Sampling, sample preparation, and analytical variability associated with testing wheat for deoxynivalenol

The variability associated with testing wheat for deoxynivalenol (DON) was measured using a 0.454 kg sample, Romer mill, 25 g comminuted subsample, and the Romer Fluoroquant analytical method. The total variability was partitioned into sampling, sample preparation, and analytical variability components. Each variance component was a function of the DON concentration and equations were developed to predict each variance component using regression techniques. The effect of sample size, subsample size, and number of aliquots on reducing the variability of the DON test procedure was also determined. For the test procedure, the coefficient of variation (CV) associated with testing wheat at 5 ppm was 13.4%. The CVs associated with sampling, sample preparation, and analysis were 6.3, 10.0, and 6.3%, respectively. For the sample variation, a 0.454 kg sample was used; for the sample preparation variation, a Romer mill and a 25 g subsample were used; for the analytical variation, the Romer Fluoroquant method was used. The CVs associated with testing wheat are relatively small compared to the CV associated with testing other commodities for other mycotoxins, such as aflatoxin in peanuts. Even when the small sample size of 0.454 kg was used, the sampling variation was not the largest source of error as found in other mycotoxin test procedures.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I₅₀ and detection limits were 0.05-2.5 and 0.1-7.5 μg L⁻¹, 0.35 (±0.04) μg L⁻¹ and 0.9 (±0.1) μg L⁻¹, 60 and 100 μg L⁻¹ in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I₅₀ in real samples was 0.2 μg L⁻¹ corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.     

Comparison of four C18 columns for the liquid chromatographic determination of deoxynivalenol in naturally contaminated wheat

Three long and 1 short reversed-phase C18 columns were compared for separation of deoxynivalenol (DON) in extracts of naturally contaminated wheat samples using liquid chromatography with ultraviolet detection and liquid chromatography/mass spectrometry (LC/MS). Among the 3 long columns used, a Symmetry C18 column with an isocratic solvent mixture of water-acetonitrile-methanol (90 + 5 + 5, v/v/v) gave the best separation for DON without interferences from other compounds in the wheat extracts. The Symmetry short (75 mm) column was comparable with the long column (250 mm) in resolving DON but significantly reduced retention time (i.e., 5.8 versus 16.3 min). Increasing the column temperature from 25 to 45 degrees C resulted in a further reduction in retention time. Identity of DON in the wheat extracts and standard solutions was confirmed by LC/MS in the positive ion mode, whereby DON appeared with an (M+1)+ ion at a mass-to-charge ratio of 297 plus fragment ions associated with loss of water and/or a 30 atomic mass unit (amu) CH2O fragment. The Symmetry short column was also capable of separating a mixture of the mycotoxins DON, 15-acetyl-DON, nivalenol, and zearalenone by use of a combination of an isocratic and gradient solvent system. The overall method showed high precision, exhibiting a relative standard deviation of 4.8%, limit of detection of 50 ng/g, and limit of quantitation of 165 ng/g. It was significantly correlated with enzyme-linked immunosorbent assay analysis, indicating its appropriateness for safety and quality assurance of wheat and related grains.     

Iodine determination in biological materials Options for sample preparation and final determination

This paper describes the development of various new acid sample decomposition methods, as well as an extraction (leaching) method and compares them with the “Sch?niger Combustion” technique. The methods have been developed as sample preparation techniques for iodine determination in biological materials, especially in solid samples. ICP-MS (inductively coupled plasma mass spectrometry) and a catalytic technique are employed and discussed for the final determination of iodine concentrations. Accuracy and reliability of the different analytical methods are shown in the examples of different CRMs (certified reference materials) available for iodine. The results of an interlaboratory comparison are specifically presented for the extraction (leaching) method. Received: 11 May 1998 / Revised: 14 June 1998 / Accepted: 16 June 1998     

Supramolecular solvent-based microextraction of ochratoxin A in raw wheat prior to liquid chromatography-fluorescence determination

