Mario Vega

Polyphenols and antioxidant activity of calafate ( Berberis microphylla ) fruits and other native berries from Southern Chile.

Calafate ( Berberis microphylla ) is a native berry grown in the Patagonian area of Chile and Argentina. In the present study the phenolic composition and antioxidant activity of its fruits were studied and also compared with data obtained for other berry fruits from southern Chile including maqui ( Aristotelia chilensis ) and murtilla ( Ugni molinae ). Polyphenolic compounds in calafate fruit were essentially present in glycosylated form, 3-glucoside conjugates being the most abundant anthocyanins. The anthocyanin content in calafate berries (17.81 +/- 0.98 micromol g(-1)) and flavonol level (0.16 +/- 0.01 micromol g(-1)) are comparable with those found in maqui (17.88 +/- 1.15 and 0.12 +/- 0.01 micromol g(-1), respectively); however, maqui shows lower flavan-3-ol concentration than calafate (0.11 +/- 0.01 and 0.24 +/- 0.03 micromol g(-1), respectively). Maqui and calafate show high antioxidant activity, which correlates highly with total polyphenol content and with anthocyanin concentration.

Alternatives for sample pre-treatment and HPLC determination of Ochratoxin A in red wine using fluorescence detection

Ochratoxin A is a mycotoxin widely studied due to its nephrotoxic, immunotoxic, teratogenic and carcinogenic effects. The European Commission has fixed maximum limits for Ochratoxin A in wines and in other foods. In order to determine Ochratoxin A levels in red wine, the present paper contrasts and discusses the results of a systematic study of analytical parameters for sample pre-treatment using different immunoaffinity cartridges as well as C-18 cartridges with three solvent combinations. The direct injection of wine into two types of C-18 chromatographic columns (conventional packed column and monolithic column) is evaluated as screening method. In all cases, the analysis was carried out using HPLC with fluorescence detection. The results show statistical differences when 3 types of immunoaffinity columns were used, while higher recoveries were obtained for C-18 cartridges using acetonitrile as extraction solvent. Repeatability and accuracy of immunoaffinity and C-18 sample pre-treatment were statistically comparable (alpha=0.05). Their sensitivity was also comparable, although more favorable detection limits were obtained using the immunoaffinity treatment (0.01 microg L(-1)) in comparison with C-18 treatment (0.09 microg L(-1)). Considering the maximal allowed concentration of Ochratoxin A in wine (2.00 microg L(-1)), both methods are suitable for its determination in wine. Both methods were applied to determine this toxin in 154 wine samples, and the quantitative results demonstrated statistic comparability (alpha=0.05). These results were also confirmed from the qualitative point of view using a GC-MS method. To find an easy screening method, based on a recent publication, a monolithic HPLC column and 2 conventional packed columns were tested for Ochratoxin A determination in real wine samples by direct injection, without previous clean-up. The results show that this procedure is not useful at the concentration levels usually found in wine and although shorter time is required when using the monolithic columns even with the chromatographic analysis. Finally, based on the results, it was concluded that the combination of C-18 cartridges with conventional particle packed columns and HPLC-FLD is the most appropriate alternative for Ochratoxin A analysis in wine. Indeed, considering cost, sensitivity and selectivity, this method can be used in broad prospective programs.

Ochratoxigenic Aspergillus species on grapes from Chilean vineyards and Aspergillus threshold levels on grapes.

This study reports the incidence of ochratoxigenic strains of Aspergillus on Chilean grapes (Vitis vinifera) and wineries, and production of OTA levels in wines with grapes having different levels of contamination with OTA-producing Aspergillus carbonarius was studied. A. carbonarius, A. niger, A. niveus, A. paradoxus, A. versicolor, A. wentii, and A. westerdijkiae were identified on apparently healthy clusters of red and white grape cultivars. However, A. carbonarius and A. niger were the most frequently identified species, more abundant on red than white grape cultivars. Aspergillus spp. populations increased between veraison and harvest, but the isolation frequencies were relatively low over the entire growing season. At the winery, A. carbonarius, A. niger and A. westerdijkiae were occasionally found in the air, exclusively during winemaking. OTA-producing strains were only found among isolates of A. carbonarius, A. niger, A. wenti, and A. westerdijkiae, producing 2 to 17 microg/L of OTA in liquid medium; however, A. westerdijkiae produced the highest OTA concentration in vitro. Red wines elaborated with 0.5% of grapes infected with an OTA-producing strain of A. carbonarius (Aspuc-SB36) exceeded the 2 microg/L of OTA tolerance established for wines by the European Community. Therefore, a threshold below 0.5% infected berries is proposed for red wines. ELISA tests proved to be useful for detecting OTA in broth culture as in wine samples.

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.

Instrumental planar chromatographic method for determination of carbamazepine in human serum

An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid-liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/microL showed correlation coefficient of 0.998. The intra-assay and inter-assay precision, expressed as the RSD, were in the range of 0.41-1.24% (n = 3) and 2.17-3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum.

