Laboratory Validation Of Method Research Articles

Forensic Evaluation of Ink Age by Raman Spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS)

Ink age analysis is a hot but challenging topic in the practice of document inspection and identification in forensic sciences. Due to the influence of ink components, paper, and storage environment on the aging process, strict validation of laboratory methods is required. In this study, Raman spectroscopy was investigated to determine ink age by analyzing the peak intensity of carbon black in neutral signature pens and has been recently used as evidence in legal documents. Through statistical analysis of Raman peak heights of inks naturally aged from 3 days to 18 months, the results demonstrate this approach is unsuitable for forensic purposes. Hence, appraisal conclusions should also be rejected by the procuratorate in actual cases. In order to further explore the identification of ink age, the volatilization rates of cyclohexanone, 1-butanol, propylene glycol, ethylene glycol, benzyl alcohol, diethylene glycol, glycerol, phenoxyethanol, and triethylene glycol commonly used in pens as a function of time were investigated. The results show that gas chromatography-mass spectrometry (GC-MS) is reliable to characterize the volatilization of these alcohols in documents. The residual levels of characteristic solvents (triethylene glycol in two samples and diethylene glycol in one) decreased significantly within 6 months and may be used to evaluate the ink age in documents.

Validation of Two Real-Time PCR Approaches for the Relative Quantitation of Pork and Horse DNA in Food Samples

Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef meat products. Test samples were prepared using raw meat admixtures or processed horse/pork in beef food products made to an industry-standard recipe. The methods were subjected to single laboratory method validation, evaluating the performance characteristics of specificity, PCR efficiency and r-squared (r2), Limit of Detection (LOD), Limit of Quantitation (LOQ), and precision and trueness. A limited UK-based inter-laboratory trial of the two methods was completed involving four participating laboratories. Full statistical analysis of the data qualified the applicability of the methods for accurate and sensitive trace-level analysis. The methods were deemed fit for purpose for reproducibly distinguishing between adventitious contamination at 0.1% (w/w), the level for further enforcement action at 1% (w/w), and a level representative of deliberate economically motivated adulteration (10% (w/w)). The data provided evidence that the precision of the two methods was applicable for qualitative and quantitative detection at topically important levels of adulteration. This work has added significant value to the current state of the art in quantitative determination of topical meat species adulteration, allowing analysts to distinguish between adventitious contamination and deliberate adulteration. The resulting methods described in this paper can easily be deployed and used by analytical laboratories for controls and due-diligence testing based on standard laboratory equipment.

Visual hemolysis Index for routine clinical chemistry laboratory parameters

Introduction: Interference substances can be significance source of error in the clinical laboratory measurements. Such errors may, in some cases represents hazards in patients. Hemolysis interferes with the Clinical Chemistry Analysis, which influence results of analytes and its leads to erroneous clinical interpretations. Hemolysis of Red cells is inevitable during the collection and handling of blood samples. In usual practice it is very difficult task to perform the rigorous analysis to evaluate interference in routine laboratories. Our objective was to assess effect of Hemolysis on routine biochemistry analysis Materials and Methods: The study followed the CLSI EP7-A2 protocol, sample were collected from 5 male subjects, aged 25-35 years, [0% (normal pool serum), serial dilution of different hemolysis grades [25% (1+coulter analyzer. The data was analyzed and ±5% difference from interferent free sample was taken as acceptable criteria.) ,50%(2+), 75%(3+) and 100%(4+)] of sample were prepared by spiking pooled serum with red cell lysate. The interference was studied up to concentrations of 0.5gm/dl for hemoglobin. Hemolysis indices graded on the basis of the appearance of the sample qualitatively ranked from “no visible hemolysis” to “4+ hemolysis. Aliquots were analyzed in triplicates using Beckman Results: The experimental design for laboratory method validation for hemolysis showed that Hemolysed samples starting from 1+ of hemolysis grade will be rejected for laboratory analysis for clinical chemistry parameters like Total bilirubin, Aspartate amino transferase, Alkaline Phosphatase, Gamaglutamyle transferase, Uric acid and serum potassium. While serum Creatinine hemolysed sample with 4+ hemolysis will not be accepted. Conclusions:Study results are useful for rejecting / to comment about the potentially misleading results in clinical chemistry parameters of clinical laboratory Keywords: Interferences, Hemolysis, Clinical laboratory analysis, Clini

