d-pinitol, saccharomyces cerevisiae, submerged fermentation, total carbohydrates

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Authors: I. Berger, N. Machour, F. Portet-Koltalo

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Polycyclic aromatic hydrocarbons (PAHs) are toxic and carcinogenic pollutants produced in majority by incomplete combustion processes in industrialized and urbanized areas. After being emitted in atmosphere, these persistent contaminants are deposited to soils or sediments. Even if persistent, some can be partially degraded (photodegradation, biodegradation, chemical oxidation) and they lead to oxygenated metabolites (oxy-PAHs) which can be more toxic than their parent PAH. Oxy-PAHs are less measured than PAHs in sediments and this study aims to compare different analytical tools in order to extract and quantify a mixture of four hydroxylated PAHs (OH-PAHs) and four carbonyl PAHs (quinones) in sediments. Methodologies: Two analytical systems – HPLC with on-line UV and fluorescence detectors (HPLC-UV-FLD) and GC coupled to a mass spectrometer (GC-MS) – were compared to separate and quantify oxy-PAHs. Microwave assisted extraction (MAE) was optimized to extract oxy-PAHs from sediments. Results: First OH-PAHs and quinones were analyzed in HPLC with on-line UV and fluorimetric detectors. OH-PAHs were detected with the sensitive FLD, but the non-fluorescent quinones were detected with UV. The limits of detection (LOD)s obtained were in the range (2-3)×10-4 mg/L for OH-PAHs and (2-3)×10-3 mg/L for quinones. Second, even if GC-MS is not well adapted to the analysis of the thermodegradable OH-PAHs and quinones without any derivatization step, it was used because of the advantages of the detector in terms of identification and of GC in terms of efficiency. Without derivatization, only two of the four quinones were detected in the range 1-10 mg/L (LODs=0.3-1.2 mg/L) and LODs were neither very satisfying for the four OH-PAHs (0.18-0.6 mg/L). So two derivatization processes were optimized, comparing to literature: one for silylation of OH-PAHs, one for acetylation of quinones. Silylation using BSTFA/TCMS 99/1 was enhanced using a mixture of catalyst solvents (pyridine/ethyle acetate) and finding the appropriate reaction duration (5-60 minutes). Acetylation was optimized at different steps of the process, including the initial volume of compounds to derivatize, the added amounts of Zn (0.1-0.25 g), the nature of the derivatization product (acetic anhydride, heptafluorobutyric acid…) and the liquid/liquid extraction at the end of the process. After derivatization, LODs were decreased by a factor 3 for OH-PAHs and by a factor 4 for quinones, all the quinones being now detected. Thereafter, quinones and OH-PAHs were extracted from spiked sediments using microwave assisted extraction (MAE) followed by GC-MS analysis. Several mixtures of solvents of different volumes (10-25 mL) and using different extraction temperatures (80-120°C) were tested to obtain the best recovery yields. Satisfactory recoveries could be obtained for quinones (70-96%) and for OH-PAHs (70-104%). Temperature was a critical factor which had to be controlled to avoid oxy-PAHs degradation during the MAE extraction process. Conclusion: Even if MAE-GC-MS was satisfactory to analyze these oxy-PAHs, MAE optimization has to be carried on to obtain a most appropriate extraction solvent mixture, allowing a direct injection in the HPLC-UV-FLD system, which is more sensitive than GC-MS and does not necessitate a previous long derivatization step.

Keywords: derivatizations for GC-MS, microwave assisted extraction, on-line HPLC-UV-FLD, oxygenated PAHs, polluted sediments

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Authors: Fangyan Li, Lin Min Lee, Hui Zhu Peh, Shoet Harn Chan