High performance thin layer chromatography determination of cellobiosan and levoglucosan in bio-oil obtained by fast pyrolysis of sawdust

In this work, high performance thin layer liquid chromatography (HTPLC) is applied to the determination of sugars in fast pyrolysis liquids (bio-oil) and fractions thereof. The proposed procedure allows the separation of anhydrosugar levoglucosan and cellobiosan, as well as glucose, arabinose, xylose and cellobiose. Pre-treatment and derivatization of samples are not necessary and volatile compounds present in bio-oil do not interfere with sugar analysis. The detrimental effect of the complex bio-oil matrix on columns and detector lifetime is avoided by using disposable HTPLC plates. Prior screening of glucose, present especially in aged and aqueous bio-oil fractions, is required to quantify cellobiosan without interference. Concentrations of levoglucosan and cellobiosan in bio-oil samples obtained from Pinus radiata sawdust were ranged between 1.27-2.26% and 0.98-1.96% respectively, while a bio-oil sample obtained from native wood contained a higher levoglucosan concentration.

Routine method for quantification of starch by planar chromatography (HPTLC)

Starch is widely distributed in diverse plant organs as a reserve carbohydrate; it is also a major source of carbohydrates in human food. Because of its importance, different methods have been developed to measure the starch content of food and feed. Planar chromatography has been used to measure starch content of cereal products. The starch was hydrolyzed using α-amylase and amyloglucosidase and the resulting glucose was separated on silica gel 60 HPTLC plates and quantified at λ = 520 nm after derivatization. The glucose calibration plot was linear between 100 and 300 ng per spot with a coefficient of determination (r2) of 0.9959. The limits of detection (LOD) and quantification (LOQ) for starch as glucose were 0.26 and 0.51 (g per 100 g), respectively. The mean concentrations of starch in wheat flour and in starch premix (an industrial product) were 74.56 ± 2.58% and 84.85 ± 1.96%, respectively. The proposed method was shown to be a precise, selective, and sensitive means of measuring starch in cereal products.

Sugars and enzyme activity in the grass Deschampsia antarctica

Deschampsia antarctica is a freezing-tolerant plant and the only native Poaceae that grows in the Maritime Antarctic. During the long days of the growing season this plant accumulates sucrose (Suc) in the leaves to 36% of the dry weight. The mechanism that leads to this high accumulation is unknown. The effect of day length and low temperature on sucrose phosphate synthase (SPS) (EC: 2.4.1.14) activity and sugar accumulation was studied in D. antarctica and compared with other Poaceae. Three different day lengths: short (SD) (8/16 h), medium (MD) (16/8 h) and long (LD) (21/3 h); and two temperatures: 4°C (cold-acclimated) and 15°C (non-acclimated) were tested. The highest contents in total soluble sugars (TSS) and Suc were reached in crowns and leaves, respectively, in cold-acclimated plants under LD. TSS and Suc contents and SPS activity with cold acclimation were higher in D. antarctica than in other agricultural (wheat, oat and barley) and non-agricultural (D. caespitosa and D. beringensis) Poaceae species. Suc/TSS ratio was higher in all Deschampsia species than in agricultural Poaceae species. SPS activity and sucrose content in leaves were positively correlated only in LD cold acclimated plants. This result shows that SPS activity is responsive to day length in D. antarctica.

Monitoring the Dose of Florfenicol in Medicated Salmon Feed by Planar Chromatography (HPTLC)

Florfenicol is a synthetic broad-spectrum antibacterial compound used in aquaculture for prevention and treatment of fish diseases. The main mode of administration used in aquaculture is through the fish feed, which makes it very important to control the concentration of the drug in the feed, to ensure application of an appropriate dose to the fish. The objective of this work was to develop a highthroughput planar chromatographic method for monitoring the dose of florfenicol in fish feed. Florfenicol was extracted with methanol and chromatography was performed on silica gel F254 HPTLC plates with ethyl acetate–n-hexane, 80 + 20 (v/v) as mobile phase. Quantitative analysis was performed by densitometry in absorbance mode at λ = 223 nm. The calibration plot was linear in the range between 20 and 80 ng per spot with a coefficient of determination (r2) of 0.9987. The limits of detection and quantification were 2.55 and 8.50 mg kg–1, respectively. Recovery at 50, 500, and 1500 mg kg–1 was 101.71 ± 2.34, 85.18 ± 2.32, and 81.91 ± 2.92 respectively. The proposed method is sensitive, accurate, precise and suitable for monitoring florfenicol dose in medicated salmon fish feed.

Validated instrumental planar chromatographic method for quantification of fluphenazine hydrochloride in injections

A simple, rapid, precise, sensitive, specific and accurate instrumental planar chromatographic method for quantification of fluphenazine hydrochloride in injections has been developed and validated. Chromatographic separation was on precoated silica gel F254 HPTLC plates. The mobile phase was methanol-purified water 9:1 (v/v). Densitometric analysis was performed at 306 nm. The calibration plots were linear in the range 100 to 500 ng μL−1 with a correlation coefficient of 0.998. The limits of detection (LOD) and quantification (LOQ) were 1.45 and 4.40 ng, respectively. Intra-assay and inter-assay precision, expressed as relative standard deviation (RSD), were in the range 0.73–1.77% (n = 3) and 1.18–1.86% (n = 9), respectively. Recovery of fluphenazine hydrochloride was between 98.29 and 101.53%, with RSD no higher than 1.87%. The method was selective for fluphenazine hydrochloride and the preservatives in the injections. Drug content was within the prescribed limits (95–110% of the labeled content of the formulations) when the method was used to quantify fluphenazine hydrochloride in real pharmaceutical samples. Because the method is sensitive, precise, accurate, and selective for the compound tested, it can be used for routine quality control testing of fluphenazine hydrochloride in injections.