On-line HPLC Method Research Articles

Determination of active radical scavenging compounds in polar fruit and vegetable extracts by an on-line HPLC method

Fruit and vegetables are believed to help fight against oxidative stress, given their natural content in radical scavenging compounds. Natural polyphenols neutralize reactive oxygen species by means of electron and hydrogen atom transfers. An HPLC method, hyphenated with a post-column reaction system relying on ABTS+ bleaching assay was applied in order to quantify the chemical activity of radical scavenging compounds in red cabbage, onion, quince, sweet cherry, strawberry, carrot and tomato. Hyphenated to the ABTS+ post-column reaction system, this method showed high antioxidant capacity notably in cherry, quince, onion, or red cabbage. Structural analysis of the compounds of interest showed the implication of several cyanidins and caffeoylquinic acids in cherry. Quince and red cabbage were found highly active through the presence of respectively caffeoylquinic acids, and an important content of diverse cyanidins variously glycosylated and acylated. The onion extract revealed a chemical core structure (quercetin) responsible for its antioxidant capacity. Moreover, our results showed that depending on the glycosylation profile of these compounds, their radical scavenging capacity can be very different.

Separation and purification of antioxidants from Ampelopsis heterophylla by counter‐current chromatography

In this study, bioactive components from Ampelopsis heterophylla were separated by counter-current chromatography (CCC). The antioxidant activity of the crude extract was initially evaluated by an online HPLC method. Five compounds in the crude extract exhibited good antioxidant activities, namely, hyperoside (1), isoquercitrin (2), rutin (3), kaempferol-3-rutinoside (4), and quercetin (5). These compounds were further separated by CCC with biphasic solvent systems and their structures were identified by MS and NMR spectroscopy. All the compounds exhibited significant 1,1-diphenyl-2-picryl-hydrazyl radical scavenging activities with IC50 values at 18.2 ± 1.3, 17.0 ± 1.4, 24.2 ± 1.2, 38.1 ± 1.7, and 9.0 ± 1.2 μM, respectively. The scavenging ratios of the compounds against hydroxyl radicals were 65 ± 5, 68 ± 4, 96 ± 2, 70 ± 4, and 98 ± 2%, respectively.

A strategy for high-speed countercurrent chromatography purification of specific antioxidants from natural products based on on-line HPLC method with radical scavenging assay

We have proposed a novel and first strategy of high-speed countercurrent chromatography (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in materials are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of target peaks would be investigated for the two-phase solvent system, and purified by HSCCC. In this study, the specific antioxidants in red cabbage, perilla and elderberry pigments were evaluated by on-line HPLC with DPPH radical scavenging assay, and purified by HSCCC technique. Specific antioxidants could be rapidly pinpointed in complex mixtures by on-line HPLC with DPPH radical scavenging assay. Then, the optimal two-phase solvent systems were investigated using these HPLC peaks. Finally, the purification of these nine antioxidants form three mixtures were performed by HSCCC. Using mass spectrometric analysis, these antioxidants were confirmed to cyanidin-based anthocyanin from red cabbage and elderberry pigments, and luteolin-based flavones from perrilla pigment. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.

Detection of antioxidant active compounds in Mori Ramulus by HPLC-MS-DPPH

To study major antioxidant active compounds in Mori Ramulus. The combination of on-line HPLC method and liquid chromatography-mass spectrometry was used to identify major antioxidant compounds and their content was determined by HPLC-DAD (detection wavelength of 320 nm). Oxyresveratrol was proved to be the major antioxidant compound in Mori Ramulus ethanolic extracts. Using the HPLC-DAD quantitative determination, Mori Ramulus oxyresveratrol had a good linear range of 14-1 260 mg x L(-1) (r = 0.999 96). The average recovery was 98.2% with RSD of 1.2%. This method is simple, rapid, accurate and sensitive. There was rich oxyresveratrol in Mori Ramulus and the content was significantly different according to the mulberry varieties. The on-line HPLC-MS-DPPH method is applicable for the determination of antioxidant compounds in Mori Ramulus.

