Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC₅₀) values of the SPR assay were 28.8 and 14.9 ng mL⁻¹ for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg⁻¹ for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg⁻¹ for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.     

Determination of multi-class pesticides in wines by solid-phase extraction and liquid chromatography-tandem mass spectrometry

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of forty-six pesticides and transformation products belonging to different chemical classes in wines. The proposed method makes use of a solid-phase extraction (SPE) procedure with Oasis HLB cartridges that combines isolation of the pesticides and sample clean-up in a single step. Analysis is performed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-MS/MS) operated in the selected reaction monitoring (SRM) mode, acquiring two specific precursor-product ion transitions per target compound. An investigation of matrix effects has been performed during method validation showing medium to low effects for the majority of the compounds. Limits of detection (LODs) were in the range 0.0003–0.003 mg L⁻¹ and limits of quantification (LOQs) were in the range 0.001–0.01 mg L⁻¹. The average recoveries, measured at two concentration levels (0.010 and 0.050 mg L⁻¹), were in the range 70–110% for most of the compounds tested with % relative standard deviations below 20%, while a value of 0.010 mg L⁻¹ has been established as the method limit of quantification (MLOQ) for all target species. Expanded uncertainty values were in the range 10–40% while the Horrat ratios were below 1. The method has been successfully applied to the analysis of 60 wine samples in the course of an annual monitoring study with carbendazim-benomyl, thiophanate-methyl and carbaryl being the most frequently determined pesticides.     

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay

In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC₅₀) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL⁻¹ and 19.97 ± 0.84 ng mL⁻¹, respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of “nanobody (N-28) - anti-DON MAb” complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 – Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.     

Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG).The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05-100 (r > 0.999) mg L⁻¹ and 4-4000 mg L⁻¹ (r > 0.999), respectively. Limits of Detection were 0.014 mg L⁻¹ for MPA and 1.85 mg L⁻¹ for MPAG. Lower Limits of Quantification were 0.05 mg L⁻¹ for MPA and 2.30 mg L⁻¹ for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1-129.7%) for MPA and 111.1% (range 73.0-139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS = 1.14 UPLC-MS/MS—0.14 [mg L⁻¹], r = 0.96, and HPLC-MS/MS = 0.77 UPLC-MS/MS + 0.50 [mg L⁻¹], r = 0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids.     

Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully ¹³C isotope labeled deoxynivalenol [(¹³C₁₅)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (¹³C₁₅)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29±6% (n=7) for wheat and 37±5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95±3% (wheat) and 99±3% (maize) when (¹³C₁₅)DON was used as an IS for data evaluation.     

Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges

A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 μg kg⁻¹ were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.     

Analysis of potassium formate in airport storm water runoff by headspace solid-phase microextraction and gas chromatography–mass spectrometry

Potassium formate was extracted from airport storm water runoff by headspace solid-phase microextraction (HS-SPME) and analyzed by GC–MS. Formate was transformed to formic acid by adding phosphoric acid. Subsequently, formic acid was derivatized to methyl formate by adding methanol. Using sodium [²H]formate (formate-d) as an internal standard, the relative standard deviation of the peak area ratio of formate (m/z 60) and formate-d (m/z 61) was 0.6% at a concentration of 208.5 mg L⁻¹. Calibration was linear in the range of 0.5–208.5 mg L⁻¹. The detection limit calculated considering the blank value was 0.176 mg L⁻¹. The mean concentration of potassium formate in airport storm water runoff collected after surface de-icing operations was 86.9 mg L⁻¹ (n = 11) with concentrations ranging from 15.1 mg L⁻¹ to 228.6 mg L⁻¹.     

