Stability of limonin glucoside in beverage matrices

BACKGROUND: We completed a study over a 200‐day period examining the stability of limonin glucoside formulated into three beverage matrices. RESULTS: Beverages containing limonin glucoside were found to contain limonin (0.13–20.10 mg L^?1) during their initial testing; however, these concentrations were directly attributable to the presence of contaminating limonin in the particular lot of limonin glucoside used to prepare the beverage and did not increase over the test period. Likewise, limonin glucoside concentrations did not vary significantly, with the exception of the beverage matrix that included vitamin B₂. Exposure of the vitamin B₂‐containing beverages to light resulted in a rapid reduction in the limonin glucoside content. Liquid chromatographic–mass spectrometric and nuclear magnetic resonance results from the analyses of pre‐ and post‐light exposed beverages suggest photooxidation of the furan moiety as the likely degradation pathway. CONCLUSION: Results from this study indicate that limonin glucoside is resistant to degradation into limonin, the stability of limonin glucoside formulated into beverages exceeds six months and that limonin glucoside should not be formulated into beverages containing vitamin B₂ unless the beverages are protected from light. Published in 2008 by John Wiley & Sons, Ltd.     

Optimisation of the determination of deoxynivalenol in wheat flour by HPLC and a comparison of four clean-up procedures

The determination of deoxynivalenol (DON) in wheat flour by liquid chromatography with photodiode array (PDA) detection was optimised. Response surface methodology (RSM) was used to determine the optimum chromatographic conditions for the determination of DON. The influence of three variables, acetonitrile (ACN) volume in mobile phase (9.5–24.5, v/v), flow rate (0.5–1.5 ml min^?1) and wavelength (215–221 nm) on DON peak area was evaluated. The best separation was achieved using a symmetry column (150 × 3.9 mm; particle size 5 µm) by isocratic elution (1.0 ml min^?1) and a mobile phase consisting of ACN/water in the ratio 17 : 83 (v/v). UV detection was performed at 218 nm. Linear calibration curves were constructed in the concentration range 1–1000 ng ml^?1. The detection limit measured as the signal-to-noise ratio (3 : 1) was 0.03 ng ml^?1. RSM results showed that the experimental data could be adequately fitted to a second-order polynomial model with multiple regression coefficients (R ²) of 0.968. The efficiency of four clean-up procedures for wheat flour extract was compared. Recovery of DON using a Mycosep #225 column was highest with a value of 99%, while that of Mycosep #227 was 65%. In contrast, DON recovery using immunoaffinity columns (IAC) and an Oasis® HLB column was only 53 and 42%, respectively. The trueness of the method using the Mycosep #225 column was established with a certified reference material CRM 379. The result obtained from three replicates was 0.66 ± 0.04 µg g^?1 and the certified value was 0.67 µg g^?1.     

Purification of citrus limonoids and their differential inhibitory effects on human cytochrome P450 enzymes

Recent studies demonstrated that citrus limonoids and flavonoids possess numerous health promoting properties. In the present study, glucosides of limonoids and flavonoids were purified from citrus molasses and limonoid aglycones from citrus seeds. Glucosides were separated on styrene (divinylbenzene), Q‐sepharose resins with increasing concentration of sodium chloride. A pH‐dependent cold precipitation was carried out for the isolation of naringin in large quantity. Major aglycones such as limonin and nomilin were isolated from seeds by direct crystallization and minor limonoids were purified by vacuum liquid chromatography. The structures of the isolated compounds were confirmed by NMR spectra. Individual limonoids were tested for O‐dealkylase and hydroxylase activities of human cytochrome P450 (CYP) isoenzymes such as CYP1A2, CYP1B1, CYP3A4 and CYP19, using ethoxyresorufin, methoxyresorufin and dibenzylfluorescein as substrates. Partial to high inhibition of CYPs was observed in dose‐dependent assays. Significant (P < 0.001) reductions in enzyme activities were observed with purified compounds above 2 µmol. Kinetic analyses indicated that limonin glucoside inhibited CYP19 competitively (IC₅₀, 7.1 µ mol L^?1), whereas Nomilinic acid glucoside inhibited it noncompetitively (IC₅₀, 9.4 µ mol^?1). Nomilinic acid glucoside was the most potent limonoid, with an overall IC₅₀ of < 10 µ mol, for all the enzymes tested. The differential inhibition of CYPs can be ascribed to structural variations of the limonoid nucleus. Limonoid inhibition of key CYPs involved in carcinogenesis supports growing evidence that citrus limonoids act as anticancer agents. Copyright © 2007 Society of Chemical Industry     