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

Persistence behaviour and safety risk evaluation of pyridalyl in tomato and cabbage

A field experiment was set up to evaluate persistence behaviour of pyridalyl in tomato, cabbage and cultivated field soil over two consecutive seasons. An analytical method was developed to analyze pyridalyl residues in different matrices and duly validated, based on single laboratory method validation criteria. Pyridalyl residues were detected and quantified using a gas chromatograph equipped with an electron capture detector. The compound exhibited low persistence in tomato, cabbage and soil. A safe waiting period of 17–18 d after final insecticide application needs to be maintained before harvesting the crop. Both dietary and soil ecological risk were assessed and it was found that the harvested vegetables were toxicologically safe for consumption. However, there was concern about insecticidal toxicity against the algal population of soil which needs to be reconfirmed by further studies.

DETERMINACIÓN DE LA DISTRIBUCIÓN DE CONTAMINANTES EMERGENTES EN AGUA INTERSTICIAL EN SEDIMENTOS DE HUMEDAL MEDIANTE LA OPTIMIZACIÓN Y VALIDACIÓN DE UN MÉTODO ANALÍTICO

The present work shows the methodology (study of a method) for the analysis of four emerging pollutants. Each studied pollutant represents a family from emerging pollutants (an estrogenic compound, an antibacterial, a pharmaceutical and a fragrance). The method was optimized to be used in a complex environmental matrix such as sediment interstitial water of a wetland with high organic matter content. The extraction of the compounds was carried out by solid phase extraction technique and quantified by gas chromatography with mass spectrometry in single ion monitoring mode. The method was validated using the “Single Laboratory Method Validation Process” through the determination of the performance parameters of the method. The optimized method was applied to the analysis of sediment interstitial water from the canals of the periurban agricultural zone of Xochimilco, Mexico. This preliminary study showed that the sampling point close to the wastewater discharge area from the secondary treatment wastewater plant had the highest concentration of emerging pollutants. Also, an inversely proportional relationship was observed between the concentrations of ibuprofen and musk with the concentration of organic matter in the sediments. The optimization of the method and the ratification with sediment interstitial water samples from the wetland, showed the worthiness of the method for the evaluation of the presence and distribution of emerging pollutants.

Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: Culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT).

IntroductionThe appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests.MethodsClinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2–4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods—pertussis culture as the “gold standard,” composite reference standard analysis (CRS), and latent class analysis (LCA).ResultsOverall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough.ConclusionsTiming of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.

Dissipation of spiromesifen and spiromesifen-enol on tomato fruit, tomato leaf, and soil under field and controlled environmental conditions.

Dissipation of spiromesifen and its metabolite, spiromesifen-enol, on tomato fruit, tomato leaf, and soil was studied in the open field and controlled environmental conditions. Sample preparation was carried out by QuEChERS method and analysis using LC-MS/MS. Method validation for analysis of the compounds was carried out as per "single laboratory method validation guidelines." Method validation studies gave satisfactory recoveries for spiromesifen and spiromesifen-enol (71.59-105.3%) with relative standard deviation (RSD) <20%. LOD and LOQ of the method were 0.0015μgmL-1 and 0.005mgkg-1, respectively. Spiromesifen residues on tomato fruits were 0.855 and 1.545mgkg-1 in open field and 0.976 and 1.670mgkg-1 under polyhouse condition, from treatments at the standard and double doses of 125 and 250ga.i.ha-1, respectively. On tomato leaves, the residues were 5.64 and 8.226mgkg-1 in open field and 6.874 and 10.187mgkg-1 in the polyhouse. In soil, the residues were 0.532 and 1.032mgkg-1 and 0.486 and 0.925mgkg-1 under open field and polyhouse conditions, respectively. The half-life of degradation of spiromesifen on tomato fruit was 6-6.5days in the open field and 8.1-9.3days in the polyhouse. On tomato leaves, it was 7-7.6 and 17.6-18.4days and in soil 5.6-7.4 and 8.4-9.5days, respectively. Metabolite, spiromesifen-enol, was not detected in any of the sample throughout the study period. Photodegradation could be the major route for dissipation of spiromesifen in the tomato leaves, whereas in the fruits, it may be the combination of photodegradation and dilution due to fruit growth. The results of the study can be utilized for application of spiromesifen in plant protection of tomato crop under protected environmental conditions.