A supramolecular solvent made up of reverse micelles of decanoic acid, dispersed in a continuous phase of THF:water, was proposed for the simple, fast and efficient microextraction of OTA in wheat prior to liquid chromatography-fluorescence determination. The method involved the stirring of 300 mg-wheat subsamples (particle size 50 μm) and 350 μL of supramolecular solvent for 15 min, subsequent centrifugation for 15 min and the direct quantitation of OTA in the extract, previous 5.7-fold dilution with ethanol/water/acetic acid (49.5/49.5/1), against solvent-based calibration curves. No clean-up of the extracts or solvent evaporation was needed. Interactions between the supramolecular solvent and major matrix components in the wheat (i.e. carbohydrates, lipids and proteins) were investigated. The reverse micelles in the extractant induced gluten flocculation but only in the coacervation region of lower analytical interest (i.e. at percentages of THF above 11%). The quantitation of OTA was interference-free. Representativity of the 300 mg-wheat subsamples was proved by analysing a reference material. OTA recoveries in wheat ranged between 84% and 95% and the precision of the method, expressed as relative standard deviation, was 2%. The quantitation limit of the method was 1.5 μg kg⁻¹ and was below the threshold limit established for OTA in raw cereals by EU directives (5.0 μg kg⁻¹). The method developed was validated by using a certified reference material and it was successfully applied to the determination of OTA in different wheat varieties from crops harvested in the South of Spain. OTA was not detected in any of the analysed samples. This method allows quick and simple microextraction of OTA with minimal solvent consumption, while delivering accurate and precise data.     

Simplified sample preparation for fluoride determination in biological material

A simple and rapid preparation method for the determination of fluoride in biological materials (blood and food) of various origins, is described. A homogenized sample was placed in a plastic diffusion cell and calcium phosphate added, it was then dried at 55 degrees C and treated with 70% HClO4 and 40% AgClO4. After digestion for 24 h at 55 degrees C, the fluorides released were fixed on the upper part of a diffusion cell containing a thin layer of NaOH. The analyses of the diffused fluoride were carried out with an ion-selective electrode. The proposed microdiffusion method, without mineralization, enables quantitative separation of the fluoride from the biological samples.     

Analytical performances of a DNA-ligand system using time-resolved fluorescence for the determination of ochratoxin A in wheat

The analytical performances of a novel DNA-ligand system using the time-resolved fluorescence (TRF) response of ochratoxin A (OTA)-terbium-DNA aptamer interaction were tested for the quantitative determination of OTA in wheat. Wheat was extracted with acetonitrile/water (60:40, v/v) followed by clean-up through affinity columns containing a DNA-aptamer-based oligosorbent. Then, OTA was detected by TRF spectroscopy after reaction with a terbium fluorescent solution containing the DNA-aptamer probe. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. The average recovery from samples spiked with 2.5-25 μg kg(-1) OTA was 77%, with a relative standard deviation lower than 6% and a quantification limit of 0.5 μg kg(-1). Comparative analyses of 29 naturally contaminated (up to 14 μg kg(-1)) wheat samples using the aptamer-affinity column/TRF method or the immunoaffinity column/high-performance liquid chromatography method showed good correlation (r = 0.985) in the range tested. The trueness of the aptamer-based method was additionally assessed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.     

Comparison of sample preparation methods for the ICP-AES determination of minor and major elements in clarified apple juices

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used for the determination of minor and major elements present in apple juices. Prior to ICP-AES measurement, samples were diluted with nitric acid or digested in a microwave assisted digestion system. The differences in the measured element concentrations after application of different types of sample preparation procedures are discussed. The direct measurement compared to closed microwave dissolution was found to be the best sample preparation procedure. Prior to the measurements the ICP-AES method was validated and optimized for the determination of elements in apple juices. For diluted apple juice samples the lowest limits of detection (LOD) were obtained for Ba and Cd (< 20 μg L^{− 1}), moderate ones for Cu, Mn, Ni, Fe, Ag, Ca, Cr, Zn, Mg, and Sr (20–100 μg L^{− 1}), and the highest LODs for K, Pb, Na, and Al (> 110 μg L^{− 1}). The results obtained for the repeatability (< 0.9%), the intermediate precision (< 4.5%), the day-to-day reproducibility (< 5.2%), and the overall uncertainty of measurement (approx. 4–7%) for all elements analyzed demonstrated the good applicability of the proposed method. Differences in major element content in fresh and commercial apple juice are discussed.     

