A high throughput method for detection of cyclooxygenase-2 enzyme inhibitors by effect-directed analysis applying high performance thin layer chromatography-bioassay-mass spectrometry.

A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N',N'-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-l-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.

Impact of molecular weight and deacetylation degree of chitosan on the bioaccessibility of quercetin encapsulated in alginate/chitosan-coated zein nanoparticles.

This work aimed at studying the effect of molecular weight (MW) and deacetylation degree (DD) of chitosan on the quercetin bioaccessibility encapsulated in alginate/chitosan-coated zein nanoparticles (alg/chiZN). The chitosan coating layer produced nanoparticulate systems with good stability parameters, high encapsulation efficiency (EE) and a higher bioaccessibilty of quercetin after in-vitro digestion. By increasing the DD of chitosan, the ζ-potential of the colloidal system significantly increased (≥27.1 mV), while low and very low MW chitosans generated systems with smaller particle sizes (≤ 277.8 nm) and polydispersity index [PDI (0.189)]. The best results, in terms of EE (≥84.44) and bioaccessibility (≥76.70), were obtained when the systems were prepared with low MW chitosan and high DD. Thus, the alg/chiZN nanocapsules may be a promising delivery system for improving the quercetin bioaccessibility or other compounds with a similar chemical nature, especially when higher DD and lower MWs are used.

Chemometric optimization of trypsin digestion method applying infrared, microwave and ultrasound energies for determination of caseins and ovalbumin in wines.

Caseins and ovalbumin are frequently used as wine fining agents to remove undesirable compounds like polymeric phenols. Their presence in wines is a subject of concern because may cause adverse effects on susceptible consumers, especially when their presence is not labeled. A key step for its determination is trypsin digestion, which is considered the bottleneck of bottom-up approach workflow because usually requires several hours. To reduce this time, the objective of this work was to carry out a chemometric optimization of trypsin digestion method applying infrared, microwave and ultrasound energies to determine caseins and ovalbumin in wines. The conditions of each accelerated digestion method were optimized using a Response Surface Methodology based on central composite design. The parameters optimized were digestion time and trypsin: protein ratio. The response variable evaluated was digestion yield, which was determined through the peak area of each protein transition determined by liquid chromatography-mass spectrometry. The most effective technique was microwave followed by ultrasound and infrared. Since optimal values of microwave and ultrasound-assisted digestion were the same, the later was chosen considering sample preparation and cost. Applying the proposed approach, a reduction of ca. 140 and 240-fold on digestion time was achieved compared with optimized and non-optimized conventional methods, respectively. With this workflow, both proteins were digested in a single 3 min process allowing its detection by liquid chromatography-mass spectrometry at µg L-1 level, which is ca. 60 times lower than the current limit of 0.25 mg L-1.

Chemometric optimisation of pressurised liquid extraction for the determination of alliin and S-allyl-cysteine in giant garlic (Allium ampeloprasum L.) by liquid chromatography tandem mass spectrometry.

INTRODUCTION: Giant garlic is a functional food that contains different kinds of bioactive molecules with beneficial effects on chronic noncommunicable diseases like diabetes and cardiovascular conditions. Considering biosynthesis pathways, abundance, and biological activity, alliin and S-allyl-cysteine were used as chemical markers of organosulphur compounds present in giant garlic. OBJECTIVE: To establish a chemometric optimisation of pressurised liquid extraction for the determination of alliin and S-allyl-cysteine in giant garlic by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODOLOGY: Samples were blanched (ca. 90°C for 10 min) to inactivate alliinase and γ-glutamyl transpeptidase enzymes and then freeze-dried. Chemometric optimisation was performed via response surface methodology based on central composite design (CCD). Organosulphur compound yields were determined applying a validated LC-MS/MS method in multiple reaction monitoring (MRM) mode using the following transitions: for alliin m/z 178 → 74 and for S-allyl-cysteine m/z 162 → 41. RESULTS: According to CCD results, under constant conditions of pressure (1500 psi) and time (20 min), the optimal conditions for pressurised liquid extraction of alliin and S-allyl-cysteine were 70.75 and 68.97% v/v of ethanol in extraction solvent and 76.45 and 98.88°C as extraction temperature, respectively. Multiple response optimisation for the simultaneous extraction of both organosulphur compounds was established via desirability function. Under these conditions, 2.70 ± 0.27 mg g-1 dry weight (DW) of alliin and 2.79 ± 0.22 mg g-1 DW of S-allyl-cysteine were extracted. CONCLUSIONS: These results clearly demonstrated that pressurised liquid extraction is an efficient green technique to extract bioactive organosulphur compounds from giant garlic. Extraction yields were significantly (p < 0.05) higher than those obtained with conventional ultra-turrax extraction.