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Advantame, a derivative of aspartame, is the latest addition to a family of low caloric and high potent dipeptide sweeteners which include aspartame, neotame and alitame. The use of advantame as a high-intensity sweetener in food was first accepted by Food Standards Australia New Zealand in 2011 and subsequently by US and EU food authorities in 2014, with the results from toxicity and exposure studies showing advantame poses no safety concern to the public at regulated levels. To our knowledge, currently there is barely any detailed information on the analytical method of advantame in food matrix, except for one report published in Japanese, stating a high performance liquid chromatography (HPLC) and liquid chromatography/ mass spectrometry (LC-MS) method with a detection limit at ppm level. However, the use of acid in sample preparation and instrumental analysis in the report raised doubt over the reliability of the method, as there is indication that stability of advantame is compromised under acidic conditions. Besides, the method may not be suitable for analyzing food matrices containing advantame at low ppm or sub-ppm level. In this presentation, a simple, specific and sensitive method for the determination of advantame in food is described. The method involved extraction with water and clean-up via solid phase extraction (SPE) followed by detection using liquid chromatography tandem mass spectrometry (LC-MS/MS) in negative electrospray ionization mode. No acid was used in the entire procedure. Single laboratory validation of the method was performed in terms of linearity, precision and accuracy. A low detection limit at ppb level was achieved. Satisfactory recoveries were obtained using spiked samples at three different concentration levels. This validated method could be used in the routine inspection of the advantame level in food.

Keywords: advantame, food, LC-MS/MS, sweetener

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Authors: Ruolan Zhang, Ryo Imai, Naoko Senda, Tomoyuki Sakai

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Enzymes have attracted increasing attentions in industrial manufacturing for their applicability in catalyzing complex chemical reactions under mild conditions. Directed evolution has become a powerful approach to optimize enzymes and exploit their full potentials under the circumstance of insufficient structure-function knowledge. With the incorporation of cell-free synthetic biotechnology, rapid enzyme synthesis can be realized because no cloning procedure such as transfection is needed. Its open environment also enables direct enzyme measurement. These properties of cell-free biotechnology lead to excellent throughput of enzymes generation. However, the capabilities of current screening methods have limitations. Fluorescence-based assay needs applicable fluorescent label, and the reliability of acquired enzymatic activity is influenced by fluorescent label’s binding affinity and photostability. To acquire the natural activity of an enzyme, another method is to combine pre-screening step and high-performance liquid chromatography (HPLC) measurement. But its throughput is limited by necessary time investment. Hundreds of variants are selected from libraries, and their enzymatic activities are then identified one by one by HPLC. The turn-around-time is 30 minutes for one sample by HPLC, which limits the acquirable enzyme improvement within reasonable time. To achieve the real high-throughput enzyme screening, i.e., obtain reliable enzyme improvement within reasonable time, a widely applicable high-throughput measurement of enzymatic reactions is highly demanded. Here, a high-throughput screening method using broadband coherent anti-Stokes Raman spectroscopy (CARS) was proposed. CARS is one of coherent Raman spectroscopy, which can identify label-free chemical components specifically from their inherent molecular vibration. These characteristic vibrational signals are generated from different vibrational modes of chemical bonds. With the broadband CARS, chemicals in one sample can be identified from their signals in one broadband CARS spectrum. Moreover, it can magnify the signal levels to several orders of magnitude greater than spontaneous Raman systems, and therefore has the potential to evaluate chemical's concentration rapidly. As a demonstration of screening with CARS, alcohol dehydrogenase, which converts ethanol and nicotinamide adenine dinucleotide oxidized form (NAD+) to acetaldehyde and nicotinamide adenine dinucleotide reduced form (NADH), was used. The signal of NADH at 1660 cm⁻¹, which is generated from nicotinamide in NADH, was utilized to measure the concentration of it. The evaluation time for CARS signal of NADH was determined to be as short as 0.33 seconds while having a system sensitivity of 2.5 mM. The time course of alcohol dehydrogenase reaction was successfully measured from increasing signal intensity of NADH. This measurement result of CARS was consistent with the result of a conventional method, UV-Vis. CARS is expected to have application in high-throughput enzyme screening and realize more reliable enzyme improvement within reasonable time.

