Ranking the efficacies of selected red wine phenolic anti-oxidants using reversed-phase HPLC

Several studies have assessed total anti-oxidant activity of wine or individual components in isolation using chemical-based assays. In this study, a quantitative approach was developed to assess the relative anti-oxidant efficacies of selected red wine phenolics via peak reduction, using reversed-phase HPLC. Both intact red wine and phenolic standard solutions were challenged with five oxidant model systems as follows: (1) hydrogen peroxide (H₂O₂); redox-active metal ions (2) Fe³⁺ and (3) Cu²⁺; and the Fenton reagents (4) H₂O₂ + Fe²⁺; and (5) H₂O₂ + Cu⁺. Treatment with oxidants (1–3) resulted in loss of 47–60% of phenolic standards, which increased to 66–89% for treatment with the Fenton systems, with quercetin exhibiting the optimal anti-oxidant activity. For intact red wine, treatment with oxidants (1–3) led to all phenolic compounds being oxidised (27–77% loss), with caffeic acid and quercetin as the most effective anti-oxidants. For both Fenton systems (4–5), activities in red wine were considerably enhanced for caffeic acid and quercetin, which exhibited the highest anti-oxidant efficacies with 100% peak reduction, while p-coumaric acid and gallic acid were less effective anti-oxidants with peak reductions of 60–68%. The ranking, facilitated by this new quantitative approach, allows comparison of the individual efficacies of the anti-oxidants in a complex matrix.     

Effect of roasting on the radical scavenging activity of cocoa beans

The free-radical scavenging activity of cocoa samples subjected to different roasting treatments has been determined. The samples (raw, pre-roasted and roasted) were separated into four molecular weight fractions per sample (>30, 30–10, 10–5, and <5 kDa). The free-radical scavenging activity was determined with the DPPH^• (1,1-dipheny-2-picrylhydrazyl), and ABTS^•+ [2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] free-radical scavenging assays for all samples. Both tests were compared in terms of sensitivity and measurement precision, at different reaction times. Comparing the results from each test, the free-radical scavenging activity trends were similar for each fraction but with notable differences in the sensitivity of the assays. Analysis of the concentration of reducing substances, such as water soluble phenolics, melanoidins, carbohydrates, etc, in these fractions by the photometric Folin–Ciocalteu assay, showed a similar pattern to the free-radical scavenging activity trend. Moreover, this comparison showed that there were significantly (P < 0.05) more reducing substances and free-radical scavenging activity in the 10–5 kDa roasted cocoa bean fraction.     

Chitosan, chitin-glucan and chitin effects on minerals (iron, lead, cadmium) and organic (ochratoxin A) contaminants in wines

Chitosan (F7), chitin (F2), chitin-glucan (F1) and chitin glucan hydrolysate (F5) of fungal origin were tested for removal of mineral (Fe or Pb and Cd) and organic (ochratoxin A: OTA) contaminants in wines. Red, white and sweet wines are spiked with either Fe (20 mg L⁻¹), or Pb (500 μg L⁻¹) and Cd (20 μg L⁻¹) or OTA (5 μg L⁻¹). The wines were then treated with F1, F2, F5 or F7 at doses of 0.1 g, 0.5 and 2 g L⁻¹. After 2 days, the levels of iron, copper, lead and cadmium were measured using a flame and graphite furnace atomic absorption spectrophotometer. Depending on the treatment red wines showed reductions of iron by 73–90%, cadmium by 29–57% and lead by 33–74%. The same treatments with white wine gave reductions for iron of 32–91%, cadmium fell by 11–23%, lead by 50–65%. In the case of sweet wines iron was reduced by 51–90%, cadmium by 17–25%, and lead by 38–84%. For wine enriched in OTA, treatments with F1, F2, F5, F7 were carried out at doses of 2 and 5 g L⁻¹. After 2 days, the levels of OTA in wines were analyzed by HPLC with fluorimetric detection. OTA was reduced by 56.7–83.4% in red wine, by 53.4–64.5% in white wine and 26.1–43.5% in sweet wine. These findings indicate that chitosan, chitin, chitin-glucan, or chitin glucan hydrolysate from fungal origin may be useful ancillaries for wine limpidity prevention by reducing levels of Fe, heavy metals (Pb, Cd) and mycotoxins (OTA) and thereby improving wine safety.     