Accelerated solvent extraction method for the quantification of polycyclic aromatic hydrocarbons in cocoa beans by gas chromatography–mass spectrometry

An accelerated solvent extraction (ASE) procedure for use with gas chromatography-mass spectrometry (GC-MS) was optimized for the determination of eight polycyclic aromatic hydrocarbons (PAHs) in cocoa beans. Plackett-Burman and rotatable central composite design (RCCD) indicated that three variables affected the recoveries of PAHs during the extraction and purification steps: agitation time in the second liquid-liquid partition, weight of silica gel in the column, and volume of hexane for PAH elution from the column. After obtaining the optimal conditions, a single laboratory method validation was performed. Linearity was demonstrated for benzo[a]pyrene in the concentration range from 0.5 to 8.0mgkg-1 of sample, corresponding to 1.25-20.0μgkg-1 of cocoa on a fat basis. For the other analytes, linearity was observed from 0.75 to 8.0μgkg-1 of sample (1.88-20.0μgkg-1 of cocoa on a fat basis). Significant matrix effects were found for chrysene and benzo[b]fluoranthene. The precision of the method was verified with relative standard deviations (RSDs) ranging from 2.57 to 14.13% and from 4.36 to 19.77% under repeatability and intermediate precision conditions, respectively. The average recoveries of the eight PAHs ranged from 74.99 to 109.73%. These parameters, limits and measurement uncertainties met the performance criteria established by European Union regulations, except for the theoretical limit of detection for chrysene. The method was applied to the analysis of samples of Brazilian cocoa beans, and only one sample was found to have a PAH content above the maximum limit defined by the European Union legislation. This optimized and validated method is intended to be used as part of the official Brazilian monitoring programs investigating contaminants and residues in food.

Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimised method using liquid chromatography-tandem mass spectrometry

Alternaria toxins and citrinin are mycotoxins produced by fungi growing on different raw materials and agricultural commodities. Maximum levels of these toxins in foods are currently under consideration by the European Commission as a risk management measure. In this study, a new quantitative method is described for the determination of five Alternaria toxins and citrinin in tomato and tomato juice samples based on LC-MS/MS detection. Samples were extracted with pure methanol, followed by a derivatisation step with 2,4-dinitrophenylhydrazine to improve the determination of tenuazonic acid and to decrease the wide polarity difference between the compounds of interest. Samples were purified on hydrophilic-modified styrene polymer solid-phase extraction cartridges. High-performance liquid chromatographic columns packed with different core–shell materials were tested for the separation of toxins and a C-18 phase was in the final method applied to achieve sufficient separation of all relevant analytes. A key element of this approach was to prove successful transferability of the method to three different triple quadrupole mass spectrometers. A full single laboratory method validation was performed on two LC-MS/MS systems and performance characteristics met the predefined requirements. Moreover, the method was used in an international proficiency test and the satisfactory z-scores obtained (−0.1 to 0.8 in tomato juice samples) demonstrated the reliability of the approach described. The method will be validated in an inter-laboratory collaborative study and if the criteria for method precision are met, the method will be proposed as a new Work Item to the European Committee for Standardisation.