Occurrence of allergen proteins in wines from Chilean market.

Casein and ovalbumin are proteins commonly used as wine fining agents that may trigger allergic reactions in susceptible individuals. Therefore, their occurrence in wines could become a health risk, moreover when their presence is not reported. The objective of this work was to determine the occurrence of casein and ovalbumin in Chilean wines. Proteins were extracted by ultrafiltration and digested applying an optimised ultrasound-assisted method. Peptides were quantified by validated tandem mass spectrometry method using stable isotope dilution analysis. Optimal digestion was achieved in 3 minutes at a 1:10 enzyme protein ratio. The method was validated following ICH guidelines showing determination coefficients R2 ≥ 0.99, repeatability, and intermediate precision with RSD values <1.95% and recoveries from 89.8% to 115.1% (RSD < 5.84%). The method was applied to analyse 60 wine samples. Fifty-six samples showed quantifiable levels, from which 28% presented a total casein and ovalbumin concentration equal or higher to the European limit (0.25 mg L-1).

Fast and selective method for biogenic amines determination in wines and beers by ultra high-performance liquid chromatography.

A new approach for biogenic amines (BA) determination in fermented beverages was developed coupling ultrafast dansylation conditions with ultra-high-performance liquid chromatography (UHPLC) method. Derivatization conditions for 8 amines analysis were optimized using D-optimal and central composite designs reducing in 88% the reaction time. Dansylated amines were separated on C18 column using a mobile phase composed of ammonium formate buffer and acetonitrile. Detection was performed by UV and fluorescence. Migration from conventional HPLC to UHPLC allowed a 60% reduction in analysis. The developed method was validated following ICH recommendations. Calibration data fitted a linear regression model with R2 were values highest to 0.984. Repeatability and intermediate precision in matrix showed relative standard deviation (RSD) values lower than 4.86 and 11.53%, respectively. Detection and quantification limits ranged from 0.03 to 0.18 and from 0.20 to 0.59 mg mg L-1. BA content in beer and wine samples were 10.53 to 73.17 mg L-1.

An improved method for a fast screening of α-glucosidase inhibitors in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin-layer chromatography-bioassay-mass spectrometry.

α-glucosidase inhibitors (AGIs) are very attractive bioactive compounds due to their therapeutic profile that includes beneficial effects over glycemic control in type 2 diabetes mellitus and viral infections. Its detection and identification in plants and fruits has gained growing attention, and certainly requires efficient screening methodologies. The objective of the present work was to develop a fast methodology to detect and identify AGIs in cherimoya fruit (Annona cherimola Mill.) applying effect-directed analysis via high-performance thin layer-chromatography (HPTLC) linked with bioassay and mass spectrometry (MS). Both, HPTLC and bioassay conditions, were optimized accomplishing 50% and 83% reduction on enzyme concentration and incubation time respectively, compared to the original method. Additionally, the contrast between inhibitory bands and purple background was also enhanced by enzyme substrate impregnation on HPTLC plate. The optimized detection conditions established were the following: 5.0 U mL-1 of enzyme solution, 1.0 mg mL-1 of 2-naphthyl-α-D-glucopyranoside substrate, 1.0 mg mL-1 of Fast Blue B salt solution and 10 min as incubation time. Applying this methodology, coupled to HPTLC-MS and ultra-high-performance liquid chromatography (UHPLC)-diode array detector (DAD)-MS/MS, it was possible for the first time to detect and identify three AGIs in cherimoya peel and seeds. Compounds were tentatively assigned as phenolamides (phenylethyl cinnamides): N-trans-feruloyl tyramine (m/z 314 [M+H]+; UV λmax 293 and 316 nm), N-trans-p-coumaroyl tyramine (m/z 284 [M+H]+; UV λmax 296 nm) and N-trans-feruloyl phenethylamine (m/z 298 [M+H]+; UV λmax 288 nm). To the best of our knowledge, the presence of latter compound is reported for the first time in cherimoya.