A Biotin–Streptavidin Amplified Enzyme-Linked Immunosorbent Assay with Improved Sensitivity for Rapid Detection of Ractopamine in muscular tissue Development and Nonspecific Adsorption Reduction

An effective biotin–streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was optimized and characterized for the rapid detection of Ractopamine (RAC) residue in muscular tissue. Purification of the RAC antiserum by protein A-Sepharose 4B followed with bovine serum albumin (BSA)-Sepharose 4B affinity chromatography enhanced the sensitivity and reduce the background adsorption. Blocking with 0.5% skimmed milk power and diluting streptavidin–HRP conjugates with 0.5% BSA/phosphate-buffered saline (PBS) effectively remove the nonspecific adsorption in biotin–streptavidin amplified ELISA system. The established method allowed RAC determination with an IC₅₀ value of 0.3 ± 0.02 ng ml⁻¹ and a limit of detection of 0.02 ± 0.003 ng ml⁻¹, more sensitive than the other reported methods. The variation coefficients of intra-assay and inter-assay were all below 7%. RAC residue in pig muscular tissue could be quantified without matrix effects after a 5-fold extraction and 2-fold dilution with PBS. Recoveries of RAC in pig muscular tissue ranged from 75% to 82.75%. The results were also compared with those from HPLC and a good correlation was obtained (r ² = 0.9822). The characters show that the established biotin–streptavidin amplified ELISA could be potentially useful in rapid detection of RAC in animal-derived foods.     

Fractionation and characterization of ɛ-poly-l-lysine from Streptomyces albulus CGMCC 1986

ɛ-Poly-l-lysine (ɛ-PLL) produced by Streptomyces albulus CGMCC 1986 was fractionated using ultra-filtration technique with 2 and 5 kDa cut-offs of membrane. The number-average molecular weight of each fraction was determined by ¹H nuclear magnetic resonance (NMR) method. The number-average molecular weights of the cutoffs of 5 and 2 kDa and the filtrate are 4,230.95, 3,687.80, and 1,900.82 Da, respectively. ¹H NMR indicates the chemical shifts of α-H, β-H, γ-H, δ-H, and ɛ-H are very similar to all the fractions. Fourier transform-infrared (FTIR) spectra showed that the ɛ-PLL solid samples obtained by freeze-drying at pH 5 with molecular weights higher than 2 kDa take on a β-turn conformation, however, the fraction with molecular weight smaller than 2 kDa adopts random coil structure. The antibacterial test proved that the fraction between 2 and 5 kDa of membranes behaves the highest antibacterial activity than other fractions for the test strains of Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, and Shigella.     

Screening of Fluoroquinolone Residues in Caprine Milk Using a 5-kg Luminescence Photometer

Fluoroquinolone (FQ) residues in caprine milk were screened by terbium-sensitized luminescence (TSL). After extraction and cleanup using Oasis HLB columns, TSL was measured at λ _ex = 300 nm and λ _em = 546 nm using a 5-kg luminescence photometer. A common threshold was established at x _F50–3σ _F50, where x _F50 and σ _F50 were the mean and standard deviation, respectively, of TSL intensities of milk samples (n = 18) spiked with flumequine at 50 ng/g, its maximum residue limits (MRL) set by the European Union. Enrofloxacin, ciprofloxacin, and danofloxacin at their respective MRLs had higher TSL responses, so could be screened below their MRLs. Among 48 blind samples, each randomly spiked with one FQ at up to 200% of its MRL, 36 were screened correctly without false negative. This rapid protocol can reduce a sample pool to a small fraction for confirmation, hence improve throughput and save assay costs.     