Keywords: Coherent Anti-Stokes Raman Spectroscopy, CARS, directed evolution, enzyme screening, Raman spectroscopy

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Authors: E. M. Condori-Peñaloza, S. S. Costa

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Eleusine indica L, known as goose-grass, is considered a troublesome weed that can cause important economic losses in the agriculture worldwide. However, this grass is used as a medicinal plant in some regions of Brazil to treat influenza and pneumonia. In Africa and Asia, it is used to treat malaria and as diuretic, anti-helminthic, among other uses. Despite its therapeutic potential, little is known about the chemical composition and bioactive compounds of E. indica. Hitherto, two major flavonoids, schaftoside and vitexin, were isolated from aerial part of the species and showed inhibitory activity on lung neutrophil influxes in mice, suggesting a beneficial effect on airway inflammation. Therefore, the aim of this study was to analyze the chemical profile of aqueous extracts from aerial parts of Eleusine indica specimens using high performance liquid chromatography (HPLC-DAD) and 1H nuclear magnetic resonance spectroscopy (NMR), with emphasis on phenolic compounds. Specimens of E. indica were collected in Minas Gerais state, Brazil. Aerial parts of fresh plants were extracted by decoction (10% p/v). After spontaneous precipitation of the aqueous extract at 10-12°C for 24 hours, the supernatant obtained was frozen and lyophilized. After that, 1 g of the extract was dissolved into 25 mL of water and fractionated on a reverse phase chromatography column (RP-2), eluted with a gradient of H2O/EtOH. Five fractions were obtained. The extract and fractions had their chemical profile analyzed by using HPLC-DAD (C-18 column: 20 μL, 256 -365 nm; gradient water 0.01% phosphoric acid/ acetonitrile. The extract was also analyzed by NMR (1H, 500 MHz, D2O) in order to access its global chemical composition. HPLC-DAD analyses of crude extract allowed the identification of ten phenolic compounds. Fraction 1, eluted with 100% water, was poor in phenolic compounds and no major peak was detected. In fraction 2, eluted with 100% water, it was possible to observe one major peak at retention time (RT) of 23.75 minutes compatible with flavonoid; fraction 3, also eluted with 100% water, showed four peaks at RT= 21.47, 23.52, 24.33 and 25.84 minutes, all of them compatible with flavonoid. In fraction 4, eluted with 50%/ethanol/50% water, it was possible to observe 3 peaks compatible with flavonoids at RT=24.65, 26.81, 27.49 minutes, and one peak (28.83 min) compatible with a phenolic acid derivative. Finally, in fraction 5, eluted with 100% ethanol, no phenolic substance was detected. The UV spectra of all flavonoids detected were compatible with the flavone subclass (λ= 320-345 nm). The 1H NMR spectra of aerial parts extract showed signals in three regions: δ 0.8-3.0 ppm (aliphatic compounds), δ 3.0-5.5 ppm corresponding to carbohydrates (signals most abundant and overlapped), and δ 6.0-8.5 ppm (aromatic compounds). Signals compatible with flavonoids (rings A and B) could also be detected in the crude extract spectra. These results suggest the presence of several flavonoids in E. indica, which reinforces their therapeutic potential. The pharmacological activities of Eleusine indica extracts and fractions will be further evaluated.

Keywords: flavonoids, HPLC, NMR, phenolic compounds

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Authors: Emad A. Shalaby

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Cancer is a disease that causes abnormal cell proliferation and invades nearby tissues. Lung cancer is the second most frequent cancer worldwide. Natural anti-cancer drugs have been developed with low side effects and toxicity. Citrus peels and extracts have been demonstrated to have significant pharmacological and physiological effects as a result of the high concentration of phenolic compounds found in citrus fruits, particularly peels. Tangerine peels can serve as an effective source of bioactive substances such as phenolics, flavonoids, and catechins, which have antioxidant, antibacterial, anticancer, and anti-inflammatory properties. Consequently, this work aims to determine the anticancer activity of ethanol extract of Tangerine peels against the A549 cell line and identify the phenolic compound profile (19 compounds) by using HPLC. Anticancer and antioxidant potentials of the extract were evaluated by MTT assay and TLC- TLC-bioautography sprayed with DPPH reagent, respectively. The obtained results revealed that tangerine peel extract showed significant activity against the A549 cell line with IC50 of 97.66 μg/mL. HPLC analysis proved that the highest concentration is naringenin 464.05 mg/g. More studies indicate that naringenin has significant anticancer potential on A549 cancer cells. The results showed that naringenin binds t0 EGFR protein in A549 with high binding affinity and thus may reduce lung cancer cell migration and enhance the apoptosis of cancer cells. From the obtained results it could be concluded that tangerine peel extract is an effective anti-cancer agent that may potentially serve as a natural therapeutic option for lung cancer treatment.

