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Identification in residue analysis based on liquid chromatography with tandem mass spectrometry: Experimental evidence to update performance criteria
Mol, J.G.J. ; Zomer, P. ; Garcia Lopez, M. ; Fussell, R.J. ; Scholten, J. ; dr. Kok, A. ; Wolheim, A. ; Anastassiades, M. ; Lozano, A. ; Fernandez Alba, A. - \ 2015
Analytica Chimica Acta 873 (2015). - ISSN 0003-2670 - p. 1 - 13.
mycotoxin analysis - veterinary drugs - food - confirmation - extraction - pesticides - matrices
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is one of the most widely used techniques for identification (and quantification) of residues and contaminants across a number of different chemical domains. Although the same analytical technique is used, the parameters and criteria for identification vary depending on where in the world the analysis is performed and for what purpose (e.g. determination of pesticides, veterinary drugs, forensic toxicology, sports doping). The rationale for these differences is not clear and in most cases the criteria are essentially based on expert opinions rather than underpinned by experimental data. In the current study, the variability of the two key identification parameters, retention time and ion ratio, was assessed and compared against requirements set out in different legal and guidance documents. The study involved the analysis of 120 pesticides, representing various chemical classes, polarities, molecular weights, and detector response factors, in 21 different fruit and vegetable matrices of varying degrees of complexity. The samples were analysed non-fortified, and fortified at 10, 50 and 200µgkg(-1), in five laboratories using different LC-MS/MS instruments and conditions. In total, over 135,000 extracted-ion chromatograms were manually verified to provide an extensive data set for the assessment. The experimental data do not support relative tolerances for retention time, or different tolerances for ion ratios depending on relative abundance of the two product ions measured. Retention times in today's chromatographic systems are sufficiently stable to justify an absolute tolerance of ±0.1min. Ion ratios are stable as long as sufficient response is obtained for both product ions. Ion ratio deviations are typically within ±20% (relative), and within ±45% (relative) in case the response of product ions are close to the limit of detection. Ion ratio tolerances up to 50% did not result in false positives and reduced the false negative rate for pesticides with product ions in the low S/N range to
The assessment of selectivity in different quadrupole-orbitrap mass spectrometry acquisition
Berendsen, B.J.A. ; Wegh, R.S. ; Meijer, T. ; Nielen, M.W.F. - \ 2015
Journal of the American Society for Mass Spectrometry 26 (2015)2. - ISSN 1044-0305 - p. 337 - 346.
performance liquid-chromatography - veterinary drugs - confirmation - metabolites - residues - plasma - food - meat
Selectivity of the confirmation of identity in liquid chromatography (tandem) mass spectrometry using Q-Orbitrap instrumentation was assessed using different acquisition modes based on a representative experimental data set constructed from 108 samples, including six different matrix extracts and containing over 100 analytes each. Single stage full scan, all ion fragmentation, and product ion scanning were applied. By generating reconstructed ion chromatograms using unit mass window in targeted MS(2), selected reaction monitoring (SRM), regularly applied using triple-quadrupole instruments, was mimicked. This facilitated the comparison of single stage full scan, all ion fragmentation, (mimicked) SRM, and product ion scanning applying a mass window down to 1 ppm. Single factor Analysis of Variance was carried out on the variance (s(2)) of the mass error to determine which factors and interactions are significant parameters with respect to selectivity. We conclude that selectivity is related to the target compound (mainly the mass defect), the matrix, sample clean-up, concentration, and mass resolution. Selectivity of the different instrumental configurations was quantified by counting the number of interfering peaks observed in the chromatograms. We conclude that precursor ion selection significantly contributes to selectivity: monitoring of a single product ion at high mass accuracy with a 1 Da precursor ion window proved to be equally selective or better to monitoring two transition products in mimicked SRM. In contrast, monitoring a single fragment in all ion fragmentation mode results in significantly lower selectivity versus mimicked SRM. After a thorough inter-laboratory evaluation study, the results of this study can be used for a critical reassessment of the current identification points system and contribute to the next generation of evidence-based and robust performance criteria in residue analysis and sports doping.