Supercritical fluid extraction of antioxidant and antimicrobial compounds from Laurus nobilis L. Chemical and functional characterization

Extraction of laurel leaves by using supercritical carbon dioxide was carried out on a supercritical fluid (SF) pilot-scale plant. The extraction pressure and temperature were set to 250 bar and 60°C, respectively, using a 4% of ethanol as modifier. The employed apparatus, owing to a two-stage separation, allowed us to obtain two different fractions (F1 and F2), whose antioxidant and antimicrobial activities were investigated. Two different methods, β-carotene bleaching test and DPPH^• free radical–scavenging assay, were carried out to determine the antioxidant activity. Moreover, antimicrobial activity of laurel fractions was tested against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10145, Escherichia coli ATCC 11775, Candida albicans ATCC 60193 and Aspergillus niger ATCC 16404. Minimum inhibitory concentration (MIC) and minimal bactericidal and fungicidal concentration (MBC) were obtained. Both fractions showed a similar antioxidant activity, although it was slightly higher for the fraction recovered in separator 2. However, antimicrobial activity against the microorganisms tested was only found when fraction 2 was used. Staphylococcus aureus was the most sensitive microorganism to this fraction, with maximal inhibition zones (25 mm) and the lowest MBC values (1.25 mg/ml), whereas the least susceptible was the fungi Aspergillus niger. In order to determine the compounds responsible for the antimicrobial activity, fraction 2 was analysed by GC–MS; results obtained showed that most of the compounds identified in the supercritical extract have been previously described to show antimicrobial activity; among them, the major compound found in the supercritical extract corresponded to a sesquiterpene lactone of the germacrolide type (6-epi-desacetyllaurenobiolide) previously described in laurel.     

In vitro synergistic anti-oxidant activities of solvent-extracted fractions from Astragalus membranaceus and Glycyrrhiza uralensis

Our study estimated the in vitro anti-oxidant activities of the solvent-extracted fractions from Astragalus membranaceus (AME), Glycyrrhiza uralensis (GU) and the combination of them (AG), using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) free radical scavenging assay and ferric reducing anti-oxidant power (FRAP) assay. Different solvent extracts have different anti-oxidant activities. As to the herb pair (AG), the highest anti-oxidant capacity and total phenolic/flavonoid content were observed in the ethyl acetate fraction, which were exhibited in different solvent fractions of the individual herbs. The ethyl acetate extract of herb pair (EA-AG) showed significantly higher anti-oxidant capacity than the theoretical sum of two single herbs, and presented better cytoprotection and induced higher activity of anti-oxidant enzymes than the single herbs. Moreover, there’s a linear correlation between the increment of total phenolics/flavonoids and anti-oxidant capacities, especially between polyphenol content and TEAC values (correlation coefficient = 0.9^∗). These indicated that the phenolic and flavonoid may be the main compounds which caused the synergistic anti-oxidant capacity and cytoprotection in the combination of AME and GU.     

Simultaneous Determination of 69 Pesticide Residues in Coffee by Gas Chromatography?CMass Spectrometry

