Fernando G. Tonin

Simple method for determination of cocaine and main metabolites in urine by CE coupled to MS

In this work, a simple method for the simultaneous determination of cocaine (COC) and five COC metabolites (benzoylecgonine, cocaethylene (CET), anhydroecgonine, anhydroecgonine methyl ester and ecgonine methyl ester) in human urine using CE coupled to MS via electrospray ionization (CE‐ESI‐MS) was developed and validated. Formic acid at 1 mol/L concentration was used as electrolyte whereas formic acid at 0.05 mol/L concentration in 1:1 methanol:water composed the coaxial sheath liquid at the ESI nozzle. The developed method presented good linearity in the dynamic range from 250 ng/mL to 5000 ng/mL (coefficient of determination greater than 0.98 for all compounds). LODs (signal‐to‐noise ratio of 3) were 100 ng/mL for COC and CET and 250 ng/mL for the other studied metabolites whereas LOQs (signal‐to‐noise ratio of 10) were 250 ng/mL for COC and CET and 500 ng/mL for all other compounds. Intra‐day precision and recovery tests estimated at three different concentration levels (500, 1500 and 5000 ng/mL) provided RSD lower than 10% (except anhydroecgonine, 18% RSD) and recoveries from 83–109% for all analytes. The method was successfully applied to real cases. For the positive urine samples, the presence of COC and its metabolites was further confirmed by MS/MS experiments.

HPLC-DAD-ESI/MS identification and quantification of phenolic compounds in Ilex paraguariensis beverages and on-line evaluation of individual antioxidant activity.

“Chimarrão” and “tererê” are maté (dried, toasted and milled Ilex paraguariensis leaves and stemlets) beverages widely consumed in South America. This paper describes the application of HPLC-DAD-ESI/MS method for the identification and quantification of caffeoylquinic acids (CQA), flavonol glycosides and purine alkaloids in these beverages. The beverage samples were prepared from commercial lots of maté from Southern Brazil. The caffeoylquinic acids, 4,5-diCQA, 3-CQA, 5-CQA, and 4-CQA were the major compounds, having 238–289, 153–242, 183–263, and 123–188 μg/mL, respectively, for chimarrão and 206–265, 122–218, 164–209, 103–169 μg/mL, respectively, for tererê. Caffeine also had high amounts while glycosides of quercetin and kaempferol were found at much lower levels. The individual antioxidant activity was also determined by an on-line system that measured their ABTS•+ radical scavenging activity, showing that the antioxidant capacity was not proportional to the concentrations of the phenolic compounds. 3-CQA, quercetina-3-O-ramnosylglucoside, and quercetina-3-O-glucoside were the major contributors to the antioxidant capacity, although the quercetin glycosides had concentrations less than 10 times that of 3-CQA.

Optimization of a method for determination of phenolic acids in exotic fruits by capillary electrophoresis

In this work, the separation of nine phenolic acids (benzoic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, protocatechuic, syringic, and vanillic acid) was approached by a 3(2) factorial design in electrolytes consisting of sodium tetraborate buffer (STB) in the concentration range of 10-50 mmol L(-1) and methanol in the volume percentage of 5-20%. Derringers desirability functions combined globally were tested as response functions. An optimal electrolyte composed by 50 mmol L(-1) tetraborate buffer at pH 9.2, and 7.5% (v/v) methanol allowed baseline resolution of all phenolic acids under investigation in less than 15 min. In order to promote sample clean up, to preconcentrate the phenolic fraction and to release esterified phenolic acids from the fruit matrix, elaborate liquid-liquid extraction procedures followed by alkaline hydrolysis were performed. The proposed methodology was fully validated (linearity from 10.0 to 100 microg mL(-1), R(2)>0.999; LOD and LOQ from 1.32 to 3.80 microg mL(-1) and from 4.01 to 11.5 microg mL(-1), respectively; intra-day precision better than 2.8% CV for migration time and 5.4% CV for peak area; inter-day precision better than 4.8% CV for migration time and 4.8-11% CV for peak area; recoveries from 81% to 115%) and applied successfully to the evaluation of phenolic contents of abiu-roxo (Chrysophyllum caimito), wild mulberry growing in Brazil (Morus nigra L.) and tree tomato (Cyphomandra betacea). Values in the range of 1.50-47.3 microg g(-1) were found, with smaller amounts occurring as free phenolic acids.