KINETICS OF THE INTERACTION OF PANCREATIC α-AMYLASE WITH A KIDNEY BEAN (PHASEOLUS VULGARIS) - AMYLASE INHIBITOR

An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min^?1 and 0.017 min^?1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10^?8 M and 10^?9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 10⁶ M^?1 min^?1 at 37°C, with 4 × 10^?8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10^?10 M at pH 5.5 and 4.4 × 10^?9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.     

Micellar liquid chromatographic determination of arbutin and hydroquinone in medicinal plant extracts and commercial cosmetic products

A simple micellar liquid chromatographic (MLC) procedure for simultaneous determination of arbutin and hydroquinone in medicinal plant extracts and commercial cosmetic products was proposed. This method was developed and validated. The chromatographic conditions were also optimized. All analyses were performed at room temperature in an isocratic mode, using a mixture of 1% (v/v) acetonitrile and 0.006 mol L^?1 Brij 35 (pH 6.0) as a mobile phase. The flow rate was set at 1.0 mL min^?1. The analytical column was a 150 × 3.9 mm Nova‐Pak C‐18 column. The effluent from the analytical column was monitored by UV detection at 280 nm. Under the optimum conditions, arbutin and hydroquinone could be determined within a concentration range of 2–50 μg mL^?1 of arbutin, and hydroquinone was obtained with the regression equations; y = 0.045x + 0.042 (r² = 0.9923) and y = 0.091x + 0.050 (r² = 0.9930) respectively. The limits of detection were found to be 0.51 μg mL^?1 and 0.37 μg mL^?1 for arbutin and hydroquinone respectively. The proposed MLC method was applied for the determination of arbutin and hydroquinone contents in medicinal plant extracts and commercial cosmetic products. This proposed MLC method is thus suitable for routine analysis of arbutin and hydroquinone in the pharmaceutical formulations, cosmetic products and raw medicinal plant extracts.     

Effect of Temperature and Moisture Content on the Kinetics of Trypsin Inhibitor Activity, Protein in vitro Digestibility and Nitrogen Solubility of Cowpea Flour

Samples of finely ground cowpea flour containing 7.5%, 19.4% and 25.5% moisture were heated in sealed tubes at 100° 125° and 150°C for periods of 0.5 to 120 min. First order rate constants for losses of trypsin inhibitor activity and nitrogen solubility ranged from 1 × 10^?2 to 18 min^?1 and from 4 × 10^?2 to 8 min^?1 respectively. In vitro protein digestibility (IVD) increased, then decreased with heating as described by sequential first order kinetics. Rate constants for increase of IVD varied from 0.13 to 12 min^?1, while for decrease in IVD the range was 5 × 10^?5 to 3 × 10^?2 min^?1. Activation energies and relationships between In k and water activity were computed.     