A method using gel permeation chromatography (GPC) combined with solid-phase extraction (SPE) cleanup followed by gas chromatography–mass spectrometry (GC-MS) has been established for quantitative determination of 69 pesticide residues in coffee. Based on an appraisal of the characteristics of GC-MS, validation experiments were conducted for 69 pesticides. In the method, 2.0 g samples were mixed with 5 ml water and 1 g sodium chloride and extracted with 5 ml of ethyl acetate by blender homogenization, centrifugation, and filtration. Evaporation was conducted and the sample was injected into a 250 mm × 10 mm S-X3 GPC column, with ethyl acetate–n-hexane (1:2 v/v) as the mobile phase at a flow rate of 3 ml/min. The 4–15 min fraction was collected for the SPE cleanup, which was Envi-Carb SPE cartridge coupled with NH₂-LC SPE cartridge with acetone–ethyl acetate (2:5 v/v) as the eluted solvent. The eluents were collected and then evaporated to dryness, which was redissolved in 0.5 ml ethyl acetate for GC-MS analysis. For the 69 pesticides determined by GC-MS, the portions collected from GPC were concentrated to 0.5 ml and exchanged with 5 ml n-hexane. In the linear range of each pesticide, the correlation coefficient was R ² ≥ 0.99. At the low, medium, and high fortification levels of 0.05–1.0 mg/kg, recoveries fell within 60–120%. The relative standard deviation was between 1.3% and 22.3% for all 69 pesticides. The limits of detection for the method were 10 μg/kg to 150 μg/kg, depending on each pesticide.     

Influence of ultrasound on mass transport during cheese brining

 The effect of ultrasound on mass transfer during cheese brining has been investigated. The rate of water removal and NaCl gain increased when ultrasound was applied in comparison with brining performed under static or dynamic conditions, suggesting that ultrasound improves both external and internal mass transfer. A simple diffusional model was developed to simulate mass transport during acoustic brining. Model parameters were estimated using experimental data from acoustic brining experiments carried out on cheese cylinders of 1.7×10^–2m radius and 3×10^–2 m height at different temperatures (5, 15 and 20  °C). Effective water (D _W) and NaCl (D _S) diffusivities estimated using the proposed model ranged from 5.0×10^–10m²/s and 8.0×10^–10 m²/s at 5  °C to 1.3×10^–9m²/s and 1.2×10^–9 m²/s at 20  °C. Both D _W and D _S varied with temperature according to the Arrhenius equation. Through the proposed model, water losses and NaCl gains of the experiments used in the parameter identification were accurately simulated (average %var=98.2%) and also of two additional acoustic experiments carried out under different conditions of temperature (10  °C) and sample size and geometry [parallelepiped of (6×2.5×1.25)×10^–2 m] to those used in the parameter identification (average %var=98.4%). Received: 22 September 1998 / Revised version: 20 November 1998     

Effect of the addition of nitrate to milk on the formation of volatile N-nitrosamines and apparent total N-nitroso compounds

 Saint-Paulin, a French semihard cheese, was manufactured from milk with and without added NO₃ ^–. Reference and experimental cheeses were analysed at different stages of ripening for the presence of apparent total N-nitroso compounds (ATNC) by chemical denitrosation and chemiluminescence detection (thermal energy analyser). The fate of NO₃ ^– and NO₂ ^–was also scrutinized. The levels of ATNC and of volatile N-nitrosamines were followed during ripening. After 60 days of ripening, ATNC were detected at concentrations of 27 μg NO-N/kg and 64 μg NO-N/kg in the cheeses obtained from milk with and without added NO₃ ^–, respectively. The NO₃ ^– contents of both cheeses were nearly the same, and below 5 mg/kg. The occurrence of NO₂ ^–was not observed. No relationship was found between the NO₃ ^– contents and the contamination of the cheeses by volatile N-nitrosamines. At the beginning of ripening, the cheeses manufactured with added NO₃ ^- showed high levels of ATNC (mean value 5800 μg N-NO/kg with large variations of 570–15210 μg NO-N/kg). These ATNC contents rapidly decreased during the first days of ripening (96%), but decreased much more slowly during the following days. An hypothetical mechanism, attempting to explain the disappearance of ATNC, involving the formation of alkylated species in the cheese was proposed. Received: 30 March 1998 / Revised version: 7 October 1998     

Variation in forage quality and chemical composition among Italian accessions of Bituminaria bituminosa (L.) Stirt.