Keywords: tangerine peel, A549 cell line, anticancer, naringenin, HPLC analysis, naringenin, TLC bioautography

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Authors: Saeeda Nadir Ali, Najma Sultana, Muhammad Saeed Arayne, Amtul Qayoom

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Present study describes a simple and a fast liquid chromatographic method using ultraviolet detector for simultaneous determination of anxiety relief medicine alprazolam with ACE inhibitors i.e; lisinopril, captopril and enalapril employing purospher star C18 (25 cm, 0.46 cm, 5 µm). Separation was achieved within 5 min at ambient temperature via methanol: water (8:2 v/v) with pH adjusted to 2.9, monitoring the detector response at 220 nm. Optimum parameters were set up as per ICH (2006) guidelines. Calibration range was found out to be 0.312-10 µg mL-1 for alprazolam and 0.625-20 µg mL-1 for all the ACE inhibitors with correlation coefficients > 0.998 and detection limits 85, 37, 68 and 32 ng mL-1 for lisinopril, captopril, enalapril and alprazolam respectively. Intra-day, inter-day precision and accuracy of the assay were in acceptable range of 0.05-1.62% RSD and 98.85-100.76% recovery. Method was determined to be robust and effectively useful for the estimation of studied drugs in dosage formulations and human serum without obstruction of excipients or serum components.

Keywords: alprazolam, ACE inhibitors, RP HPLC, serum

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Authors: Supakorn Tantisriyanurak, Hussaya Maneesuwan, Thanyalak Chaisuwan, Sujitra Wongkasemjit

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TUD-1 is a siliceous mesoporous material with a three-dimensional amorphous structure of random, interconnecting pores, large pore size, high surface area (400-1000 m2/g), hydrothermal stability, and tunable porosity. However, the significant disadvantage of the mesoporous silicates is few catalytic active sites. In this work, a series of bimetallic Fe and Ti incorporated into TUD-1 framework is successfully synthesized by sol–gel method. The synthesized Fe,Ti-TUD-1 is characterized by various techniques. To study the catalytic activity of Fe, Ti–TUD-1, phenol hydroxylation was selected as a model reaction. The amounts of residual phenol and oxidation products were determined by high performance liquid chromatography coupled with UV-detector (HPLC-UV).

Keywords: iron, phenol hydroxylation, titanium, TUD-1

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Authors: Poonam Malik, Ravi Bhushan

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This work describes a very efficient approach for synthesis of activated ester of (S)-naproxen which was characterized by UV, IR, ¹HNMR, elemental analysis and polarimetric studies. It was used as a C-N bond forming chiral derivatizing reagent for further synthesis of diastereomeric amides of (RS)-salbutamol (a β₂ agonist that belongs to the group β-adrenolytic and is marketed as racamate) under microwave irradiation. The diastereomeric pair was separated by achiral phase HPLC, using mobile phase in gradient mode containing methanol and aqueous triethylaminephosphate (TEAP); separation conditions were optimized with respect to pH, flow rate, and buffer concentration and the method of separation was validated as per International Council for Harmonisation (ICH) guidelines. The reagent proved to be very effective for on-line sensitive detection of the diastereomers with very low limit of detection (LOD) values of 0.69 and 0.57 ng mL⁻¹ for diastereomeric derivatives of (S)- and (R)-salbutamol, respectively. The retention times were greatly reduced (2.7 min) with less consumption of organic solvents and large (α) as compared to literature reports. Besides, the diastereomeric derivatives were separated and isolated by preparative HPLC; these were characterized and were used as standard reference samples for recording ¹HNMR and IR spectra for determining absolute configuration and elution order; it ensured the success of diastereomeric synthesis and established the reliability of enantioseparation and eliminated the requirement of pure enantiomer of the analyte which is generally not available. The newly developed reagent can suitably be applied to several other amino group containing compounds either from organic syntheses or pharmaceutical industries because the presence of (S)-Npx as a strong chromophore would allow sensitive detection.This work is significant not only in the area of enantioseparation and determination of absolute configuration of diastereomeric derivatives but also in the area of developing new chiral derivatizing reagents (CDRs).