Determination of arginine in dietary supplements

We present a rapid and sensitive flow injection method for the determination of arginine in dietary supplements. Detection was based on the chemiluminescence reaction of arginine with alkaline hypobromite. The response is proportional to analyte concentration over the calibration range, from 2.5 × 10^?6 to 1 × 10^?4 M , and a relative standard deviation of 1% was calculated (1 × 10^?5 M , n = 12). Samples required only aqueous dilution prior to analysis, and over 100 samples could be analysed per hour, which is superior to that achieved with conventional colorimetric and enzymatic procedures. The results obtained with the flow injection methodology were concordant with those achieved using high‐performance liquid chromatography employing pre‐column derivatisation and fluorescence detection. Copyright © 2005 Society of Chemical Industry     

Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves

A fast isocratic HPLC method for analysis of steviol glycosides from Stevia rebaudiana leaves was developed with a high selectivity for nine known steviol glycosides and low eluent consumption. The analytical method was performed on a Purospher^? STAR RP-18 endcapped 3???m Hibar^? RT 250-4.6 column at 50?°C with an eluent composition of water (65 vol.?%) and acetonitrile (35 vol.?%). With a flow rate of 1?mL?min^?1, nine known steviol glycosides were detected selectively after 15?min. Method validation for rebaudioside A showed a LOD of 0.0004?mg?mL^?1 and a LOQ of 0.0038?mg?mL^?1. Particularly with regard to moderate solubility, the method is linear up to a concentration of rebaudioside?A of 4.8?mg?mL^?1. The linear calibration curve was obtained with a coefficient of determination of 0.9997?±?0.0002 and a total error of 2.01?% RSD (n?=?12). The accuracy of the method was determined by the percentage mean recovery rate to 100.99?±?2.01?%. The intra-day precision was in a range of 0.12 to 1.96?%?RSD and the inter-day precision varies from 0.02 to 1.89?% RSD. Small changes in operating conditions like eluent composition (65?±?2 vol.?%), temperature (50?±?10?°C) or flow rate (1?±?0.2?mL?min^?1) do not affect the performance of the analytical method. The reliable and robust proposed HPLC method can be applied for quantification of steviol glycosides in Stevia rebaudiana Bertoni leaves in laboratories and quality control in industry.     

Spray drying of low‐phenylalanine skim milk: optimisation of process conditions for improving solubility and particle size

The optimisation of the spray drying process for low‐phenylalanine skim milk as a dietary supplement for phenylketonurics was studied. The effects of basic parameters including the inlet air temperature (100, 150 and 200 °C), feed flow rate (5, 10 and 15 mL min^?1) and air flow rate (400, 600 and 800 L h^?1) on the solubility and particle size of the low‐phenylalanine skim milk powder were determined using response surface methodology. The optimum conditions have been obtained with inlet air temperature of 133 °C, feed flow rate of 5 mL min^?1, and air flow rate of 800 L h^?1. With the optimum parameters, the predicted values for the solubility and mean diameter were 95.33% and 5.34 μm, respectively, and experimentally were 94.36 ± 1.62% and 5.50 ± 0.44 μm, respectively. The experimental and predictive values were closely related showing predictive accuracy of the models.     

A colorimetric study of oenin copigmented by procyanidins

A colorimetric method was used to analyse the influence of procyanidin structure on colour changes of malvidin 3‐O‐glucoside (oenin) solution resulting from copigmentation. The study was performed in hydroalcoholic citrate/phosphate buffer solution (120 g L^?1) at pH 3.6 and ionic strength 0.2 mol L^?1. Chromatic L*, a* and b* coordinates (CIELAB, D65/10° illuminant/observer condition) obtained from spectral curves recorded between 360 and 830 nm allowed the calculation of lightness L*, chroma C* and hue angle h_ab. In general, addition of copigment induced colour enhancement (loss of lightness and increased chroma). The prevailing parameters affecting colour changes were lightness and chroma for monomers and lightness and hue for procyanidins B5 and B8 (C4–C6 dimers). A small blueing effect was observed only for catechin monomer‐copigmented solutions. For procyanidin copigments, as the structural complexity of the copigment increased, the hue angle moved to yellower values. The ester gallate of dimer B2 produced the strongest modification of colour attributes of oenin solution. Copyright © 2006 Society of Chemical Industry     

Sorbic Acid and Potassium Sorbate Permeability of an Edible Methylcellulose-Palmitic Acid Film: Water Activity and pH Effects