Evaluation of X-ray fluorescence spectroscopy as a method for the rapid and direct determination of sodium in cheese

Cheese manufacturers indirectly determine Na in cheese by analysis of Cl using the Volhard method, assuming that all Cl came from NaCl. This method overestimates the actual Na content in cheeses when Na replacers (e.g., KCl) are used. A direct and rapid method for Na detection is needed. X-ray fluorescence spectroscopy (XRF), a mineral analysis technique used in the mining industry, was investigated as an alternative method of Na detection in cheese. An XRF method for the detection of Na in cheese was developed and compared with inductively coupled plasma optical emission spectroscopy (ICP-OES; the reference method for Na in cheese) and Cl analyzer. Sodium quantification was performed by multi-point calibration with cheese standards spiked with NaCl ranging from 0 to 4% Na (wt/wt). The Na concentration of each of the cheese standards (discs: 30mm×7mm) was quantified by the 3 methods. A single laboratory method validation was performed; linearity, precision, limit of detection, and limit of quantification were determined. An additional calibration graph was created using cheese standards made from natural or process cheeses manufactured with different ratios of Na:K. Both Na and K calibration curves were linear for the cheese standards. Sodium was quantified in a variety of commercial cheese samples. The Na data obtained by XRF were in agreement with those from ICP-OES and Cl analyzer for most commercial natural cheeses. The XRF method did not accurately determine Na concentration for several process cheese samples, compared with ICP-OES, likely due to the use of unknown types of Na-based emulsifying salts (ES). When a calibration curve was created for process cheese with the specific types of ES used for this cheese, Na content was successfully predicted in the samples. For natural cheeses, the limit of detection and limit of quantification for Na that can be determined with an acceptable level of repeatability, precision, and trueness was 82 and 246mg/100g of cheese, respectively. Calibration graphs should be created with standards that reflect the concentration range, ratio, and salt type present in the cheeses. This XRF method can be successfully used for the rapid and direct measurement of Na content in a wide variety of natural cheeses. Commercial process cheese manufacturers use proprietary blends of ES. We did find that the XRF technique worked for process cheese when the calibration graphs were created with the specific types of ES actually used.

The role of domestic tap water on Acanthamoeba keratitis in non-contact lens wearers and validation of laboratory methods.

Acanthamoeba is increasingly recognized as an important cause of keratitis in non-contact lens wearers while contact lens wear is the leading risk factor for Acanthamoeba keratitis (AK). It is unlikely that the Acanthamoeba colonization is a feature which is effective only in patient's homes with infectious keratitis since the organism has been isolated from domestic tap water. Two hundred and thirty-one (231) corneal scrapings were taken from infectious keratitis cases, and four contact lens solutions and domestic tap waters were taken from 22 out of 44 AK-diagnosed patient's homes. Microscopic examination, culture, PCR, real-time PCR and DNA sequencing analyses were used for AK-diagnosed samples. The real-time PCR was the most sensitive (100 %) one among the methods used in diagnosis of AK. The 44 (19.0 %) out of 231 corneal scrapings, 4/4 (100 %) contact lens solution and 11/22 (50 %) of domestic tap water samples were found to be positive by real-time PCR for Acanthamoeba. A. griffini (T3), A. castellanii (T4) and A. jacobsi (T15) genotypes were obtained from corneal scrapings, contact lens solutions and domestic tap water samples taken from the patient's homes diagnosed with AK. The isolation of Acanthamoeba containing 6/22 (27.3 %) A. griffini (T3), 14/22 (63.6 %) A. castellanii (T4) and 2/22 (9.1 %) A. jacobsi (T15) from the domestic tap water outlets of 22 of 44 (50 %) of patient's homes revealed that is a significant source of these organisms. A. griffini (T3) and A. jacobsi (T15) genotypes have not been determined from AK cases in Turkey previously. Thus, we conclude that Acanthamoeba keratitis is associated with exposition of patients who has ocular trauma or ocular surface disease to domestic tap water in endemic or potentially endemic countries.