Keywords: chiral derivatizing reagent, naproxen, salbutamol, synthesis

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Authors: Milica Z. Karadzic, Lidija R. Jevric, Sanja Podunavac-Kuzmanovic, Strahinja Z. Kovacevic, Anamarija I. Mandic, Katarina Penov-Gasi, Andrea R. Nikolic, Aleksandar M. Okljesa

Abstract:

In this study, a set of 29 newly synthesized steroid derivatives were investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) as a first step in preselection of drug candidates. This analysis presents an experimental determination of chromatographic lipophilicity, and it was conducted to obtain physicochemical characterization of these molecules. As the most widely used bonded phases in RP-HPLC, octadecyl (C18) and octyl (C8) were used. Binary mixtures of water and acetonitrile or methanol were used as mobile phases. Obtained results were expressed as retention factor values logk and they were correlated with logP values. The results showed that both columns provide good estimations of the chromatographic lipophilicity of the molecules included in this study. This analysis was conducted in order to characterize newly synthesized steroid derivatives for further investigation regarding their antiproliferative and antimicrobial activity. This article is based upon work from COST Action (CM1306), supported by COST (European Cooperation in Science and Technology).

Keywords: antiproliferative activity, chromatographic lipophilicity, liquid chromatography, steroids

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Authors: Nirmala Deenadayalu, Kwanele B. Mazibuko, Lethiwe D. Mthembu

Abstract:

Sugarcane bagasse is the residue obtained after the extraction of sugar from the sugarcane. The main aim of this work was to produce catechol from sugarcane bagasse. The optimization of catechol production was investigated using a Box-Behnken design of experiments. The sugarcane bagasse was heated in a Parr reactor at a set temperature. The reactions were carried out at different temperatures (100-250) °C, catalyst loading (1% -10% KOH (m/v)) and reaction times (60 – 240 min) at 17 bar pressure. The solid and liquid fractions were then separated by vacuum filtration. The liquid fraction was analyzed for catechol using high-pressure liquid chromatography (HPLC) and characterized for the functional groups using Fourier transform infrared spectroscopy (FTIR). The optimized condition for catechol production was 175 oC, 240 min, and 10 % KOH with a catechol yield of 79.11 ppm. Since the maximum time was 240 min and 10 % KOH, a further series of experiments were conducted at 175 oC, 260 min, and 20 % KOH and yielded 2.46 ppm catechol, which was a large reduction in catechol produced. The HPLC peak for catechol was obtained at 2.5 min for the standards and the samples. The FTIR peak at 1750 cm⁻¹ was due to the C=C vibration band of the aromatic ring in the catechol present for both the standard and the samples. The peak at 3325 cm⁻¹ was due to the hydrogen-bonded phenolic OH vibration bands for the catechol. The ANOVA analysis was also performed on the set of experimental data to obtain the factors that most affected the amount of catechol produced.

Keywords: catechol, sugarcane bagasse, lignin, hydrothermal liquefaction

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Authors: Parvaneh Hajeb, Shahram Shakibazadeh, Md. Zaidul Islam Sarker

Abstract:

High-quality fish oil for human consumption requires low levels of toxic elements. The aim of this study was to develop a method to extract oil from fish wastes with the least toxic elements contamination. Supercritical fluid extraction (SFE) was applied to detoxify fish oils from toxic elements. The SFE unit used consisted of an intelligent HPLC pump equipped with a cooling jacket to deliver CO2. The freeze-dried fish waste sample was extracted by heating in a column oven. Under supercritical conditions, the oil dissolved in CO2 was separated from the supercritical phase using pressure reduction. The SFE parameters (pressure, temperature, CO2 flow rate, and extraction time) were optimized using response surface methodology (RSM) to extract the highest levels of toxic elements. The results showed that toxic elements in fish oil can be reduced using supercritical CO2 at optimum pressure 40 MPa, temperature 61 ºC, CO2 flow rate 3.8 MPa, and extraction time 4.25 hr. There were significant reductions in the mercury (98.2%), cadmium (98.9%), arsenic (96%), and lead contents (99.2%) of the fish oil. The fish oil extracted using this method contained elements at levels that were much lower than the accepted limits of 0.1 μg/g. The reduction of toxic elements using the SFE method was more efficient than that of the conventional methods due to the high selectivity of supercritical CO2 for non-polar compounds.