Antioxidant Properties of Ethanolic Extract from Ramulus mori (Sangzhi)

The antioxidant properties and total phenolic contents of four fractions of ethanolic extract from Ramulus mori were examined. Various experimental models including superoxide radical, hydroxyl radical, 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) scavenging activity, metal chelating activity, and reducing power were used for characterization of their antioxidant activity. The four fractions showed various degrees of efficacy in each assay in a dose-dependent manner. The third fraction with the highest amount of total phenolics was the most potent antioxidant in all assays used. In addition, the most powerful compound (oxyresveratrol) was isolated and identified followed by on-line HPLC method and characterized by different spectral analysis. Oxyresveratrol exhibited impressive antioxidant activities in scavenging the superoxide radical, hydroxide radical, and DPPH. On the basis of the results obtained, Ramulus mori may serve as a potential source of natural antioxidant due to its significant antioxidant activity and oxyresveratrol may be the most powerful antioxidant in ethanolic extracts of Ramulus mori.

Measurement and correlation of solute solubility in HFA-134a/ethanol systems

The goals of this study were to determine the solubility values of solid organic solutes in pure HFA-134a and in HFA-134a/ethanol cosolvent systems (0–20%, w/w), and to investigate the relationship between these solubilities and a solute's physico-chemical properties. A direct inject on-line HPLC method was used to determine the solubility of 21 solutes in HFA-134a/ethanol. The samples were allowed to equilibrate for at least 48 h. The filtered sample was injected directly on an analytical HPLC column through a manual injector interface, and analyzed at an appropriate solute wavelength via HPLC. The solutes display diverse physico-chemical properties and yielded solubility values that ranged over four orders of magnitude. In general, a linear–linear solubility relationship was observed as the fraction of ethanol increased. The effects on solubilization ranged from 1.3 to 99.4 times when 20% (w/w) ethanol was introduced, relative to pure HFA-134a. A regression equation utilizing a solute's hydrogen bonding potential resulted in a significant correlation to the slope obtained from a linear model for solubility in HFA-134a with 0–20% (w/w) ethanol, and may be useful for pre-formulation studies.

On-line high-performance liquid chromatography method for analyte quantitation from pressurized metered dose inhalers

A sensitive and rapid, on-line reversed-phase high-performance liquid chromatographic method for quantitation of compounds at low concentrations in pressurized metered dose inhaler (MDI) systems was developed. Traditional methods for the quantitation of compounds in MDI formulations involve the opening of the MDI vial along with sample dilution prior to quantitation. The new method, reported in this study, involves a direct injection from the MDI vial into the needle injector port of a manual injector. Since there is no dilution step involved, this method can be used to quantitate low concentrations of compounds in MDIs with excellent precision. In addition, since the method requires a small injection volume of 5 μl, repeated analyses can be performed in order to generate multiple data points using the same MDI vial. Validation of the method was performed using ethanol–1,1,1,2-tetrafluoroethane (134a)-based MDIs. Beclomethasone dipropionate (BDP), a corticosteroid used for the treatment of asthma, was used as a model compound. Phase separation studies were conducted to investigate the miscibility of the ethanol–134a mixtures with different mobile phase solvent compositions. For the MDI systems in this study, an acetonitrile–water (90:10, v/v) mobile phase at a flow rate of 0.9 ml/min was found to give acceptable chromatography for BDP on a Apollo C18 5 μm, 150 mm×4.6 mm column (Alltech Associates, Deerfield, IL, USA). Ultraviolet detection was done at 240 nm and the retention time of BDP was 2.7 min. The on-line HPLC method was characterized to be accurate, precise, sensitive, and specific.

On-line clean-up by multidimensional liquid chromatography–electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine

We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher ® ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5–2.0 μg/l urine). First results on a small non-exposed group ( n=8) ranged for 5OH-MEHP from 0.59 to 124 μg/l, for 5oxo-MEHP from <LOQ to 73.0 μg/l, and for MEHP from <LOQ to 41.1 μg/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 μg/l, for MBzP of 7.19 μg/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.