Enzymatic biosensor of horseradish peroxidase immobilized on Au-Pt nanotube/Au-graphene for the simultaneous determination of antioxidants

A new electrochemical method has been proposed for the simultaneous determination of butylated hydroxyanisole (BHA) and propyl gallate (PG) in food matrices based on enzymatic biosensors. Spiny Au-Pt nanotubes (SAP NTs) was first synthesized and demonstrated to exhibit intrinsic peroxidase and catalase-like activity. The structure of SAP NTs provides large surface area and favorable medium for electron transfer, on which HRP were immobilized and acted as enzymatic biosensor for the simultaneous detection of BHA and PG. The results revealed that BHA and PG both have well-defined oxidation waves with peak potentials of 624 and 655 mV, respectively. Under the optimal conditions, the method behaved satisfactory analytical performance towards BHA and PG with a wide linear range of 0.3–50 mg L⁻¹ and 0.1–100 mg L⁻¹, as well as a detection limit of 0.046 mg L⁻¹ and 0.024 mg L⁻¹ (3σ/slope), respectively. Besides, the proposed method exhibits good sensitivity, stability and reproducibility, providing an alternative to fabricate electrode and construct sensitive biosensors.     

A green analytical procedure for flow-injection determination of nitrate in natural waters

Nitrate determination in waters is generally carried out with cadmium filings and carcinogenic reagents or by reaction with phenolic compounds in highly concentrated sulfuric acid medium. In this work, it was developed a green analytical procedure for nitrate determination in natural waters based on direct spectrophotometric measurements in ultraviolet, using a flow-injection system with an anion-exchange column for separation of nitrate from interfering species. The proposed method employs only one reagent (HClO₄) in a minimum amount (equivalent to 18 μL concentrated acid per determination), and allowed nitrate determination within 0.50-25.0 mg L⁻¹, without interference of up to 200.0 mg L⁻¹ humic acid; 1.0 mg L⁻¹ NO₂⁻; 200.0 mg L⁻¹ PO₄³⁻; 75.0 mg L⁻¹ Cl⁻; 50.0 mg L⁻¹ SO₄²⁻ and 15.0 mg L⁻¹ Fe³⁺. The detection limit (99.7% confidence level) and the coefficient of variation (n = 20) were estimated as 0.1 mg L⁻¹ and 0.7%, respectively. The results obtained for natural water samples were in agreement with those achieved by the reference method based on nitrate reduction with copperized cadmium at the 95% confidence level.     

Direct quantification of deoxynivalenol glucuronide in human urine as biomarker of exposure to the <Emphasis Type="Italic">Fusarium</Emphasis> mycotoxin deoxynivalenol

The direct quantification of deoxynivalenol glucuronide (DON-GlcA) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application as a biomarker of exposure to the Fusarium mycotoxin deoxynivalenol (DON) is reported. Usually, DON exposure is estimated from dietary average intakes or by measurement of the native toxin in urine after enzymatic hydrolysis with β-glucuronidase. These methods are time-consuming, expensive, and fail to determine the ratio of DON to DON-GlcA in a simple one-step procedure. One of the main reasons for the use of indirect methods is the unavailability of DON-GlcA standards. Consequently, DON-3-O-glucuronide (D3GlcA) was synthesized and used to develop a method allowing quantification of both DON and D3GlcA by a simple “dilute and shoot” approach without the need for any cleanup. Limit of detection and apparent recovery of D3GlcA was 3 μg l⁻¹ and 88%, respectively. The identity of D3GlcA in human urine was confirmed by comparison with LC-MS/MS measurements of the synthetically produced D3GlcA standard which was also used for external calibration. The applicability of the method was demonstrated through the analysis of urine samples obtained from a volunteer during regular and cereal-restricted diet, respectively. In regular-diet urine samples, D3GlcA was quantified in concentrations >30 μg l⁻¹ by this approach.     

Evaluation of alternate lines of Fe for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry

The usefulness of the secondary line at 252.744 nm and the approach of side pixel registration were evaluated for the development of a method for sequential multi-element determination of Cu, Fe, Mn and Zn in soil extracts by high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS). The influence of side pixel registration on the sensitivity and linearity was investigated by measuring at wings (248.325, 248.323, 248.321, 248.329, and 248.332 nm) of the main line for Fe at 248.327 nm. For the secondary line at 252.744 nm or side pixel registration at 248.325 nm, main lines for Cu (324.754 nm), Mn (279.482 nm) and Zn (213.875 nm), sample flow-rate of 5.0 mL min⁻¹ and calibration by matrix matching, analytical curves in the 0.2-1.0 mg L⁻¹ Cu, 1.0-20.0 mg L⁻¹ Fe, 0.2-2.0 mg L⁻¹ Mn, 0.1-1.0 mg L⁻¹ Zn ranges were obtained with linear correlations better than 0.998. The proposed method was applied to seven soil samples and two soil reference materials (IAC 277; IAC 280). Results were in agreement at a 95% confidence level (paired t-test) with reference values. Recoveries of analytes added to soil extracts containing 0.15 and 0.30 mg L⁻¹ Cu, 7.0 and 14 mg L⁻¹ Fe, 0.60 and 1.20 mg L⁻¹ Mn, 0.07 and 0.15 mg L⁻¹ Zn, varied within the 94-99, 92-98, 93-101, and 93-103% intervals, respectively. The relative standard deviations (n = 12) were 2.7% (Cu), 1.4% (Fe - 252.744 nm), 5.7% (Fe - 248.325 nm), 3.2% (Mn) and 2.8% (Zn) for an extract containing 0.35 mg L⁻¹ Cu, 14 mg L⁻¹ Fe, 1.1 mg L⁻¹ Mn and 0.12 mg L⁻¹ Zn. Detection limits were 5.4 μg L⁻¹ Cu, 55 μg L⁻¹ Fe (252.744 nm), 147 μg L⁻¹ Fe (248.325 nm), 3.0 μg L⁻¹ Mn and 4.2 μg L⁻¹ Zn.     

Measurement of trace levels of antibiotics in river water using on-line enrichment and triple-quadrupole LC-MS/MS

This study presents the development of an automated on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 23 antibiotics in environmental water samples. After optimisation of LC-MS/MS conditions, SPE parameters such as sorbent type, sample pH or sample volume were optimised. Antibiotic recoveries ranged from 64% to 98% and compared favourably with those achieved using off-line SPE. Limits of detection were in the range 0.5-13.7 ng L⁻¹.This on-line SPE-LC-MS/MS procedure was applied to the analysis of water samples taken in three rivers within the Seine River basin, near Paris (France). The obtained results revealed the occurrence of 12 antibiotics, including tylosin, erythromycin, tetracycline, amoxicillin, trimethoprim, sulfamethoxazole, oxolinic acid, flumequine, norfloxacin, ciprofloxacin, ofloxacin, and vancomycin (2-1435 ng L⁻¹).     

Sensitive determination of hydrazine in water by gas chromatography–mass spectrometry after derivatization with ortho-phthalaldehyde

A gas chromatography–mass spectrometric (GC–MS) method has been established for the determination of hydrazine in drinking water and surface water. This method is based on the derivatization of hydrazine with ortho-phthalaldehyde (OPA) in water. The following optimum reaction conditions were established: reagent dosage, 40 mg mL⁻¹ of OPA; pH 2; reaction for 20 min at 70 °C. The organic derivative was extracted with methylene chloride and then measured by GC–MS. Under the established condition, the detection and the quantification limits were 0.002 μg L⁻¹ and 0.007 μg L⁻¹ by using 5.0-mL of surface water or drinking water, respectively. The calibration curve showed good linearity with r² = 0.9991 (for working range of 0.05–100 μg L⁻¹) and the accuracy was in a range of 95–106%, and the precision of the assay was less than 13% in water. Hydrazine was detected in a concentration range of 0.05–0.14 μg L⁻¹ in 2 samples of 10 raw drinking water samples and in a concentration range of 0.09–0.55 μg L⁻¹ in 4 samples of 10 treated drinking water samples.     

Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg⁻¹. The detection capability (CCβ) of the assay was determined to be ≤0.85 mg kg⁻¹ for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg⁻¹. The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.     

