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Tanaka H.,Japan National Institute of Health Sciences | Takino M.,Agilent Technologies | Sugita-Konishi Y.,Japan National Institute of Health Sciences | Tanaka T.,Kobe Institute of Health | And 3 more authors.
Rapid Communications in Mass Spectrometry | Year: 2010

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with immunoaffinity extraction is described. A clean-up was carried out using a DZT MS-PREP® immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean-up were compared. The results with the DZT MS-PREP® IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent-tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS-PREP® IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03-0.33 ng g-1. From these studies, it is suggested that use of an IAC is effective in the clean-up of each mycotoxin, and, when combined with LC/ESI-MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity. © 2010 John Wiley & Sons, Ltd.


Blackburn M.A.,Covance | Kapron J.,Thermo Fisher Scientific | Mackie J.,R BiopharmRhone Ltd. | Firth J.,Covance | And 2 more authors.
Bioanalysis | Year: 2011

Background: The determination of pharmacokinetic parameters requires accurate and reliable bioanalytical methods. Even using highly selective MS/MS, interferences can occur. This paper describes the source of some of these interferences with an example discussed involving the problem of a ketamine interference in a plasma assay. Results: The introduction of field asymmetric waveform ion mobility spectrometry (FAIMS) removed the interference, enhanced signal-to-background and met GLP acceptance criteria. Relative to the non-FAIMS method, assay calibration characteristics were improved. The FAIMS source gave optimal performance following the introduction of a split in order to reduce the inlet flow to approximately 0.4 ml/min. Conclusion: The introduction of ion-mobility separation into a bioanalytical LC-MS/MS method can remove unexpected isobaric interferences without the need to redevelop the chromatography. © 2011 Future Science Ltd.


Senyuva H.Z.,Middle East Technical University | Gilbert J.,Middle East Technical University | Turkoz G.,Turizm San. ve Tic. Ltd STI | Leeman D.,R Biopharm Rhone Ltd | Donnelly C.,R Biopharm Rhone Ltd
Journal of AOAC International | Year: 2012

A comparison has been made of an LC/MS/MS method using direct analysis of acetonitrile extracts of feed and cereal samples and a method using acetonitrile extraction and subsequent immunoaffinity column (IAC) cleanup. Naturally contaminated samples containing one or more of deoxynivalenol, zearalenone, T-2, and HT-2 toxins were analyzed together with test materials containing known toxin levels. LC/MS/MS ion ratios and peak profiles, repeatability, and LOQs were used as the basis for comparing the two approaches. The method without cleanup had poorer performance than the method with IAC cleanup in terms of identification based on ion ratios compared to standards. Without cleanup, there was more evidence of background interference, and monitored ions were invariably seen against a noisy background. Nevertheless, quantification of samples analyzed without cleanup gave reasonable agreement with the levels found in the same samples that had received IAC cleanup. Repeatability was poorer with no cleanup, and LOQ values were higher for HT-2 and T-2 toxins, but there was no evidence of any adverse effects on MS performance with repeated injections of crude extracts. Overall, it was concluded that LC/MS/MS analysis of samples with no cleanup is adequate for screening, but for definitive measurements (e.g., for food regulatory control purposes) IAC cleanup remains essential.


Rhemrev R.,R Biopharm Rhone Ltd | Pazdanska M.,R Biopharm Rhone Ltd | Marley E.,R Biopharm Rhone Ltd | Biselli S.,Eurofins | Staiger S.,Eurofins
Journal of AOAC International | Year: 2015

A novel reusable immunoaffinity cartridge containing monoclonal antibodies to aflatoxins coupled to a pressure resistant polymer has been developed. The cartridge is used in conjunction with a handling system inline to LC with fluorescence detection to provide fully automated aflatoxin analysis for routine monitoring of a variety of food matrixes. The handling system selects an immunoaffinity cartridge from a tray and automatically applies the sample extract. The cartridge is washed, then aflatoxins B1, B2, G1, and G2 are eluted and transferred inline to the LC system for quantitative analysis using fluorescence detection with postcolumn derivatization using a KOBRA® cell. Each immunoaffinity cartridge can be used up to 15 times without loss in performance, offering increased sample throughput and reduced costs compared to conventional manual sample preparation and cleanup. The system was validated in two independent laboratories using samples of peanuts and maize spiked at 2, 8, and 40 μg/kg total aflatoxins, and paprika, nutmeg, and dried figs spiked at 5, 20, and 100 μg/kg total aflatoxins. Recoveries exceeded 80% for both aflatoxin B1 and total aflatoxins. The between-day repeatability ranged from 2.1 to 9.6% for aflatoxin B1 for the six levels and five matrixes. Satisfactory Z-scores were obtained with this automated system when used for participation in proficiency testing (FAPAS®) for samples of chilli powder and hazelnut paste containing aflatoxins.


Marley E.,R Biopharm Rhone Ltd | Brown P.,R Biopharm Rhone Ltd | Mackie J.,R Biopharm Rhone Ltd | Donnelly C.,R Biopharm Rhone Ltd | And 3 more authors.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2015

A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg−1 to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg−1. For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg−1 the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02  and 0.6 µg kg−1 respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS. © 2015 Taylor & Francis.


PubMed | Catholic University of the Sacred Heart, R Biopharm Rhone Ltd and Fera Science Ltd.
Type: Journal Article | Journal: Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment | Year: 2015

A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 g kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 g kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 g kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 g kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.