Detection and identification of acetylcholinesterase inhibitors in Annona cherimola Mill. by effect-directed analysis using thin-layer chromatography-bioassay-mass spectrometry.

INTRODUCTION: Acetylcholinesterase (AChE) inhibitors are considered an important strategy in the treatment of neurological disorders such as Alzheimer's disease. A simple and fast planar chromatography-bioassay methodology has been established to detect bioactive molecules in cherimoya fruit. OBJECTIVE: Detect and identify AChE inhibitors in cherimoya by high-performance thin-layer chromatography (HPTLC)-bioassay-mass spectrometry (MS) and related techniques. METHODOLOGY: Effect-directed analysis by planar chromatography-bioassay-mass spectrometry was applied to detect and identify AChE inhibitors in pulp, peel and cherimoya seed. Bioassay was optimised establishing the following conditions: enzymatic solution (1.0 U mL-1 ), 1-naphtyl acetate substrate (1.5 mg mL-1 ) and Fast Blue B salt (1.0 mg mL-1 ). TLC-MS interface was used to directly elute the active zones into a mass spectrometer or to a micro-vial for further off-line studies. RESULTS: Two AChE inhibitory bands were detected in peel extracts. An analysis via HPTLC-MS and high-performance liquid chromatography diode array detector tandem mass spectrometry (HPLC-DAD-MS/MS) allowed to characterise three potential AChE inhibitors: anonaine (m/z 266 [M + H]+ ; UV λmax = 269.6 nm), glaucine (m/z 256 [M + H]+ ; UV λmax = 282.9 and 300.6 nm) and xylopine (m/z 296 [M + H]+ ; UV λmax = 278.5 nm). CONCLUSIONS: The application of this optimised high throughput method allowed to establish the presence of three potential AChE inhibitors in cherimoya peel. For the first time AChE inhibitory capacity of these alkaloids is reported.

Evaluation of biogenic amines content in chilean reserve varietal wines.

Biogenic amines play important roles in many physiological functions, but when they are ingested in high concentrations may produce severe adverse effects. The aim of this research was to evaluate the biogenic amine content in Chilean reserve varietal wines. A high performance liquid chromatography method was optimized and validated to quantify histamine, tyramine, spermine, spermidine, putrescine, cadaverine and phenylethylamine in Chilean wines. Derivatization and chromatographic conditions were optimized using a central composite design. Sixty reserve wines of the most important Chilean grape varieties were analyzed, i.e. Cabernet Sauvignon (n=11), Merlot (n=11), Carménère (n=11), Syrah (n=10) and Sauvignon Blanc (n=10), as well as organic wines (n=7). Biogenic amines content ranged from 2.19 to 65.09 mg L(-1), no significant difference (P>0.05) was observed between Cabernet Sauvignon, Merlot and Carménère but all showed statistically higher (P<0.05) concentrations than Sauvignon Blanc. Syrah wines showed no difference (P>0.05) with Cabernet Sauvignon, higher concentrations (P<0.05) than Sauvignon Blanc and lower than Merlot and Carménère. Regarding biogenic amines profile, putrescine showed the highest concentration in all grape varieties.