Determination of catechins in green tea infusions by reduced flow micellar electrokinetic chromatography

A sulfated-β-cyclodextrin (s-β-CD) modified reduced flow micellar electrokinetic chromatography (RF-MEKC) method was developed and validated for the determination of catechins in green tea. The optimal electrolyte consisted of 0.2% triethylamine, 50mmol/L SDS and 0.8% s-β-CD (pH=2.9), allowing baseline separation of five catechins in 4min. The samples and standards were injected at 0.6psi for 5s under constant voltage of -30kV. Sample preparation simply involved extraction of 2g of tea with 200mL water at 95°C under constant stirring for 5min. The method demonstrated excellent performance, with limits of detection (LOD) and quantification (LOQ) of 0.02-0.1 and 0.1-0.5μg/mL, respectively, and recovery percentages of 94-101%. The method was applied to six samples of Brazilian green tea infusions. Epigallocatechin gallate (23.4-112.4μg/mL) was the major component, followed by epigallocatechin (18.4-78.9μg/mL), epicatechin gallate (5.6-29.6μg/mL), epicatechin (4.6-14.5μg/mL) and catechin (3.2-8.2μg/mL).

Simultaneous analysis of aspartame, cyclamate, saccharin and acesulfame-K by CZE under UV detection

An alternative methodology for simultaneous analysis of aspartame (ASP), cyclamate (CYC), saccharin (SAC) and acesulfame-K (ACE) by capillary zone electrophoresis was developed and validated. Optimum separation conditions were achieved by evaluation of the effective mobility curve followed by background electrolyte (BGE) optimization through a full 32 factorial design. The optimized electrolyte composed of 20.0 mmol L−1 sodium tetraborate, 15.0 mmol L−1 Tris and 7.5 mmol L−1 benzoic acid (pH 9.15) was suitable for simultaneous direct (ASP, ACE and SAC) and indirect (CYC) UV detection at 215 nm. Method performance was evaluated for linearity (r > 0.992), precision (RSD%: <4.0%, ASP; <3.0%, CYC; <4.5%, SAC and <4.5%, ACE), accuracy (mean recovery range: 101.2%, ASP; 102.2%, CYC; 91.9%, SAC and 94.4%, ACE), detection limit (expressed in mg L−1: 6.80, ASP; 12.0, CYC; 0.50, SAC and 3.30, ACE) and quantification limit (expressed in mg L−1: 22.0, ASP; 40.0, CYC; 1.60, SAC and 10.0, ACE). Method applicability was demonstrated by analysis of lemon tea sachet samples containing ASP, CYC, SAC and ACE.

Applications of capillary electrophoresis to the analysis of compounds of clinical, forensic, cosmetological, environmental, nutritional and pharmaceutical importance

Since its inception in the 80s, capillary electrophoresis has matured into a well-established separation technique, actually encompassing a family of electrodriven techniques with distinct separation mechanisms and selectivity, performed in a single capillary column. In this work, the versatility of capillary electrophoresis in handling materials from a diversity of chemical classes and complex sample matrices is illustrated by representative applications in the clinical, forensic, cosmetological, environmental, nutritional and pharmaceutical areas, grouping together our own research interests and results.

Determination of Urinary Biomarkers for Assessment of Short-Term Human Exposure to Aflatoxins in São Paulo, Brazil

In the present study, a longitudinal assessment was carried out to evaluate the short-term human exposure to aflatoxins in Pirassununga region, São Paulo, Brazil, by determination of urinary aflatoxins by a liquid chromatography coupled to mass sprectrometry (UPLC-MS/MS) method. Sixteen volunteers with ages ranging from 14 to 55 years old were instructed to collect the early morning first urine four times every three months, from June 2011 to March 2012, totaling 64 samples. Aflatoxin M1 (AFM1) was found in 39 samples (61%) at levels ranging from 0.19 to 12.7 pg·mg−1 creatinine (mean: 1.2 ± 2.0 pg·mg−1 creatinine). Residues of aflatoxins B1, B2, G1, G2 and aflatoxicol were not identified in any urine sample. No significant difference was found among the AFM1 mean levels in urine samples collected in the four sampling periods. The levels of AFM1 found in urine samples indicate a low short-term exposure of the population studied to aflatoxins through the diet, although further investigations are needed to assess other long-term biomarkers of exposure to AFB1.