A record of Pleistocene diatom preservation from the Amundsen Sea, West Antarctica with possible implications on silica leakage

article i nfo Accumulation of fossil diatoms in marine sediments is variably affected by the production and dissolution of diatom frustules across space and time. No quantitative proxy exists for estimating the amount of silica lost to dissolution in Southern Ocean sediment cores. To address this gap, cultured valves of Fragilariopsis kerguelensis, the dominant planktonic diatom in the Pleistocene to recent Southern Ocean, were dissolved progressively under controlled laboratory conditions. Expansion, and eventual conjoining, of the areolae of F. kerguelensis advances through several phases, which are presented herein as a measure of diatom dissolution. In order to evaluate the validity of laboratory methods, this proxy is applied to sediments from piston core PS58/254 from the Amundsen Sea. Dissolution-induced degradation morphologies from laboratory experiments are seen to be identical to those preserved naturally in sediments, thus suggesting the laboratory conditions accurately reflect the natural process of dissolution. These resultsrepresentthe firstapplication of afullyquantitativeproxyfordiatomdissolution toSouthernOcean sediments, revealing that meaningful paleoceanographic data can be generated via taphonomic analysis of diatom valves. Intensity of diatom dissolution in core PS58/254 does not correspond to the relative abundance of any diatom species, clay mineralogy, or linear sedimentation rates. However, a correlation does exist between glacial cyclicity and diatom dissolution, indicating that variation in average wall thickness and susceptibility to dissolution reflects primary conditions during formation of the silica wall, transit through the water column, and eventual deposition. Higher overall silica dissolution is observed during each glacial period and enhanced silica preservation during interglacials. This result is consistent with the predictions of the silicic acid leakage hypothesis (SALH), whereby reduced rates of Si usage in the Southern Ocean lead to shifts in marine primary productivity in the tropics from coccolithophoriddominance duringinterglacialstodiatomdominanceduringglacials.Thiswouldfuela decrease in atmospheric pCO2duringglacials. Silica acidleakage isdrivenbya decreaseinthe Si:Nratioofdiatoms,caused by increased iron input to glacial-aged Southern Ocean waters. This resulted in more lightly silicified diatom valves in the Southern Ocean, which dissolved more quickly in the upper water column, thereby maintaining the high silica content of glacial-aged SO waters, and allowing for export of Si to upwelling zones in the lower latitudes.

Single laboratory method validation for cyanide in beans with insufficient levels of β-glucosidase activity

Methods to determine cyanide in beans, such as the AOAC OMA 915.03 utilise endogenous enzyme linamarase (β-glucosidase) to liberate hydrogen cyanide (HCN). However, several beans are found to contain insufficient levels of β-glucosidase activity to evaluate their levels of cyanide. This implies some beans indicate false negative using official methods. Therefore, we developed a method to analyse cyanide in beans which lack linamarase activity, and a single laboratory validation was performed. This method used 3 U of linamarase and samples were maintained at 38 °C for 24 h to hydrolyse the cyanogenic glucoside in the beans. After hydrolysis, water and antifoam were added, then, this mixture was distilled to trap cyanide in an alkaline solution. The trapped cyanide was then quantified using a 4-pyridinecarboxylic acid-pyrazolone colouring method. To validate this method, non-cyanide containing blank bean (cowpea) was spiked with potassium cyanide (KCN) from 1.0 to 500 mg/kg and linamarin from 5.0 to 25 mg/k...

Detection Dicyandiamide in Environmental Water by Liquid Chromatography–Mass Spectrometry Combined with Solid Phase Extraction Method

A new analytical method has been developed to determine dicyandiamide in environmental water. Residues of dicyandiamide were extracted from the environmental water samples by solid phase extraction using Waters Sep-Pak AC2 Cartridge. The extraction procedure condition (included loading, washing, and eluting) of flow rate of 1.0 mL/min, and dicyandiamide was eluted with 20 mL of acetonitrile and methanol (v/v = 60:40) mixture solution, and preconcentrated using nitrogen evaporation followed by analysis with liquid chromatography–mass spectrometry. Laboratory method validation data was collected in water samples at concentration down to 1.0 ng/mL. Average recovery and relative standard deviation using spiked calibration standard curve were 89% and 2.9%, respectively. The method provides a effective approach to monitor the contaminant in environmental water samples.