The (un)certainty of selectivity in liquid chromatography tandem mass spectrometry
Berendsen, B.J.A. ; Stolker, A.A.M. ; Nielen, M.W.F. - \ 2013
Journal of the American Society for Mass Spectrometry 24 (2013). - ISSN 1044-0305 - p. 154 - 163.
drug residues - lc-ms - identification - confirmation - spectra - library - fragmentation - system - food
We developed a procedure to determine the "identification power" of an LC-MS/MS method operated in the MRM acquisition mode, which is related to its selectivity. The probability of any compound showing the same precursor ion, product ions, and retention time as the compound of interest is used as a measure of selectivity. This is calculated based upon empirical models constructed from three very large compound databases. Based upon the final probability estimation, additional measures to assure unambiguous identification can be taken, like the selection of different or additional product ions. The reported procedure in combination with criteria for relative ion abundances results in a powerful technique to determine the (un)certainty of the selectivity of any LC-MS/MS analysis and thus the risk of false positive results. Furthermore, the procedure is very useful as a tool to validate method selectivity. Figure
Advantages of Atmospheric Pressure Chemical Ionization in Gas Chromatography Tandem Mass Spectrometry: Pyrethroid Insecticides as a Case Study
Portolés, T. ; Mol, J.G.J. ; Sancho, J.V. ; Hernández, F. - \ 2012
Analytical Chemistry 84 (2012)22. - ISSN 0003-2700 - p. 9802 - 9810.
solid-phase extraction - human breast tissues - pesticide-residues - gc-ms - liquid-chromatography - samples - water - vegetables - quantification - confirmation
Gas chromatography coupled to mass spectrometry (GC/MS) has been extensively applied for determination of volatile, nonpolar, compounds in many applied fields like food safety, environment, or toxicology. The wide majority of methods reported use electron ionization (EI), which may result in extensive fragmentation of analytes compromising selectivity and sensitivity. This might also complicate the application of tandem MS due to lack of specific/abundant precursor ions. Pyrethroids are examples of compounds with this behavior. In this work, the potential of atmospheric pressure chemical ionization (APCI), a softer form of ionization, combined with GC and a triple quadrupole mass analyzer was investigated, taking pyrethroids as a case study and their determination in fruit and vegetables as example application. Ionization and fragmentation behavior of eight pyrethroids (bifenthrin, cyfluthrin, cypermethrin, permethrin, ¿-cyhalothrin, fluvalinate, fenvalerate, and deltamethrin) by APCI were studied. The formation of a highly abundant (quasi) molecular ion was the main goal because of the enhanced selectivity when used as precursor ion in tandem MS. The addition of water as a modifier was tested to promote the generation of protonated molecules, resulting in notable improvement of sensitivity and selectivity for most compounds. The excellent detectability (low detection limits (LODs)
Qualitative Aspects in the Analysis of Pesticide Residues in Fruits and Vegetables Using Fast, Low -Pressure Gas Chromatography-Time-of-Flight Mass Spectrometry
Lehotay, S.J. ; Koesukwiwat, U. ; Kamp, H.J. van der; Mol, J.G.J. ; Leepipatpiboon, N. - \ 2011
Journal of Agricultural and Food Chemistry 59 (2011)14. - ISSN 0021-8561 - p. 7544 - 7556.
gc-ms - spectral deconvolution - identification system - multiresidue method - quechers - confirmation - extraction - food
Quantitative method validation is a well-established process to demonstrate trueness and precision of the results with a given method. However, an assessment of qualitative results is also an important need to estimate selectivity and devise criteria for chemical identification when using the method, particularly for mass spectrometric analysis. For multianalyte analysis, automatic instrument software is commonly used to make initial qualitative identifications of the target analytes by comparison of their mass spectra against a database library. Especially at low residue levels in complex matrices, manual checking of results is typically needed to correct the peak assignments and integration errors, which is very time-consuming. Low-pressure gas chromatography-mass spectrometry (LP-GC-MS) has been demonstrated to increase the speed of analysis for GC-amenable residues in various foods and provide more advantages over the traditional GC-MS approach. LP-GC-MS on a time-of-flight (ToF) instrument was used, which provided high sample throughput with
A quantitative trait locus for a primary antibody response to keyhole limpet hemocyanin on chicken chromosome 14-Confirmation and candidate gene approach
Siwek, M. ; Slawinska, A. ; Nieuwland, M.G.B. ; Witkowski, A. ; Zieba, G. ; Minozzi, G. ; Knol, E.F. ; Bednarczyk, M. - \ 2010
Poultry Science 89 (2010)9. - ISSN 0032-5791 - p. 1850 - 1857.