Comparison of sample preparation methods for determination of free carbon in boron carbide by X-ray powder diffraction

Several sample preparation methods were evaluated for determination of free carbon in boron carbide powders by quantitative X-ray diffraction method, including ultrasonication, wet ball milling and dry ball milling–wet mixing. Quantitation was based on measuring the integral peak area ratio of the diffraction lines of graphite (002) to boron carbide (012) in samples spiked with pure graphite. The dry milling–wet mixing method provided the best precision and accuracy in all the measurements as well as in determination of free carbon in a boron carbide reference material. There was a linear relationship between the integral peak area ratios and graphite added to boron carbide samples which were purified from their free carbon content. The method provided a low detection limit of 0.05 wt% free carbon.     

Applications of laboratory robotics in spectrophotometric sample preparation and experimental optimization

Optimal experimental conditions for spectrophotometric solution-preparation procedures for determinations of iron(III) and phosphate were determined by a completely robotic application of the SIMPLEX algorithm, factorial design methods, and response surface fitting. The overall reliability of the approach, the precision observed and its significance are discussed.     

Comparison of direct solid sampling and slurry sampling for the determination of cadmium in wheat flour by electrothermal atomic absorption spectrometry

Two analytical methods for the determination of cadmium in wheat flour by electrothermal atomic absorption spectrometry without prior sample digestion have been compared: direct solid sampling analysis (SS) and slurry sampling (SlS). Besides the conventional modifier mixture of palladium and magnesium nitrates (10 μg Pd + 3 μg Mg), 0.05% (v/v) Triton X-100 has been added to improve the penetration of the modifier solution into the solid sample, and 0.1% H₂O₂ in order to promote an in situ digestion for SS. For SlS, 30 μg Pd, 12 μg Mg and 0.05% (v/v) Triton X-100 have been used as the modifier mixture. Under these conditions, and using a pyrolysis temperature of 800 °C, essentially no background absorption was observed with an atomization temperature of 1600 °C. About 2 mg of sample have been typically used for SS, although as much as 3-5 mg could have been introduced. In the case of SlS multiple injections had to be used to achieve the sensitivity required for this determination. Calibration against aqueous standards was feasible for both methods. The characteristic mass obtained with SS was 0.6 pg, and that with SlS was 1.0 pg. The limits of detection were 0.4 and 0.7 ng g⁻¹, the limits of quantification were 1.3 and 2.3 ng g⁻¹ and the relative standard deviation (n = 5) was 6-16% and 9-23% for SS and SlS, respectively. The accuracy was confirmed by the analysis of certified reference materials. The two methods were applied for the determination of cadmium in six wheat flour samples acquired in supermarkets of different Brazilian cities. The cadmium content varied between 8.9 ± 0.5 and 13 ± 2 ng g⁻¹ (n = 5). Direct SS gave results similar to those obtained with SlS using multi-injections; the values of both techniques showed no statistically significant difference at the 95% confidence level. Direct SS was finally adopted as the method of choice, due to its greater simplicity, the faster speed of analysis and the better figures of merit.     

High-performance liquid chromatographic determination of ochratoxin A in artificially contaminated cocoa beans using automated sample clean-up

A HPLC method is described for the analysis of ochratoxin A at low-ppb levels in samples of artificially contaminated cocoa beans. The samples are extracted in a mixture of methanol–water containing ascorbic acid, adjusted to pH and evaporated to dryness. Samples in this state are then placed onto a Benchmate sample preparation workstation where C₁₈ solid-phase extraction operations are performed. The resulting materials are evaporated to dryness and analyzed by reversed-phase HPLC with fluorescence detection. The method was evaluated for accuracy and precision with R.S.D.s for multiple injections of sample and standard calculated to be 1.1% and 2.5% for sample and standard, respectively. Recoveries of ochratoxin A added to cocoa beans ranged from 87–106% over the range of the assay.     