New procedure of selected biogenic amines determination in wine samples by HPLC

A new procedure for determination of biogenic amines (BA): histamine, phenethylamine, tyramine and tryptamine, based on the derivatization reaction with 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (CNBF), is proposed. The amines derivatives with CNBF were isolated and characterized by X-ray crystallography and ¹H, ¹³C, ¹⁹F NMR spectroscopy in solution. The novelty of the procedure is based on the pure and well-characterized products of the amines derivatization reaction. The method was applied for the simultaneous analysis of the above mentioned biogenic amines in wine samples by the reversed phase-high performance liquid chromatography. The procedure revealed correlation coefficients (R²) between 0.9997 and 0.9999, and linear range: 0.10–9.00 mg L⁻¹ (histamine); 0.10–9.36 mg L^-1 (tyramine); 0.09–8.64 mg L⁻¹ (tryptamine) and 0.10–8.64 mg L⁻¹ (phenethylamine), whereas accuracy was 97%–102% (recovery test). Detection limit of biogenic amines in wine samples was 0.02–0.03 mg L⁻¹, whereas quantification limit ranged 0.05–0.10 mg L⁻¹. The variation coefficients for the analyzed amines ranged between 0.49% and 3.92%. Obtained BA derivatives enhanced separation the analytes on chromatograms due to the inhibition of hydrolysis reaction and the reduction of by-products formation.     

Automatic flow system for the sequential determination of copper in serum and urine by flame atomic absorption spectrometry

In this work, a fully automated flow system exploiting the advantages of the association of multi-pumping, multicommutation, binary sampling and merging zones, to accomplish the sequential determination of copper in serum and urine by flame atomic absorption spectrometry, is described. The developed flow system allowed multiple tasks, such as serum samples preparation (samples and standard solutions viscosity adjustment), serum copper (SCu) measurement, urine copper (UCu) pre-concentration and its subsequent elution and measurement, to be carried out sequentially. The implemented flow manifold presented a modular configuration consisting on two quasi-independent modules, each one accountable for a specific sample manipulation and whose combined operation under computer control enabled the determination of copper in a wide concentrations range.Once optimised and with a sample consumption of about 0.250 mL of serum and 7 mL of urine, the developed flow system allowed linear calibration plots up to 5 mg L⁻¹ with a detection limit of 0.035 mg L⁻¹ for SCu and linear calibration plots up to 300 μg L⁻¹ with a detection limit of 0.67 μg L⁻¹ for UCu. The sampling rate varied according to the module employed and was about 360 determinations h⁻¹ (SCu module), 12 determinations h⁻¹ (UCu module) or 24 determinations h⁻¹ (12 urine and 12 serum samples; UCu and SCu modules simultaneously). Repeatability studies (R.S.D.%, n = 10) showed good precision for UCu at concentrations of 25 μg L⁻¹ (2.54%), 50 μg L⁻¹ (0.90%) and 100 μg L⁻¹ (1.62%) as well as for SCu at concentrations of 0.25 mg L⁻¹ (8.11%), 1 mg L⁻¹ (3.11%) and 5 mg L⁻¹ (0.90%). A comparative evaluation showed a good agreement between the results obtained in the analysis of UCu and SCu (n = 18) by both the developed methodology and the reference procedures. Accuracy was further evaluated by means of the analysis of reference samples (Seronorm™ Trace Elements Urine and Seronorm™ Trace Elements Serum) and the obtained results complied with the certified values.     

Study on different pre-treatment procedures for metal determination in Orujo spirit samples by ICP-AES

In this work several pre-treatment methods were studied for metal (Na, K, Mg, Cu and Ca) determination in Orujo spirit samples using inductively coupled plasma atomic emission spectrometry (ICP-AES). Dilution, digestion, evaporation, and cryogenic desolvatation techniques were comparatively evaluated. Because of their analytical characteristics, digestion and evaporation with nitrogen current were found to be appropriate procedures for the determination of metals in alcoholic spirit samples. Yet, if simplicity and application time are to be considered, the latter—evaporation in a water bath with a nitrogen current—stands out as the optimum procedure for any further determinations in Orujo samples by ICP-AES. Low detection levels and wide linear ranges (sufficient to determine these metals in the samples studied) were achieved for each metal. The recoveries (in the 97.5-100.5% range) and the precision (R.S.D. lower than 5.6%) obtained were also satisfactory. The selected procedure was applied to determine the content of metals in 80 representative Galician Orujo spirit samples with and without a Certified Brand of Origin (CBO) which had been produced using different distillation systems. The metal concentrations ranged between 0.37 and 79.7 mg L⁻¹ for Na, <LOD to 12.4 mg L⁻¹ for K, 0.02-4.83 mg L⁻¹ for Mg content, <LOD to 37.3 mg L⁻¹ for Cu and 0.03-13.10 mg L⁻¹ for Ca.     