Determination of myo-inositol in infant formulae and milk powders using capillary gas chromatography with flame ionisation detection

A gas chromatography–flame ionisation detection method was developed to establish the content of myo-inositol in milk powders and infant formulations subsequent to formation of the volatile trialkylsilyl derivative. Samples were prepared by acid digestion, thereby releasing inositol from its multiple bound forms. A digestion time of 4 h at 114 °C was sufficient to hydrolyse potentially interfering carbohydrates and quantitatively recover bound inositol from milk-based products. Single laboratory method validation showed a within-day relative standard deviation (RSDr) of 5.9% and between-day relative standard deviation (RSDiR) of 9.6%. A between-laboratory (n = 7) collaborative study yielded an average reproducibility (RSDR) of 12.8%, considered fit-for-purpose as a quality control method for milk-based infant formulations. Soy-based products, containing significant inositol hexaphosphate (IP6, phytic acid), required 32 h for release of all inositol. Although current regulations do not specify the inclusion of IP6 in an inositol measurement, the current method allows for its discrimination if present.

Single laboratory method validation comparing MS3 with FI/MS fingerprinting, and quantitation strategies for the accurate determination of ochratoxins in beer

The current mycotoxin safety concerns demand the availability of reliable and selective control systems to measure the content of mycotoxins present in daily food. To address this, we report a prospective analytical tool involving the application of a flow injection mass spectrometry (FI/MS) tandem artificial neural network (ANN) strategy to predict the amount of ochratoxin A and B (OTA and OTB) in beer. Triple stage mass spectrometry (MS3) aided by chromatographic separation was applied as a reference method for comparison. 0.1% formic acid and methanol were used to convert ochratoxins into their respective ions under negative MS polarity. For experiments involving MS3, ochratoxins were separated by reversed-phase liquid chromatography in a 6 min run, ionised using electrospray ionisation, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge ratios were used to perform quantitation in MS3 mode. For experiments involving FI/MS, no chromatographic separation was performed. Approximately 2% of the mass spectra was used for model construction. ANN models representing each ochratoxin were individually trained and validated using three sets of matrix-matched and matrix-free calibration curves at nine concentration levels of 2.5, 10, 25, 50, 100, 200, 300, 400 and 500 μg/l. Quintuplicate analyses were made in FI/ MS mode providing a total of 270 spectra for both OTA and OTB. Single measurement was made for each sample in MS3 mode. A root-mean-square error value of &lt;1% was reported for both ochratoxin models in beer. Limits of quantitation were determined to be 0.2 μg/kg for both MS3 and FI/MS mode. Recovery assessment was performed over two days using beer blanks (n=6) spiked at three concentration levels of 5, 100 and 200 μg/kg. Extraction using acetonitrile provided excellent recovery ranges of 88 to 102% for both MS techniques. Relative standard deviations of 10% or better were achieved for interday spike recovery experiments. The successful utilisation of FI/MS without performing chromatographic separation implies the availability of a new analytical tool relevant to the field of mycotoxin analysis, possibly offering analyte specificity exceeding the capability of MS3 through chemometry.

Single laboratory method validation comparing MS&lt;sup&gt;3&lt;/sup&gt; with FI/MS fingerprinting, and quantitation strategies for the accurate determination of ochratoxins in beer

