An improved and fast UHPLC-PDA methodology for determination of L-ascorbic and dehydroascorbic acids in fruits and vegetables: evaluation of degradation rate during storage

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultrahigh-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm×2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intraday precision. Validation experiments revealed very good recovery rate of 96.6±4.4% for L-AA and 103.1±4.8 % for total vitamin C, good linearity with r2-values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.

A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 μg mL−1 to 0.58 μg mL−1, and from 0.019 μg mL−1 to 1.94 μg mL−1, for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n = 9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.

Use of novel biomarkers (Homocysteine, vitamin B6, B9 and B12) on the assessing the progression of cardiovascular disease

A large number of evidences correlate elevated levels of homocysteine (Hcys) with a higher cardiovascular diseases (CVDs) risk, especially, atherosclerosis. Similarly, abnormal low levels of the vitamins B6, B9 and B12 are associated to an instability in the methionine cycle with an over production of Hcys. Thus, biomedical sciences are looking forward for a cheaper, faster, precise and accurate analytical methodology to quantify these compounds in a suitable format for the clinical environment. Therefore the objective of this study was the development of a simple, inexpensive and appropriate methodology to use at the clinical level. To achieve this goal, a procedure integrating a digitally controlled (eVol®) microextraction by packed sorbent (MEPS) and an ultra performance liquid chromatography (UPLC) coupled to a photodiode array detector (PDA) was developed to identify and quantify Hcys vitamins B6, B9 and B12. Although different conditions were assayed, we were not able to combine Hcys with the vitamins in the same analytical procedure, and so we proceeded to the optimization of two methods differing only in the composition of the gradient of the mobile phase and the injected volume. It was found that MEPS did not bring any benefit to the quantification of the Hcys in the plasma. Therefore, we developed and validate an alternative method that uses the direct injection of treated plasma (reduced and precipitated). This same method was evaluated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect and precision (intra-and inter-day) and applied to the determination of Hcys in a group composed by patients presenting augmented CVD risk. Good results in terms of selectivity and linearity (R2> 0.9968) were obtained, being the values of LOD and LOQ 0.007 and 0.21 mol / L, respectively. The intra-day precision (1.23-3.32%), inter-day precision (5.43-6.99%) and the recovery rate (82.5 to 93.1%) of this method were satisfactory. The matrix effect (>120%) was, however, higher than we were waiting for. Using this methodology it was possible to determine the amount of Hcys in real plasma samples from individuals presenting augmented CVD risk. Regarding the methodology developed for vitamins, despite the optimization of the extraction technique and the chromatographic conditions, it was found that the levels usually present in plasma are far below the sensitivity we obtained. Therefore, further optimizations of the methodology developed are needed. As conclusion, part of the objectives of this study was achieved with the development of a quick, simple and cheaper method for the quantification of Hcys.

Otimização evalidação de métodos analíticos para a determinação simultânea de tuberculostáticos (4-FDC) por CLAE/DAD e CLUE/ DAD

Tuberculosis is a serious disease, but curable in practically 100% of new cases, since complied the principles of modern chemotherapy. Isoniazid (ISN), Rifampicin (RIF), Pyrazinamide (PYR) and Chloride Ethambutol (ETA) are considered first line drugs in the treatment of tuberculosis, by combining the highest level of efficiency with acceptable degree of toxicity. Concerning USP 33 - NF28 (2010) the chromatography analysis to 3 of 4 drugs (ISN, PYR and RIF) last in average 15 minutes and 10 minutes more to obtain the 4th drug (ETA) using a column and mobile phase mixture different, becoming its industrial application unfavorable. Thus, many studies have being carried out to minimize this problem. An alternative would use the UFLC, which is based with the same principles of HPLC, however it uses stationary phases with particles smaller than 2 μm. Therefore, this study goals to develop and validate new analytical methods to determine simultaneously the drugs by HPLC/DAD and UFLC/DAD. For this, a analytical screening was carried out, which verified that is necessary a gradient of mobile phase system A (acetate buffer:methanol 94:6 v/v) and B (acetate buffer:acetonitrile 55:45 v/v). Furthermore, to the development and optimization of the method in HPLC and UFLC, with achievement of the values of system suitability into the criteria limits required for both techniques, the validations have began. Standard solutions and tablets test solutions were prepared and injected into HPLC and UFLC, containing 0.008 mg/mL ISN, 0.043 mg/mL PYR, 0.030 mg.mL-1 ETA and 0.016 mg/mL RIF. The validation of analytical methods for HPLC and UFLC was carried out with the determination of specificity/selectivity, analytical curve, linearity, precision, limits of detection and quantification, accuracy and robustness. The methods were adequate for determination of 4 drugs separately without interfered with the others. Precise, due to the fact of the methods demonstrated since with the days variation, besides the repeatability, the values were into the level required by the regular agency. Linear (R> 0,99), once the methods were capable to demonstrate results directly proportional to the concentration of the analyte sample, within of specified range. Accurate, once the methods were capable to present values of variation coefficient and recovery percentage into the required limits (98 to 102%). The methods showed LOD and LOQ very low showing the high sensitivity of the methods for the four drugs. The robustness of the methods were evaluate, facing the temperature and flow changes, where they showed robustness just with the preview conditions established of temperature and flow, abrupt changes may influence with the results of methods