Mackie J.,R Biopharm Rhone Ltd | Marley E.,R Biopharm Rhone Ltd | Donnelly C.,R Biopharm Rhone Ltd
Journal of AOAC International | Year: 2013

A single-laboratory validation was conducted to establish the effectiveness of an immunoaffinity column (IAC) cleanup procedure followed by LC/MS/MS for the determination of chloramphenicol (CAP) in honey and prawns. Honey is dissolved in buffer solution and centrifuged, and an aliquot applied to an IAC. For prawns, a portion of the homogenized sample is shaken with buffer and centrifuged, and an aliquot similarly applied to an IAC. For both matrix extracts, CAP is removed from the IAC with neat methanol, then directly analyzed by electrospray LC/MS/MS in the negative ionization mode using m/z 321 as a precursor ion and m/z 257 and 152 as qualifier and quantifier ions, respectively. Test portions of blank honey and prawns were fortified with CAP to give levels of 0. 3, 1.0, and 5.0 μg/kg. Recoveries of CAP on 3 consecutive days ranged from 83-103% for honey and 84-108% for prawns. Based on results for fortified blank matrixes (triplicate at three levels), the RSD for repeatability (RSDr) averaged 8.4% for honey and 4.8% for prawns. The method LOD was 0.05 for prawns and 0.16 μg/kg for honey, both well below the minimum required method performance limit for CAP. The accuracy of the method was demonstrated by participation in proficiency testing, where satisfactory Z-scores were obtained for CAP in incurred samples of both honey and prawns. The method was shown to be applicable to a wide range of other matrixes, including milk, egg, royal jelly, meat, and seafood products.


Wilcox J.,R Biopharm Rhone Ltd | Donnelly C.,R Biopharm Rhone Ltd | Leeman D.,R Biopharm Rhone Ltd | Marley E.,R Biopharm Rhone Ltd
Journal of Chromatography A | Year: 2015

This paper describes the use of two immunoaffinity columns (IACs) coupled in tandem, providing selective clean-up, based on targeted mycotoxins known to co-occur in specific matrices. An IAC for aflatoxins+ochratoxin A+fumonisins (AOF) was combined with an IAC for deoxynivalenol+zearalenone+T-2/HT-2 toxins (DZT); an IAC for ochratoxin A (O) was combined with a DZT column; and an aflatoxin+ochratoxin (AO) column was combined with a DZT column. By combining pairs of columns it was demonstrated that specific clean-up can be achieved as required for different matrices. Samples of rye flour, maize, breakfast cereal and wholemeal bread were analysed for mycotoxins regulated in the EU, by spiking at levels close to EU limits for adult and infant foods. After IAC clean-up extracts were analysed by LC-MS/MS with quantification using multiple reaction monitoring. Recoveries were found to be in range from 60 to 108%, RSDs below 10% depending on the matrix and mycotoxin combination and LOQs ranged from 0.1ng/g for aflatoxin B1 to 13.0ng/g for deoxynivalenol. Surplus cereal proficiency test materials (FAPAS®) were also analysed with found levels of mycotoxins falling within the satisfactory range of concentrations (Z score≤±2), demonstrating the accuracy of the proposed multi-mycotoxin IAC methods. © 2015 Elsevier B.V..


Marley E.,R Biopharm Rhone Ltd | Brown P.,R Biopharm Rhone Ltd | Leeman D.,R Biopharm Rhone Ltd | Donnelly C.,R Biopharm Rhone Ltd
Journal of AOAC International | Year: 2016

The analysis of citrinin in various cereals (wheat, oats, maize, rice, and rye and multigrain breakfast cereal), red yeast rice (dietary supplement and traditional medicine), distillers dried grain with solubles, and barley (animal feed) was carried out using a citrinin immunoaffinity column (IAC) for sample cleanup before LC analysis with fluorescence detection (LC-fluorescence). To establish method performance characteristics, wheat was spiked with citrinin at levels of 10-200 g/kg, whereas red yeast rice was spiked at levels of 100-3000 g/kg. Methanol-water (75 + 25, v/v) was used for the extraction of cereals and animal feed, and extraction was with 100% methanol for red yeast rice. Cleanup used a commercial citrinin IAC, followed by LC-fluorescence (ex, 330 nm; em, 500 nm). Recoveries ranged from 80 to 110%, with r from 0.7 to 4.3%. The LOQ for citrinin in both wheat and red yeast rice was 10 g/kg, with an LOD of 3 g/kg. Satisfactory performance was demonstrated in a proficiency testing exercise for a sample of maize contaminated with both citrinin and ochratoxin A. It was concluded that the commercial citrinin IAC was capable of providing an efficient and effective cleanup of complex food and feed matrixes to enable citrinin to be reliably determined with the specific LC-fluorescence system used.


This paper describes the use of two immunoaffinity columns (IACs) coupled in tandem, providing selective clean-up, based on targeted mycotoxins known to co-occur in specific matrices. An IAC for aflatoxins+ochratoxin A+fumonisins (AOF) was combined with an IAC for deoxynivalenol+zearalenone+T-2/HT-2 toxins (DZT); an IAC for ochratoxin A (O) was combined with a DZT column; and an aflatoxin+ochratoxin (AO) column was combined with a DZT column. By combining pairs of columns it was demonstrated that specific clean-up can be achieved as required for different matrices. Samples of rye flour, maize, breakfast cereal and wholemeal bread were analysed for mycotoxins regulated in the EU, by spiking at levels close to EU limits for adult and infant foods. After IAC clean-up extracts were analysed by LC-MS/MS with quantification using multiple reaction monitoring. Recoveries were found to be in range from 60 to 108%, RSDs below 10% depending on the matrix and mycotoxin combination and LOQs ranged from 0.1n g/g for aflatoxin B1 to 13.0 ng/g for deoxynivalenol. Surplus cereal proficiency test materials (FAPAS()) were also analysed with found levels of mycotoxins falling within the satisfactory range of concentrations (Z score 2), demonstrating the accuracy of the proposed multi-mycotoxin IAC methods.

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