The apparent permeability constants for potassium sorbate and sorbic acid through an edible film composed of methylcellulose and palmitic acid (weight ratio 3:1) were evaluated as a function of water activity (a_w) and pH. For films with thickness 55–66 μm, potassium sorbate permeability increased from 2.3 × 10^?10 to 2.0 × 10^?8 (mg/sec cm²)(cm)/(mg/mL) as a_w increased from 0.65 to 0.80. Films were not stable at a_w levels above 0.80. Permeability of the film to sorbic acid at a_w 0.8 decreased from 3.3 × 10^?8 to 9.1 × 10^?10 (mg/sec cm²)(cm)/ (mg/mL) as pH increased from 3 to 7. At pH 3 the undissociated acid was 97.5% and at pH 7 it was 0.4%.     

Preparative separation and purification of phenolic compounds from Canarium album L. by macroporous resins

BACKGROUND: Canarium album L. (also called Chinese olive) is a traditional medicine material in China, and phenolic compounds from C. album possess great pharmacological activities. To obtain high‐purity phenolics from C. album, a crude extract of C. album phenolics was prepared by ethanol extraction. The use of macroporous resins for further separation and purification of phenolics in the extract was studied. RESULTS: Through static adsorption and desorption tests, AB‐8 resin was chosen for the separation of phenolics because of its higher adsorption capacity and desorption ratio than other resins. Then, dynamic adsorption and desorption experiments were carried out on an AB‐8 resin packed column to obtain optimal separation parameters. The highest adsorption capacity of AB‐8 was achieved when variables including initial concentration (C₀), feed flow rate and feed volume were 10 mg mL^?1, 2 mL min^?1 and 9 bed volumes (BV), respectively, and saturated resin was first washed with 5 BV of water to remove impurities, then a purified product containing more than 85% of C. album phenolics was obtained by desorbing the resin with 2.5 BV of 70% (v/v) aqueous ethanol at flow rate of 1 mL min^?1, and the recovery of phenolics was up to 75%. In addition, five phenolic compounds in the product were identified as gallic acid, ellagic acid, corilagin, hyperin and kaempferol‐3‐glucopyranoside by UV and LC–ESI–MS analysis. CONCLUSION: The results in this study could provide scientific references for the large‐scale production of phenolics from C. album. Copyright © 2007 Society of Chemical Industry     

Response surface optimisation of spray dryer operational parameters for low‐phenylalanine skim milk powder

The aim of this study was to optimise the spray‐drying process for low‐phenylalanine (low‐Phe) milk. Influence of inlet air temperature (100, 150 and 200 °C), feed flow rate (5, 10 and 15 mL min^?1) and air flow rate (400, 600 and 800 L h^?1) on the solubility, yield, water activity and moisture content (MC) of the milk powder were assessed using response surface methodology. The following optimum process parameters were determined: inlet air temperature of 160 °C, feed flow rate of 8 mL min^?1 and air flow rate of 400 L h^?1. The predicted values for the solubility, yield, water activity and MC were 98.77%, 88.08%, 0.263% and 5.48%, respectively. With the optimum parameters, the experimental values for the solubility, yield, water activity and MC were 97.56 ± 0.03%, 87.00 ± 0.10%, 0.26 ± 0.01% and 5.53 ± 0.08%, respectively. The similarity of the experimental results to the predicted values verified the models.     