On-line HPLC detection of tocopherols and other antioxidants through the formation of a phosphomolybdenum complex.

An on-line method to detect and quantify antioxidant species in complex extracts has been developed as a combination of conventional HPLC separation and a postcolumn reaction with phosphomolybdenum reagent at acidic pH. Sample analytes were chromatographed by HPLC, and the postcolumn formation of a phosphate/Mo(V) complex was detected at 598 nm with an on-line absorbance detector. An optimized instrumental system was set up using pure alpha-tocopherol, and it was successfully tested with complex food extracts including lettuce, tomato, red pepper, and soybean seed, where several tocopherols and carotenoids were identified. A potential application of this detection method to quantitatively determine different antioxidants was considered, and a specific application to the determination of tocopherols was developed. The new method was characterized with respect to linearity interval, repeatability, and reproducibility, the quantitative results obtained were validated by comparison with a conventional HPLC method with fluorometric detection, and it was applied to the determination of tocopherols in different foods. The results suggest that the proposed on-line HPLC method can be a powerful instrument for the detection, purification, and characterization of natural antioxidants.

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS•+ assay

The ABTS/H2O2/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples – conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) – provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.

Application of ABTS radical cation for selective on-line detection of radical scavengers in HPLC eluates.

The radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate), (ABTS*+) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react postcolumn with the preformed ABTS*+, and the induced bleaching is detected as a negative peak by an absorbance detector at 734 nm. An optimized instrumental and experimental setup is presented. The method is suitable for both isocratic and gradient HPLC runs using mobile phases containing 100% organic solvent or its solution in water, weak acids, or buffers (pH 3-7.4). The method is sensitive, selective, relatively simple, applicable to compounds of different chemical natures; uses common instruments and inexpensive reagents; and has a time-saving, nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The minimum detectable concentration varied from 0.02 to 0.13 microg/mL, depending on the compound tested. The method can be applied to perform kinetic studies, which is illustrated by determination of Trolox equivalent antioxidant capacities (TEAC) of several known antioxidants in flow injection mode.

An on-line HPLC method for detection of radical scavenging compounds in complex mixtures.

A rapid on-line method for screening of complex mixtures for radical scavenging components was developed using a methanolic solution of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) stable free radical. The HPLC-separated analytes react postcolumn with the DPPH solution, and the induced bleaching is detected as a negative peak by an absorbance detector at 517 nm. An optimized instrumental setup is presented. The method is suitable for both isocratic and gradient HPLC runs with mobile-phase compositions ranging from 10 to 90% organic solvent in water or buffer (pH 3-6). The method is simple, has a broad applicability, and uses common instruments, inexpensive and stable reagents, and a time-saving and nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The limits of detection were 0.33-94 microg/mL, depending on the compound tested.

On-line HPLC method for clean-up and analysis of hydrocortisone and sulconazole nitrate in a cream

The application of a new on-line high-performance liquid Chromatographic method for the clean-up and analysis of drugs in semi-solid formulations was investigated. A short column of a chemically bonded stationary phase (10 μm octadecylsilane) mounted into the sample loop position of the high pressure injection valve was used as the primary clean-up step. The elution behavior of commonly used cream excipients on the loop column as a function of various combinations of water, methanol and THF was studied using a refractive index detector. Typically, samples of the cream were dissolved in methanol-THF and injected onto the loop column. The drug substances were eluted onto the analytical column with predetermined volumes of the mobile phase. The relatively non-polar cream excipients were retained on the loop column. At this time the injection valve was switched, isolating the loop column and the retained excipients were eluted off to waste with a strong solvent (methanol-THF). Loss of efficiency due to the insertion of the loop column was negligible. This technique was applied to the simultaneous analysis of a corticosteroid (hydrocortisone) and a hydrophobic amine (sulconazole nitrate) in a topical cream formulation.