Keywords: food safety, toxic elements, fish oil, supercritical carbon dioxide

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Authors: Meri Khakhutaishvili, Indira Djaparidze, Maia Vanidze, Aleko Kalandia

Abstract:

Cultivation of blueberry in Georgia started in 21st century. There are more than 20 species of blueberry cultivated in this region from all other the world. The species are mostly planted on acidic soil, previously occupied by tea plantations. Many of the plantations have pretty good yield. It is known that changing the location of a plant to a new soil or climate effects chemical compositions of the plant. However, even though these plants are brought from other countries, no research has been conducted to fully examine the blueberry fruit cultivated in Georgia. Shota Rustaveli National Science Foundation Grant FR/335/10-160/14, gave us an opportunity to continue our previous works and conduct research on several berries, among them of course the chemical composition of stored Blueberry. We were able to conduct the first study that included examining qualitative and quantitative features of bioactive compounds in Georgian Blueberry. This experiments were held in the ‘West Georgia Regional Chromatography center’ (Grant AP/96/13) of our university, that is equipped with modern equipment like HPLC UV-Vis, RI-detector, HPLC-conductivity detector, UPLC-MS-detector. Biochemical analysis was conducted using different physico-chemical and instrumental methods. Separation-identification and quantitative analysis were conducted using UPLC-MS (Waters Acquity QDa detector), HPLC (Waters Brceze 1525, UV-Vis 2489 detectors), pH-meters (Mettler Toledo). Refractrometer -Misco , Spectrometer –Cuvette Changer (Mettler Toledo UV5A), C18 Cartridge Solid Phase Extraction (SPE) Waters Sep-Pak C18 (500 mg), Chemicals – stability radical- 2,2-Diphenil-1-picrilhydrazyl (Aldrich-germany), Acetonitrile, Methanol, Acetic Acid (Merck-Germany), AlCl3, Folin Ciocalteu reagent (preparation), Standarts –Callic acid, Quercetin. Carbohydrate HPLC-RI analysis used systems acetonitrile-water (80-20). UPLC-MS analysis used systems- solvent A- Water +1 % acetic acid და solvent -B Methanol +1% acetic acid). It was concluded that the amount of sugars was in range of 5-9 %, mostly glucose and fructose. Also, the amount of organic acids was 0.2-1.2% most of which was malic and citric acid. Anthocians were also present in the sample 200-550mg/100g. We were able to identify up to 15 different compounds, most of which were products of delphinidine and cyanide. All species have high antioxidant level(DPPH). By rapidly freezing the sample and then keeping it in specific conditions allowed us to keep the sample for 12 months.

Keywords: antioxidants, bioactive, blueberry, storage

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Authors: Chahla Rahal, Philippe Refait

Abstract:

Oleuropein-rich extract from olive leaf and acid hydrolysates, rich in hydroxytyrosol and elenolic acid was prepared under different experimental conditions. These phenolic compounds may be used as a corrosion inhibitor. The inhibitive action of these extracts and its major constituents on the corrosion of copper in 0.5 M NaCl solution has been evaluated by potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and weight loss measurements. The product of extraction was analyzed with high performance liquid chromatography (HPLC), whose analysis shows that olive leaf extract are greatly rich in phenolic compounds, mainly Oleuropeine (OLE), Hydroxytyrosol (HT) and elenolic acid (EA). After the acid hydrolysis and high temperature of extraction, an increase in hydroxytyrosol concentration was detected, coupled with relatively low oleuropeine content and high concentration of elenolic acid. The potentiodynamic measurements have shown that this extract acts as a mixed-type corrosion inhibitor, and good inhibition efficiency is observed with the increase in HT and EA concentration. These results suggest that the inhibitive effect of olive leaf extract might be due to the adsorption of the various phenolic compounds onto the copper surface.