Determination of deoxynivalenol in whole wheat flour and wheat bran

A liquid chromatographic (LC) method was developed for determining deoxynivalenol (DON) in whole wheat flour and wheat bran. A 15 g test sample was extracted with acetonitrile-water (84 + 16, v/v) and applied to a Romer MycoSep cleanup column. The eluate was dried and then reconstituted in a 0.1 M phosphate buffer, pH 7.0, and applied to a Vicam DONtest-LC cleanup column. The methanol eluate was chromatographed with a methanol-water (17 + 83, v/v) mobile phase on a C18 column with UV detection at 220 nm. Five replicates at each of 5 fortification levels (0.25, 0.50, 1.0, 2.0, and 4.0 ppm), plus 5 controls, were determined for both whole wheat flour and wheat bran. For flour, the average recoveries were 72.2-91.5% with relative standard deviations (RSDs) of 4.9-18.4%. The intra-assay flour recovery was 82.4% with 9.8% RSD. A 5 replicate sample of naturally incurred wheat had an average of 1.1 ppm DON with 6.7% RSD. For bran, average recoveries of fortified samples were 69.5-99.7% with RSDs of 1.7-18.8%. The intra-assay bran recovery was 81.5% with 8.9% RSD. The limit of detection (about 3x noise) for the method is 0.05 ppm; the correlation coefficient (linearity) was >0.9995. The DON peak was clearly identified and easily integrated in the chromatograms.     

Sediment sample preparation for the determination of Pu and Am isotopes

Summary Sediment samples from the Romanian sector of the Danube River and the Black Sea coast were analyzed for Pu and Am. Three different ways of bringing the samples into solution were tested: acid attack, microwave digestion and alkaline fusion. A conventional anion-exchange resin was used to separate plutonium from other radionuclides and several variants were tried to improve the separation of americium. The preparation of thin sources for alpha-spectrometry was tested through electrodeposition and coprecipitation with Nd(III). Discussion and recommendations for the dissolution step, the americium separation and preparation of alpha sources are made.     

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography

Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC-FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC-FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC-FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C18 using methanol-acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1-1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.     

Comparison of sample preparation methods applied for determination of atrazine in freshwater biofilms by gas chromatograph-mass spectrometer system

A sample preparation procedure was developed for the determination of atrazine in freshwater biofilms by gas chromatography-mass spectrometry. Different methods of sample preservation (immediate extraction, deep-freezing, cooling, vacuum-drying, freeze-drying) were tested for atrazine pretreated freshwater biofilms, followed by combined liquid- and solid-phase extraction (SPE). The immediate extraction of atrazine with acetonitrile, and a SPE, proved to be the best sample preparation procedure. Applying our method, atrazine pollution of the Lake Velence (Hungary) could be detected in the biofilms. At the same time in the lake freshwater samples, prepared by SPE and analyzed the same way, the atrazine concentration did not exceed the limit of detection.     

Development of a selective sample clean-up method based on immuno-ultrafiltration for the determination of deoxynivalenol in maize

This paper describes the development of a highly selective analytical method for the determination of deoxynivalenol (DON) in maize. The developed method is based on immuno-ultrafiltration (IUF) and is the first application of IUF as a clean-up strategy in food analysis. Quantification of DON was carried out by high-performance liquid chromatography with ultraviolet detection. In contrast to immunoaffinity chromatography, in IUF the antibodies are not bound to a solid support material but used in free form, thus making it possible to avoid the critical immobilisation step. Sample clean-up by IUF proved to be as selective as clean-up using commercially available immunoaffinity columns. The limit of detection (S/N=3) of the analytical method was found to be 74 ng DON/g maize. Repeated analysis of a certified maize reference material on four different days resulted in a mean recovery of 93% with a standard deviation of 10%.