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

LC Evaluation of In Vitro Release of AZT from Microemulsions

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Total polyphenols from Syzygium cumini (L.) Skeels fruit extract

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caffeine Determination at a Carbon Fiber Ultramicroelectrodes by Fast-Scan Cyclic Voltammetry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Novel Diagnostic Target in the Hepatitis C Virus Genome

BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (59-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.Methods and FindingsIn this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.ConclusionThis study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Levels of selected persistent organic pollutants in blood from delivering women in seven selected areas of São Paulo State, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens

The pharmacokinetics of enrofloxacin (ENRO), a fluoroquinolone antimicrobial agent, was studied in male broiler chickens (Cobb) after single oral administration of 10 mg of ENRO/kg b.w. A high-performance liquid chromatography-photodiode array detector (DAD) (HPLC-DAD) method was developed and validated and used for quantitation of ENRO and its major metabolite ciprofloxacin in plasma. The HPLC analyses were carried out using a cationic-octadecyl mixed column and 0.05 mol/L phosphate buffer (pH 2.5)/acetonitrile as mobile phase. The sample preparation of plasma consisted of the precipitation of proteins followed by solid phase extraction on cationic-octadecyl mixed cartridges. The method was validated considering linear range, linearity, selectivity, sensitivity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precisions and accuracy. The LOD and LOQ for both fluoroquinolones were 60 and 200 ng/mL for plasma. The plasma concentration vs. time graph was characteristic of a two-compartment open model. The maximal plasma concentration of 1.5 +/- 0.2 mg/mL was achieved at 9 +/- 2 h. The elimination half-life and the mean residence time of ENRO were 1.5 +/- 0.2 and 15.64 h, respectively. The area under the concentration-time curve was calculated as 35 +/- 4 mg(.)h/mL.

Determination of selenium by GFAAS in slurries of fish feces to estimate the bioavailability of this micronutrient in feed used in pisciculture

This paper presents a simple, fast and sensitive method to determine selenium in samples of feces and of fish feed by graphite furnace atomic absorption spectrometry (GFAAS) through the direct introduction of slurries of the samples into the spectrometer's graphite tube. The limits of detection (LOD) and quantification (LOQ calculated for 20 readings of the blank of the standard slurries (0.50% m/v of feces or feed devoid of selenium) were 0.31 mu g 1(-1) and 1.03 mu g 1(-1), respectively, for the standard feces slurries and 0.35 mu g 1(-1) and 1.16 mu g 1(-1), respectively, for the standard feed slurries. The proposed method was applied in studies of bioavailability of selenium in different fish feeds and the results proved consistent with that obtained from samples mineralized by acid digestion using the microwave oven. (C) 2007 Elsevier Ltd. All rights reserved.

Determination of chromium by GFAAS in slurries of fish feces to estimate the apparent digestibility of nutrients in feed used in pisciculture

This paper presents a simple, fast and sensitive method to determine chromic oxide (used as a biological marker of fish feed) in samples of fish feces by GFAAS through the direct introduction of slurries of the samples into the spectrometer's graphite tube. The standard samples of feces and of fish feed containing 0.10-1.00 mg kg(-1) of Cr2O3 were pre-frozen for I min in liquid nitrogen and then ground a cryogenic mill for 2 min, which reduced the samples' grain size to less than 60 mu m. The standard slurries were prepared by mixing 20 mg of standard samples of fish feed or feces with I mL of a solution containing 0.05% (v/v) of Triton X-100 and 0.50% (v/v) of suprapure HNO3 directly in the spectrometer's automatic sampling glass. The final concentrations of Cr2O3 present in the standard slurries were 2, 4, 8, 16 and 20 mu g L-1. After sonicating the mixture for 20s, 10 mu L of standard slurries were injected into the graphite tube, whose internal wall was lined with a metallic palladium film that acted as a permanent chemical modifier. The limits of detection (LOD) and quantification (LOQ) calculated for 20 readings of the blank of the standard slurries (2%, m/v of feces or feed devoid of minerals) were 0.81 and 2.70 mu g L-1 of Cr2O3 for the standard feces slurries, 0.84 and 2.83 mu g L-1 of Cr2O3 for the standard feed slurries. The proposed method was applied in studies of nutrient digestibility of different fish feeds and its results proved compatible with the results obtained from samples pre-mineralized by acid digestion. (c) 2005 Elsevier B.V. All rights reserved.

Tributyltin in Crustacean Tissues: Analytical Performance and Validation of Method

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mapping the BDA20 Gene to BTAX

The lipocalin family is a large group of proteins that exhibits great structural and functional variation both within and among species, including a significant number of animal-derived aeroallergens, such as the bovine BDA20 (major cow dander allergen). This protein is classified as an occupational allergen causing asthma and other work-related allergic disorders among dairy farmers. Using a somatic cell panel the BDA20 gene was assigned to the bovine X chromosome (BTAX) with a significant concordant value of 97% to the previously mapped reference marker MAF45. A radiation hybrid (RH) mapping approach confirmed the assignment of BDA20 to BTAX. Two-point LOD scores showed that BDA20 is linked to XBM451 with a LOD score of 22.1 for a 0 value of 0.03.