Keywords: olive leaf extract, oleuropein, voltammetry, copper, corrosion, HPLC, EIS

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Authors: Rashmi Tomar, A. Srinivasan, Nayan K. Mohanty, Arun K. Jain

Abstract:

Phthalates have been used as additives in industrial products since the 1930s, and are universally considered to be ubiquitous environmental contaminants. The general population is exposed to phthalates through consumer products, as well as diet and medical treatments. Animal studies showing the existence of an association between some phthalates and testicular toxicity have generated public and scientific concern about the potential adverse effects of environmental changes on male reproductive health. Unprecedented declines in fertility rates and semen quality have been reported during the last half of the 20th century in developed countries and increasing interest exists on the potential relationship between exposure to environmental contaminants, including phthalates, and human male reproductive health Studies. Phthalates may be associated with altered endocrine function and adverse effects on male reproductive development and function, but human studies are limited. The aim of the present study was detection of phthalate compounds, estimation of their metabolites in infertile & fertile male. Blood and urine samples were collected from 150 infertile patients & 75 fertile volunteers recruited through Department of Urology, Safdarjung Hospital, New Delhi. Blood have been collected in separate glass tubes from the antecubital vein of the patients, serum have been separate and estimate the phthalate level in serum samples by Gas Chromatography / Mass Spectrometry using NIOSH / OSHA detailed protocol. Urine of Infertile & Fertile Subjects was collected & extracted using solid phase extraction method, analysis by HPLC. In conclusion, to the best of our knowledge the present study based on human is first to show the presence of phthalate in human serum samples and their metabolites in urine samples. Significant differences were observed between several phthalates in infertile and fertile healthy individuals.

Keywords: Gas Chromatography, HPLC, male infertility, phthalates, serum, toxicity, urine

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Authors: Sharareh Sharifi, Pargol Ghavam Mostafavi, Ali Mashinchian Moradi, Mohammad Hadi Givianrad, Hassan Niknejad

Abstract:

Marine organisms such as soft coral, sponge, ascidians, and tunicate containing rich source of natural compound have been studied in last decades because of their special chemical compounds with anticancer properties. The aim of this study was to investigate anti-cancer property of ethyl acetate extracted from marine sea pen Virgularia gustaviana found from Persian Gulf coastal (Bandar Abbas). The extraction processes were carried out with ethyl acetate for five days. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used for qualitative identification of crude extract. The viability of HeLa and MDA-Mb-231 cancer cells was investigated using MTT assay at the concentration of 25, 50, and a 100 µl/ml of ethyl acetate is extracted. The crude extract of Virgularia gustaviana demonstrated ten fractions with different Retention factor (Rf) by TLC and Retention time (Rt) evaluated by HPLC. The crude extract dose-dependently decreased cancer cell viability compared to control group. According to the results, the ethyl acetate extracted from Virgularia gustaviana inhibits the growth of cancer cells, an effect which needs to be further investigated in the future studies.

Keywords: anti-cancer, Hela cancer cell, MDA-Md-231 cancer cell, Virgularia gustavina

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Authors: A. M. Tarola, R. Preti, A. M. Girelli, P. Campana

Abstract:

Safeguarding, studying and enhancing biodiversity play an important and indispensable role in re-launching agriculture. The ancient local varieties are therefore a precious resource for genetic and health improvement. In order to protect biodiversity through the recovery and valorization of autochthonous varieties, in this study we analyzed 12 samples of four ancient apple cultivars representative of Friuli Venezia Giulia, selected by local farmers who work on a project for the recovery of ancient apple cultivars. The aim of this study is to evaluate the polyphenolic profile and the antioxidant capacity that characterize the organoleptic and functional qualities of this fruit species, besides having beneficial properties for health. In particular, for each variety, the following compounds were analyzed, both in the skins and in the pulp: gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, coumaric acid, ferulic acid, rutin, phlorizin, phloretin and quercetin to highlight any differences in the edible parts of the apple. The analysis of individual phenolic compounds was performed by High Performance Liquid Chromatography (HPLC) coupled with a diode array UV detector (DAD), the antioxidant capacity was estimated using an in vitro essay based on a Free Radical Scavenging Method and the total phenolic compounds was determined using the Folin-Ciocalteau method. From the results, it is evident that the catechins are the most present polyphenols, reaching a value of 140-200 μg/g in the pulp and of 400-500 μg/g in the skin, with the prevalence of epicatechin. Catechins and phlorizin, a dihydrohalcone typical of apples, are always contained in larger quantities in the peel. Total phenolic compounds content was positively correlated with antioxidant activity in apple pulp (r² = 0,850) and peel (r² = 0,820). Comparing the results, differences between the varieties analyzed and between the edible parts (pulp and peel) of the apple were highlighted. In particular, apple peel is richer in polyphenolic compounds than pulp and flavonols are exclusively present in the peel. In conclusion, polyphenols, being antioxidant substances, have confirmed the benefits of fruit in the diet, especially as a prevention and treatment for degenerative diseases. They demonstrated to be also a good marker for the characterization of different apple cultivars. The importance of protecting biodiversity in agriculture was also highlighted through the exploitation of native products and ancient varieties of apples now forgotten.