Assessment of aflatoxin exposure using serum and urinary biomarkers in Sao Paulo, Brazil: A pilot study.

The aim of this study was to evaluate the human exposure of individuals from Pirassununga, Brazil, to dietary aflatoxins B1 (AFB1) and M1 (AFM1) by determination of serum AFB1-lysine and urinary aflatoxin biomarkers (AFM1 and AFB1-N(7)-guanine). The participants were recruited among employees from a Campus of the University of São Paulo, which provided food samples from their homes, as well as serum and urine samples four times every three months, from June 2011 until March 2012. The probable daily intake (PDI) of aflatoxin was estimated by using the results from analysis of food products collected by the time of samples collection, and data from a 24-hour dietary recall questionnaire. Analyses of AFB1 and AFM1 in food samples were conducted by high-performance liquid chromatography with fluorescence detection. Biomarkers in serum and urine were determined by tandem mass spectrometry. AFB1 and AFM1 were detected in 38 samples of cereals (28%, N=136) and 31 milk products (36%, N=86), respectively. AFB1-lysine and AFB1-N(7)-guanine and were not detected in serum or urine samples, respectively. However, AFM1 was found in 74 urine samples (65%), at mean levels in the 4 sampling times ranging from 0.37±0.23 to 1.70±2.88pg/mg creatinine. The mean PDI varied among different sampling times, ranging from 0.09±0.09 to 1.35±5.98ng/kg body weight/day. A modest though significant correlation (r=0.45; p=0.03; N=23) was found for the first time in Brazil between the AFM1 concentration in urine and the PDI for total aflatoxins (AFB1+AFM1) in sampling 1 (June 2011). Urinary AFM1 was confirmed as very sensitive for monitoring the human exposure to dietary aflatoxin. Further studies using serum and urinary biomarkers are needed to estimate the aflatoxin exposure of populations in higher risk areas in Brazil.

Synthesis and purification of the aflatoxin B1-lysine adduct

Abstract This work reports the chemical synthesis of aflatoxin B1 (AFB1)-lysine based on procedures available in the literature, but using lysine without a protection group in the α-amine group. AFB1-exo-8,9-epoxide was obtained by epoxidation of AFB1 with chloroperoxybenzoic acid in dichloromethane and phosphate buffer. Purification and identification of the AFB1-lysine were conducted by liquid chromatography (LC), and its structure was confirmed by LC with mass spectrometer and diode-array detection. The preparation of AFB1-lysine using lysine without a protection group in the α-amine group was completed in 24 h, being a practical modification of available methods that can be reproduced in analytical laboratories.

The Antioxidant Capacity of Rosemary and Green Tea Extracts to Replace the Carcinogenic Antioxidant (BHA) in Chicken Burgers

The present study aimed to evaluate the effect of natural extracts (rosemary and green tea extracts) in frozen storage of chicken burgers. Chicken burger treatments were prepared as follows: control (CON), 20 mg BHA/kg (BHA20), 10 mg green tea extract/kg (GT10), 38 mg green tea extract/kg (GT38), 18.6 mg rosemary extract/kg (RO18), and 480 mg rosemary extract/kg (RO480). Analysis of physicochemical parameters, color, TBAR index, and sensory acceptance were performed at 0, 30, 60, and 120 days of storage at −18°C in burgers packaged in LDPE plastic bags. The addition of natural antioxidants did not affect ( ) the color and physicochemical parameters of the chicken burgers. After 120 days at −18°C, the RO480 sample showed a TBAR index similar ( ) to BHA20 (0.423 and 0.369 mg, resp.). Sensory acceptance did not differ ( ) among the treatments throughout the storage period ( ).