Extraction of polycyclic aromatic hydrocarbons from cookies: A comparative study of ultrasound and microwave-assisted procedures

The chromatographic determination of 15 polycyclic aromatic hydrocarbons (PAHs) in cookies has been improved in order to obtain a fast method with a low limit of detection through the combination of microwave-assisted extraction (MAE), oil saponification and solid-phase extraction clean-up before the injection of purified extracts in a C18 201TP52 (5 µm, 250 ×?2.1 mm) column. Using acetonitrile–water as mobile phase, with a 50% to 95% w/w acetonitrile gradient for a fixed flow of 0.250 ml min^?1, 15 PAHs were separated in 45 min. The column temperature was maintained at 15°C; and fluorimetric detection was made at a fixed excitation wavelength of 264 nm and emission measurements at the best wavelength for each analyte, from 352 nm for 11H-benzo[b]fluorene to 500 nm for indeno[1,2,3-c,d]pyrene. Recoveries for all 15 PAHs varied between 96 ±?4 and 105% ±?4%; and the limits of detection ranged from 0.015 ng g^?1 for chrysene to 0.7 ng g^?1 for phenantrene. Results were compared with those obtained by conventional Soxhlet extraction during 8-h refluxing with toluene, demonstrating that the methodology proposed is appropriate to quantify PAHs in cookies. Furthermore, the microwave-assisted method was faster and used less solvent than the conventional and ultrasound-assisted methods. The extraction time was reduced to 9 min compared with the 8 h required for Soxhlet extraction and 60 min required for ultrasound-assisted treatment, and the solvent consumption has been reduced to 25 ml compared with the 155 and 90 ml required using Soxhlet and ultrasound, respectively.     

The UPLC–ESI–QqQLIT–MS/MS method for quantitative determination of phytochemicals in ethanolic extracts of different parts of eight Ficus species: Development and validation

Ficus and validation of the ultra performance liquid chromatography–electrospray ionization hybrid triple quadrupole–linear ion trap–tandem mass spectrometry (UPLC–ESI–QqQ_LIT–MS/MS) method in a multiple-reaction monitoring (MRM) mode for the quantitative determination of 19 phytochemicals. The chromatographic separation of targeted phytochemicals was performed using the Waters ACQUITY UPLC BEH? C18 column (1.7 μm, 2.1 mm × 50 mm) with 0.1% formic acid with water and acetonitrile as a mobile phase at a flow rate of 0.25 mL/min. The validation parameters showed the overall recoveries from 95.78?101.44% (RSD ≤ 3.25%), precision (intra-day: RSD ≤ 2.96%; inter-day: RSD ≤ 2.89%), linearity (R² ≥ 0.9982), limit of detection (8.60 × 10^–10?2.18 × 10^–6 mg/mL), and the limit of quantitation (2.60 × 10^–9–6.63 × 10^–6 mg/mL) in the concentration range from 0.5 to 1000 × 10^–6 mg/mL. This method was successfully applied in ethanolic extracts of different parts (fruits, leaves, and barks) of selected eight Ficus species. Quinic acid was predominant followed by rutin and chlorogenic acid among the studied nineteen phytochemicals. Ficus benjamina showed the maximum total content in fruits and leaves. The UPLC–ESI–QqQ_LIT–MS/MS method combined with principal component analysis (PCA) was successfully used for Ficus species discrimination on the basis of the contents of 15 compounds. The UPLC–ESI–QqQ_LIT–MS/MS method combined with PCA could be used for quality control.     

Quantification of malachite green in fish feed utilising liquid chromatography-tandem mass spectrometry with a monolithic column

The purpose of this study was to develop a rapid and sensitive method for the quantification of malachite green (MG) in fish feed using LC-ESI-MS/MS with a monolithic column as stationary phase. Fish feed was cleaned using ultrasonic assisted liquid–liquid extraction. The separation was achieved on a Chromolith^® Performance RP-18e column (100 × 4.6 mm) using gradient mobile phase composition of methanol and 0.1% formic acid at the flow rate of 1.0 ml min^–1. The analyte was ionised using electrospray ionisation in positive mode. Mass spectral transitions were recorded in selected reaction monitoring (SRM) mode at m/z 329.78 → m/z 314.75 with a collision energy (CE) of 52% for MG. The system suitability responses were calculated for reproducibility tests of the retention time, number of theoretical plates and capacity factor. System validation was evaluated for precision, specificity and linearity of MG. The linearity and calibration graph was plotted in the range of 15.0–250 ng ml^?1 with the regression coefficient of >0.997. The lower limits of detection and quantification for MG were 0.55 and 1.44 ng ml^?1, respectively, allowing easy determination in fish feed with accuracy evaluated as a percentage recovery of 92.1% and precision determined as % CV of < 5. The method was also extended to the determination of MG in an actual fish feed. The sensitivity and selectivity of LC-ESI-MS/MS using monolithic column offers a valuable alternative to the methodologies currently employed for the quantification of MG in fish feeds.     