Native Bituminaria bituminosa (L.) Stirt. accessions from central and southern Italy were evaluated for chemical composition to assess their nutritional value as forages. The germplasm was evaluated subjectively ex situ for intensity of ‘oil’ smell and analysed for fibre fraction, crude protein and water‐soluble carbohydrate contents as well as furanocoumarin (psoralen and angelicin) concentration. Total phenolics and their antioxidant activity were also evaluated. The quality parameters indicated this species as a good forage source, showing nutritive values similar to those of other wild legume species. Psoralen and angelicin contents ranged from 2.8 to 5.4 and from 2.3 to 4.7 mg g^?1 dry weight respectively, while total phenolic concentrations were between 11.2 and 13.5 mg g^?1 dry weight. Correlations among forage quality parameters, chemical components and climatic features at sites of origin were also assessed. Copyright © 2007 Society of Chemical Industry     

Effect of Low Temperature Sonication on Orange Juice Quality Parameters using Response Surface Methodology

Freshly squeezed orange juice samples were sonicated at a constant frequency of 20 kHz for a range of processing temperatures (10–30 °C), amplitude levels (40–100%) and time (2–10 min) with pulse durations of 5 s on and 5 s off. Hunter colour values (L*, a* and b*), pH, ^oBrix, titratable acidity, cloud, non-enzymatic browning and ascorbic acid content were measured. Response surface methodology (RSM) based upon a three-factor, three-level Box–Behnken experimental design was used to determine the effect of independent variables. Under process conditions used in this study, no significant difference (p < 0.05) in pH, ^oBrix or titratable acidity was observed. Model predictions developed for Hunter colour values, cloud value, non-enzymatic browning and ascorbic acid content were closely correlated (R ² > 0.92) to experimental data. Box–Behnken design and RSM was demonstrated to be an effective technique to model the effect of sonication on juice quality while minimising the number of experiments required.     

Identification of a new phenolic compound from Chinese olive (<Emphasis Type="Italic">Canarium album</Emphasis> L.) fruit

Chinese olive (Canarium album L.), a native and a well-known tropical fruit tree in the southeast of China, contains large amount of phenolics and possesses great pharmacological activities. In this study, phenolics were extracted from Chinese olive fruit pulp using 80% (v/v) aqueous acetone, and acetone extracts were further fractioned with petroleum ether, ethyl acetate and n-butanol sequentially. From n-butanol fraction, a new phenolic compound was isolated and purified through AB-8 adsorption resin column chromatography, polyamide column chromatography and TSK Toyopearl HW-40 (S) column chromatography, and the structure of the new compound was established as 3-O-galloyl quinic acid butyl ester by electrospray ionization mass spectrometry (ESI-MS), 1D- and 2D-NMR (DEPT, COSY, HMBC, HMQC) and UV–vis techniques.     

In vitro evaluation of the antioxidant activities in the differentially processed seeds from underutilized legume, Bauhinia vahlii Wight & Arn

Antioxidant potential and total phenolics content of 70% acetone extracts of the raw and processed seeds of Bauhinia vahlii were evaluated. The extract of raw seeds contained higher levels of total phenolics (30.8 g/100 g) and tannins (19.6 g/100 g) compared to dry heated and soaking followed by autoclaving seed extracts. Extracts were screened for antioxidant and free radical scavenging activities using various chemical and in vitro model systems. In all the models, except DPPH radical scavenging activity, the extract from raw seeds manifested the strongest antioxidant activity than that from processed seeds. In β-carotene/linoleic acid emulsion system and superoxide scavenging activity, the raw seed extract registered more activity when compared to the standards (butylated hydroxyanisole and α-tocopherol). Whereas, the extract from dry heated seed exhibited higher DPPH^· scavenging activity (IC₅₀ 70.77 μg/mL) than the raw seeds (IC₅₀ 74.4 μg/mL). This study has to some extent validated the antioxidant potential of the seeds of B. vahlii.     