Keywords: apple, biodiversity, polyphenols, antioxidant activity, HPLC-DAD, characterization

Procedia PDF Downloads 116

Authors: Chahla Rahal, Philippe Refait

Abstract:

Oleuropein-rich extract from olive leaf and acid hydrolysates, rich in hydroxytyrosol and elenolic acid was prepared under different experimental conditions. These phenolic compounds may be used as a corrosion inhibitor. The inhibitive action of these extracts and its major constituents on the corrosion of copper in 0.5 M NaCl solution has been evaluated by potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and weight loss measurements. The product of extraction was analyzed with high performance liquid chromatography (HPLC), whose analysis shows that olive leaf extract are greatly rich in phenolic compounds, mainly Oleuropeine (OLE), Hydroxytyrosol (HT) and elenolic acid (EA). After the acid hydrolysis and high temperature of extraction, an increase in hydroxytyrosol concentration was detected, coupled with relatively low oleuropeine content and high concentration of elenolic acid. The potentiodynamic measurements have shown that this extract acts as a mixed-type corrosion inhibitor, and good inhibition efficiency is observed with the increase in HT and EA concentration. These results suggest that the inhibitive effect of olive leaf extract might be due to the adsorption of the various phenolic compounds onto the copper surface.

Keywords: Olive leaf extract, Oleuropein, hydroxytyrosol, elenolic acid , Copper, Corrosion, HPLC/DAD, Polarisation, EIS

Procedia PDF Downloads 226

Authors: Zahid Iqbal, Ijaz Javed, Riaz Hussain, Ibadullah Jan, Amir Ali Khan

Abstract:

This study was conducted to investigate the disposition kinetics of ciprofloxacin and calculate its optimal dosage in Pakistani sheep of Lohi breed. Injectable preparation of ciprofloxacin was given intramuscularly to eight sheep at a dose of 5 mg/Kg. Before administration of drug blood sample was drawn from each animal. Post drug administration, blood samples were also drawn at various predetermined time periods. Drug concentration in the blood samples was assessed through high performance liquid chromatograph (HPLC). Data were best described by two compartment open model and different pharmacokinetic (PK) parameters were calculated. Cmax of 1.97 ± 0.15 µg/ml was reached at Tmax of 0.88 ± 0.09 hours. Half life of absorption (t1/2 abs) was observed to be 0.63 ± 0.16 hours while t1/2 α (distribution half life) and t1/2 ß (elimination half life) were found to be 0.46 ± 0.05 and 2.93 ± 0.45 hours, respectively. Vd (apparent volume of distribution) was calculated as 2.89 ± 0.30 L/kg while AUC (area under the curve) was 7.19 ± 0.38 µg.hr/mL and CL (total body clearance) was 0.75 ± 0.04 L/hr/kg. Using these parameters, an optimal intramuscular dosage of ciprofloxacin in adult Lohi sheep was calculated as 21.43 mg/kg, advised to be repeated after 24 hours. From this, we came to the conclusion that calculated dose was much higher than the dose advised by the foreign manufacturer and to avoid antimicrobial resistance, it is advised that this locally investigated dosage regimen should be strictly followed in local sheep.

Keywords: pharmacokinetics, dosage regimen, ciprofloxacin, HPLC, sheep

Procedia PDF Downloads 505