Sensitive and ultra-fast determination of arsenic(III) by gas-diffusion flow injection analysis with chemiluminescence detection

A novel chemiluminescence gas-diffusion flow injection system for the determination of arsenic(III) in aqueous samples is described. The analytical procedure involves injection of arsenic(III) samples and standards into a 0.3 mol L⁻¹ hydrochloric acid carrier stream which is merged with a reagent stream containing 0.2% (w/v) sodium borohydride and 0.015 mol L⁻¹ sodium hydroxide. Arsine, generated in the combined carrier/reagent donor stream, diffuses across the hydrophobic Teflon membrane of the gas-diffusion cell into an argon acceptor stream and then reacts with ozone in the flow-through chemiluminescence measuring cell of the flow system. Under optimal conditions, the method is characterized by a wide linear calibration range from 0.6 μg L⁻¹ to 25 mg L⁻¹, a detection limit of 0.6 μg L⁻¹ and a sample throughput of 300 samples per hour at 25 mg L⁻¹ and 450 samples per hour at 25 μg L⁻¹.     

Optimization and validation of a method for the direct determination of catechin and epicatechin in red wines by HPLC/fluorescence

The present paper describes a direct procedure for the determination of catechin and epicatechin concentrations in red wines employing reverse-phase high performance liquid chromatography (RP HPLC) and detection by fluorescence. The method was performed using a sample volume of 10 µL without dilution. The separation process employed a Chromolith performance RP-18e (100 mm × 4.6 mm) column, and the mobile phase was composed of solvent A: methanol-acetic acid-water (90:8:2) and solvent B: water-acetic acid-methanol (10:2:88) at a flow rate of 1.0 mL min^− 1. Linearity was observed in the range of 1 to 30 mg L^− 1, with limits of detection and quantification of 0.27 and 0.89 mg L^− 1, respectively, for catechin and 0.33 and 1.01 mg L^− 1, respectively, for epicatechin. The precisions estimated by the relative standard deviation were 3.34 and 1.09% for catechin concentrations of 0.5 and 20 mg L^− 1 respectively and 2.82 and 0.49% for epicatechin concentrations of 0.5 and 20 mg L^− 1, respectively. The evaluation of the accuracy was done using an addition/recovery assay. Four wine samples were used, and the recoveries varied from 105 to 108% for catechin and from 97 to 119% for epicatechin. The method was applied to the analysis of red wine samples collected from the São Francisco region, Bahia State, Brazil. Nine samples were analyzed, and the catechin and epicatechin concentrations varied from 7.51 to 73.20 and from 5.08 to 43.32 mg L^− 1, respectively. The concentrations found agree with data reported in the literature.     

Determination of trace trichlorfon by high performance liquid chromatography with UV detection based on its catalytic effect on sodium perborate oxidizing benzidine

Trichlorfon has the capacity to catalyze the oxidation of benzidine (4,4′-diamino-biphenyl) to 4-amino-4′-nitro biphenyl in the presence of sodium perborate. The product of the catalyzed reaction was validated by LC-MS method. Reversed-phase high performance liquid chromatography with 365 nm UV detection was used for separation and quantification of 4-amino-4′-nitro biphenyl. It can be proven there is a linear relationship between the peak areas of 4-amino-4′-nitro biphenyl and trichlorfon in the concentration range of 0.02-0.5 mg L⁻¹ (r = 0.9988). Limit of detection was 2.0 μg L⁻¹. A method for the indirect determination of trichlorfon using HPLC was developed based on catalytic effect of trichlorfon. Method validation was performed on samples spiked at three levels (0.5, 1.0, 1.5 mg kg⁻¹), the recoveries ranged from 67.5 to 82.1%, with relative standard deviations between 4.5 and 7.3%.0.01 mol L⁻¹ sodium dodecyl sulphate (SDS) solution was used to extract trichlorfon from samples and solid-phase extraction was used to isolate and concentrate trichlorfon in SDS solution. The recoveries of trichlorfon obtained with percolating the extraction through a SPE system were essentially in agreement with those obtained by liquid-liquid extraction. This new isolation technique decreases the use of toxic solvents and satisfies the requirements of Green Analytical Chemistry.