Ethyl acetate leaf fraction of Cnidoscolus aconitifolius (Mill.) I. M. Johnst: antioxidant potential,inhibitory activities of key enzymes on carbohydrate metabolism,cholinergic, monoaminergic,purinergic, and chemical fingerprinting

In this study, the antioxidant, carbohydrate metabolism, cholinergic, monoaminergic, and purinergic enzyme activities of ethyl acetate leaf fraction of Cnidoscolus aconitifolius were evaluated with the high-performance liquid chromatography (HPLC) analysis. The ethyl acetate fraction of the plant leaf was tested for antioxidant properties, key carbohydrate metabolism, cholinergic, monoaminergic, and purinergic enzyme activities using standard procedures, while the chemical composition of the fraction was evaluated using HPLC. The results revealed that the fraction has higher phenolic compounds than flavonoid and exhibited the ability to scavenge iron chelation and ABTS. The fraction also inhibited the activities of α-amylase and α-glucosidase, acetylcholinesterase, butyrylcholinesterase, monoamine oxidase, tyrosinase, arginase, Ecto-5’-nucleotidase, phosphodieterase-5, angiotensin-I-converting enzyme and encouraged the activity of Na⁺/K⁺-ATPase. The HPLC analysis revealed that the ethyl acetate fraction contained coumaric acid, amentoflavone, hesperidin, protocatechuic acid, kaempferol, dihydromyricetin, quercetin,and rutin. The obtained results in this study suggest that the ethyl acetate fraction obtained from aqueous extracts of Cnidoscolus aconitifolius leaf possessed outstanding antioxidative potentials. Thus, excellent enzyme inhibitory activities probably due to bioactive compounds are also observed in the leaf.     

Sensitive Determination of Decoquinate in Milk by High-Performance Liquid Chromatographic Coupled to Laser-Induced Fluorescence Detection

A new and sensitive method based on high performance liquid chromatography with laser-induced fluorescence detection has been developed for the determination of decoquinate in milk. Laser source was obtained with a He–Cd laser using a continuous excitation wavelength at 325 nm. Decoquinate exhibits moderate fluorescence, but it is increased using Ca(NO₃)₂ in the mobile phase. The chromatographic separation was performed on a Luna C₁₈ 5-mm reversed phase column, which solves the broadening of peaks and peak tailing compared with other columns tested. The mobile phase, delivered at 1 ml min⁻¹, consisted of methanol–calcium nitrate (0.025 M)–acetonitrile (83/13/4 v/v/v). Decoquinate was successfully cleaned up from milk by solid-phase extraction using C₁₈ cartridges. The method was found to be linear between 0.16 and 16.33 ng ml⁻¹. The results of recovery studies were found to be satisfactory; an average recovery rate of 88.7% was obtained. The LOQ of decoquinate in milk was 0.16 ng ml⁻¹. The intraday relative standard deviation (RSD) was 4%, and interday assay gave an RSD of 4.4%.     

Effects of variety on chemical composition,in situ nutrient disappearance and in vitro gas production of spineless cacti

This study determined the chemical composition, in situ ruminal nutrient disappearance and in vitro gas production kinetics of three cactus varieties grown in northeastern Brazil. The varieties were Gigante, IPA‐20 and Miúda. Results of the chemical analysis showed no significant differences in ash, ether extract, crude protein (CP) and neutral detergent fibre (NDF) between the cactus varieties. However, acid detergent fibre was highest (P < 0.05) for IPA‐20, intermediate (P < 0.05) for Gigante, and lowest (P < 0.05) for Miúda. Fractionation of carbohydrate and true protein based on rates of ruminal degradation indicated that the main carbohydrate component was the rapidly degradable fraction, whereas the main true protein component was the intermediately degradable fraction. No differences in carbohydrate or protein fractions were observed between the cactus varieties. Results of the in situ experiment showed no differences in ruminal dry matter (DM, average 803 g kg^?1 of DM), CP (900 g kg^?1 of CP) and NDF (611 g kg^?1 of NDF) disappearance between the three cactus varieties after 48 h of ruminal incubation. Potential gas production at the end of 48 h of incubation was higher (P < 0.05) for Gigante than for the Miúda or IPA‐20 variety. However, rate of gas production and lag time in gas production were similar for the three cactus varieties at 6.8% h^?1 and 0.6 h respectively. Our results showed little or no differences in chemical composition or ruminal nutrient degradabilities between the three cactus varieties. Copyright © 2003 Society of Chemical Industry     