The current mycotoxin safety concerns demand the availability of reliable and selective control systems to measure the content of mycotoxins present in daily food. To address this, we report a prospective analytical tool involving the application of a flow injection mass spectrometry (FI/MS) tandem artificial neural network (ANN) strategy to predict the amount of ochratoxin A and B (OTA and OTB) in beer. Triple stage mass spectrometry (MS3) aided by chromatographic separation was applied as a reference method for comparison. 0.1% formic acid and methanol were used to convert ochratoxins into their respective ions under negative MS polarity. For experiments involving MS3, ochratoxins were separated by reversed-phase liquid chromatography in a 6 min run, ionised using electrospray ionisation, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge ratios were used to perform quantitation in MS3 mode. For experiments involving FI/MS, no chromatographic separation was performed. Approximately 2% of the mass spectra was used for model construction. ANN models representing each ochratoxin were individually trained and validated using three sets of matrix-matched and matrix-free calibration curves at nine concentration levels of 2.5, 10, 25, 50, 100, 200, 300, 400 and 500 μg/l. Quintuplicate analyses were made in FI/ MS mode providing a total of 270 spectra for both OTA and OTB. Single measurement was made for each sample in MS3 mode. A root-mean-square error value of <1% was reported for both ochratoxin models in beer. Limits of quantitation were determined to be 0.2 μg/kg for both MS3 and FI/MS mode. Recovery assessment was performed over two days using beer blanks (n=6) spiked at three concentration levels of 5, 100 and 200 μg/kg. Extraction using acetonitrile provided excellent recovery ranges of 88 to 102% for both MS techniques. Relative standard deviations of 10% or better were achieved for interday spike recovery experiments. The successful utilisation of FI/MS without performing chromatographic separation implies the availability of a new analytical tool relevant to the field of mycotoxin analysis, possibly offering analyte specificity exceeding the capability of MS3 through chemometry.

Determination of fusarium mycotoxins in wheat, maize and animal feed using on-line clean-up with high resolution mass spectrometry

An automated method involving on-line clean-up and analytical separation in a single run using TurboFlow™ reversed phase liquid chromatography coupled to a high resolution mass spectrometer has been developed for the simultaneous determination of deoxynivalenol, T2 toxin, HT2 toxin, zearalenone and fumonisins B1 and B2 in maize, wheat and animal feed. Detection was performed in full scan mode at a resolution of R = 100,000 full width at half maximum with high energy collision cell dissociation for the determination of fragment ions with a mass accuracy below 5 ppm. The extract from homogenised samples, after blending with a 0.1% aqueous mixture of 0.1% formic acid/acetonitrile (43:57) for 45 min, was injected directly onto the TurboFlow™ (TLX) column for automated on-line clean-up followed by analytical separation and accurate mass detection. The TurboFlow™ column enabled specific binding of target mycotoxins, whereas higher molecular weight compounds, like fats, proteins and other interferences with different chemical properties, were removed to waste. Single laboratory method validation was performed by spiking blank materials with mycotoxin standards. The recovery and repeatability was determined by spiking at three concentration levels (50, 100 and 200% of legislative limits) with six replicates. Average recovery, relative standard deviation and intermediate precision values were 71 to 120%, 1 to 19% and 4 to 19%, respectively. The method accuracy was confirmed with certified reference materials and participation in proficiency testing.

Coupled Turbulent Flow Chromatography: LC–MS/MS Method for the Analysis of Pesticide Residues in Grapes, Baby Food and Wheat Flour Matrices

This paper describes an on-line sample preparation method for the simultaneous determination of 48 pesticides in grapes, baby food and wheat flour matrices. Target pesticides were selected to represent a wide variety of chemical structures and three typical matrices were selected. Turbulent flow chromatography was applied for on-line sample cleanup directly coupled to LC–MS/MS. The aim of the method was to reduce total analysis time, eliminate manual laboratory work, provide clean extracts and achieve reproducible results. Single laboratory method validation was conducted establishing limits of detection between 0.8 and 6.0 ng g−1 for baby food, and 0.8–10.3 ng g−1 for other matrices. Within-day precision values varied between 4 and 18 %, while between-day precisions were in the range 5–22 %. Method recovery ranged from 67 to 124 %, and method accuracy was demonstrated by analysis of external quality control samples. The method was also tested on 24 different survey samples from both bio and organic origin. The method was shown to be convenient, fast and fit for purpose in meeting regulatory requirements for pesticide residue monitoring.