red-blood-cells - infectious pancreatic necrosis - salmon salmo-salar - unselected traits - disease virus - laying hens - major qtl - lines - confirmation - resistance
A QTL involved in the primary antibody response toward keyhole limpet hemocyanin (KLH) was detected on chicken chromosome 14 in the experimental population, which was created by crossing commercial White Leghorn and a Polish native chicken breed (green-legged partridgelike). The current QTL location is a validation of previous experiments pointing to the same genomic location for the QTL linked to a primary antibody response to KLH. An experimental population was typed with microsatellite markers distributed over the chicken chromosome 14. Titers of antibodies binding KLH were measured for all individuals by ELISA. Statistical models applied in the Grid QTL Web-based software were used to analyze the data: a half-sib model, a line-cross model, and combined analysis in a linkage disequilibrium and linkage analysis model. Candidate genes that have been proposed were genotyped with SNP located in genes exons. Statistical analyses of single SNP associations were performed pointing out 2 SNP of an axis inhibitor protein (AXIN1) gene as significantly associated with the trait of an interest
Validation of the QTL on SSC4 for meat and carcass quality traits in a commercial crossbred pig population
Slawinska, A. ; Siwek, M. ; Knol, E.F. ; Roelofs-Prins, D.T. ; Wijk, H.J. van; Dibbits, B.W. ; Bednarczyk, M. - \ 2009
Journal of Animal Breeding and Genetics 126 (2009)1. - ISSN 0931-2668 - p. 43 - 51.
influencing economic traits - sus-scrofa chromosome-4 - genome scan analysis - landrace cross - loci - fatness - growth - confirmation - identification - linkage
Porcine chromosome 4 harbours many quantitative trait loci (QTL) affecting meat quality, fatness and carcass composition traits, detected in resource pig populations previously. However, prior to selection in commercial breeds, QTL identified in an intercross between divergent breeds require confirmation, so that they can be segregated. Consequently, the objective of this study was to validate several QTL on porcine chromosome 4 responsible for meat and carcass quality traits. The experimental population consisted of 14 crossbred paternal half-sib families. The region of investigation was the q arm of SSC4 flanked by the markers S0073 and S0813. Regression analysis resulted in the validation of three QTL within the interval: Minolta a* loin, back fat thickness and the weight of trimmed ham. The results were additionally confirmed by factor analysis. Candidate genes were proposed for meat colour, which was the most evident QTL validated in this study.
A generic method for the quantitative analysis of aminoglycosides (and spectinomycin) in animal tissue using methylated internal stan dards and liquid chromatography tandem mass spectrometry
Holthoon, F.L. van; Essers, M.L. ; Mulder, P.P.J. ; Stead, D.E. ; Caldow, M. ; Ashwin, H.M. ; Sharman, M. - \ 2009
Analytica Chimica Acta 637 (2009)1-2. - ISSN 0003-2670 - p. 135 - 143.
solid-phase extraction - precolumn derivatization - fluorescence detection - performance - antibiotics - confirmation - gentamicin - resistance - sulfate - feeds
Aminoglycosides (AGs) are a large and diverse group of antibiotics. Although AGs may cause side effects of nephrotoxicity and ototoxicity, they are still occasionally being used for the treatment of serious infections. In this study the development of a method is described for the quantitative determination and confirmation of seven aminoglycosides (and relevant isomers) and spectinomycin in animal tissues. The extraction was based on an extraction followed by a concentration and clean-up step using weak cation exchange solid phase extraction. The separation was performed by ion-pair liquid chromatography on a C18 column followed by mass spectrometric detection. The method was validated according to the EU requirements for a quantitative confirmatory method. Permethylated aminoglycosides (in-house synthesised internal standards) were used for accurate quantification. The accuracy of the analyses of AGs in kidney ranged from 94 to 111%, intra-day precision ranged between 2.5 and 7.4% (R.S.D.r) and inter-day precision ranged between 2.2 and 17.3% (R.S.D.RL, n = 21, MRL level). Accuracy (muscle tissue) varied from 83 to 128% with an intra-day precision between 2.2 and 17.3% (R.S.D.r, n = 7, MRL level). From the results it was concluded that the method was able to monitor MRL levels which ranged from 750 to 20,000 µg kg-1 for kidney and from 50 to 10,000 µg kg-1 for muscle tissue
Elimination kinetic of 17B-estradiol 3-benzoate and 17B-nandrolone laureate ester metabolites in calves' urine
Pinel, G. ; Rambaud, L. ; Cacciatore, G. ; Bergwerff, A. ; Elliott, C. ; Nielen, M.W.F. - \ 2008
Journal of Steroid Biochemistry and Molecular Biology 110 (2008)1-2. - ISSN 0960-0760 - p. 30 - 38.
tandem mass-spectrometry - anabolic-steroids - bovine urine - nandrolone metabolites - gas - 19-nortestosterone - confirmation - residues - cattle - horse
Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.