HPLC determination of γ‐aminobutyric acid in Chinese rice wine using pre‐column derivatization

γ‐Aminobutyric acid (GABA) is a major functional ingredient in Chinese rice wine. A new HPLC method for determining GABA using pre‐column derivatization with dansyl chloride was developed. The HPLC operating conditions were as follows: a Hypersil ODS2 C₁₈ column; mobile phase, methanol and water (1:1); gradient elution; absorption at 254 nm; flow rate, 1 mL/min; pH 9; and column temperature, 30 °C. Under optimal HPLC conditions, the linear equation was y = 3.5 × 10^?7x ? 0.014. The precision (relative standard deviation, RSD) was 0.54%, the stability (RSD) was 0.21%, the recovery ratio was 97.4% and reproducibility (RSD) was 1.57%. This new method appears to be sensitive, accurate, convenient, steady and relatively fast. The results suggest that the new method is also more reliable. Copyright © 2015 The Institute of Brewing & Distilling     

高效液相色谱法测定柑橘中的柠檬苦素类似物

建立了同时测定柑橘中柠檬苦素和诺米林2种苷元的高效液相色谱方法。色谱柱为Phenomenex C18(250mm×4.6mm,5μm),流动相为体积分数45%乙腈,流速为1.0mL/min,柱温为25℃,检测波长在210nm。经考察该方法可行性强,柠檬苦素和诺米林的平均回收率分别99.91%和98.28%,RSD分别为2.55%和2.30%。因此用该法测定了柑橘中的柠檬苦素和诺米林的含量,并取得了理想的结果。     

Kinetics of Thermal Inactivation of Peroxidase and Color Degradation of African Cowpea (Vigna unguiculata) Leaves

Cowpea leaves form an important part of the diet for many Kenyans, and they are normally consumed after a lengthy cooking process leading to the inactivation of peroxidase (POD) that could be used as an indicator for the potential shelf life of the vegetables. However, color degradation can simultaneously occur, leading to poor consumer acceptance of the product. The kinetics of POD in situ thermal (for thermal treatments in the range of 75 to 100 °C/120 min) inactivation showed a biphasic first‐order model, with Arrhenius temperature dependence of the rate constant. The kinetic parameters using a reference temperature (T_ref) of 80 °C were determined for both the heat‐labile phase (k_ref = 11.52 ± 0.95 × 10^?2 min^?1 and E_a of 109.67 ± 6.20 kJ/mol) and the heat‐stable isoenzyme fraction (k_ref = 0.29 ± 0.07 × 10^?2 min^?1 and E_a of 256.93 ± 15.27 kJ/mol). Color degradation (L*, a*, and b* value) during thermal treatment was investigated, in particular as the “a*” value (the value of green color). Thermal degradation (thermal treatments between 55 and 80 °C per 90 min) of the green color of the leaves followed a fractional conversion model and the temperature dependence of the inactivation rate constant can be described using the Arrhenius law. The kinetic parameters using a reference temperature (T_refC = 70 °C) were determined as k_refC = 13.53 ± 0.01 × 10^?2 min^?1 and E_aC = 88.78 ± 3.21 kJ/mol. The results indicate that severe inactivation of POD (as an indicator for improved shelf life of the cooked vegetables) is accompanied by severe color degradation and that conventional cooking methods (typically 10 min/100 °C) lead to a high residual POD activity suggesting a limited shelf life of the cooked vegetables.