Sorption Isotherms of Barnyard Millet Grain and Kernel

The moisture sorption isotherms of grain and kernel of barnyard millet (Echinochloa frumentacea) were determined at 20, 30, 40, and 50 °C. A gravimetric static method was used under 0.112–0.964 water activity (a _w) range for the determination of sorption isotherms. The models were compared using the coefficient of determination (r ²), reduced chi-square (χ ²) values, and on the basis of residual plots. In grain, modified Chung–Pfost (r ² > 0.99; χ ² < 0.7) and modified Oswin (r ² > 0.99; χ ² < 0.55) models were found suitable for predicting the M _e –a _w relationship for adsorption and desorption, respectively. Modified Henderson model was found to give the best fit (r ² > 0.99 and χ ² < 0.55) for describing the adsorption and desorption of the kernel. The isosteric heat, calculated using Clausius–Clapeyron equation, was varied between 46.76 and 61.71 kJ g⁻¹ mol⁻¹ at moisture levels 7–21% (d.b.) for grain and 47.11–63.52 kJ g⁻¹ mol⁻¹ at moisture level between 4% and 20% (d.b.) for kernel. The monolayer moisture content values ranged from 4.3% to 6% d.b. in the case of adsorption of barnyard millet grain and 5.2–6.6% d.b. in the case of desorption at the temperature ranges of 50–20 °C. The monolayer moisture values of barnyard millet kernel ranged from 4.4% to 6.67% d.b. in adsorption and 4.6% to 7.3% d.b. in desorption in the temperature ranges of 50–20 °C.     

Optimization and Validation of a UV–HPLC Method for Vitamin C Determination in Strawberries (<Emphasis Type="Italic">Fragaria ananassa</Emphasis> Duch.), Using Experimental Designs

Vitamin C or total ascorbic acid (TAA) in fruits can be assumed as ascorbic acid (AA) plus dehydroascorbic acid (DHA) content. The aim of this work was to optimize and validate, using experimental designs, a new high-performance liquid chromatographic method for vitamin C determination in strawberries. The mobile phase (MP) consisted of a 0.03 M sodium acetate/acetic acid buffer, 5% methanol. For optimization, a Box–Behnken design was used (three factors at three levels: (a) pH of MP, 3.8–5.8; (b) wavelength, 240–270 nm; and (c) flow rate, 0.5–1.2 ml min⁻¹). Responses were: AA and TAA areas, peak widths, and retention times. A global optimization was performed using the Derringer desirability function, and a value of 0.84 was reached for the combination of design factors: A = 5.8, B = 251 nm, and C = 1.15 ml min⁻¹. Method validation, using AA standard solutions, included: linearity study, limits of detection and quantification, and calibration and analytical sensitivity quantifications. Precision and accuracy were studied in strawberry extracts. The coefficients of variation (percent) were: AA, 1.5%; TAA, 1.8%, and DHA, 4.9%. Accuracy was evaluated with AA standard spiked in 30–150% range of the expected amount of analyte in real samples. The joint confidence elliptical region test and t test were employed for the study of the difference between recoveries (percent) and the ideal 100%. The robustness was analyzed using a fractional factorial design (3⁴⁻²), and an AA recovery study after slight changes in operative variables was performed. The results indicate that the optimized method was linear, sensible, precise, accurate, and robust.