COMMENT pubs.acs.org/ac
Comment on “Effect of Uncontrolled Factors in a Validated Liquid Chromatography Tandem Mass Spectrometry Method Question Its Use As a Reference Method for Marine Toxins: Major Causes for Concern” Arjen Gerssen,* Hester J. van den Top, and Hans P. van Egmond RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands
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n a recent paper in Analytical Chemistry by Otero and coworkers,1 an attempt was made to show the effects of uncontrolled factors in a validated liquid chromatography tandem mass spectrometric method for marine biotoxins. The authors questioned if this method can be used as a reference method for marine biotoxins. In our opinion, the paper contains results of experiments where technical errors were made. In addition there are inconsistencies and inaccuracies, while some relevant information is lacking. Therefore, the results presented in this paper are questionable and the conclusions of the authors are not justified. Below we have summarized our main criticisms. (1) The authors wrongly refer to the official EU reference method and subsequent validation study, on which agreement would exist within the EU. In this respect an European Reference Laboratory (EURL) standard operating procedure (SOP) is mentioned, and reference is made to a German validation study performed by These et al. This EURL SOP version 2, reference 20 in the original paper does not contain validation data. When we read legislation (15/2011/EC), the reference method can be found through the Web site of the EURL on Marine Biotoxins (EURL-MB).2,3 The publication of These et al. does not contain validation data on which agreement is on in the EURL-National Reference Laboratory (NRL) network.3,4 (2) In the section Materials and Methods, numerous errors and questionable settings occur. Important information is missing in the paper, such as, are the same settings used for positive and negative electrospray ionization (as this will effect detectability)? The described observation that less ion transitions will improve detectability should have been supported with details about the dwell times used and the amount of data points obtained over a peak to prove that the correct settings were applied to draw this conclusion. It is also essential to know how the optimal settings for the transitions were determined to clearly understand the sections on results and discussion. In the discussion, the focus is on quantifying okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) against each other and it is important that the same settings are used for ionization and fragmentation of these toxins. Figure 1 leaves the impression that not the same settings are used. When, for example, DTX1 (MET, MP P) is quantified against OA, an overestimation of approximately 40% is observed (Figure 1C). Under controlled conditions, we would then expect an underestimation of approximately 40% when OA (MET, MP P) is quantified against DTX1 (Figure 1G). However, this seems not the case: again an overestimation of 70% is reported! The same phenomena can be observed in other occasions in Figure 1, r 2011 American Chemical Society
and not only in the ultraperformance liquid chromatography tandem mass spectrometry (UPLC MS/MS) experiments but also in the LC MS/MS experiments (e.g., compare parts E and I of Figure 1). On the basis of these results, the authors should have addressed these contradictive findings in detail considering the conclusions presented with respect to the factors studied (mobile phase, dilution solvent, UPLC or LC, and MS method). (3) The authors indicate that no detectable peaks were observed with the QTrap when polarity switching was used even at concentrations of 300 ng/mL. Do the authors have an explanation for this? Were the appropriate dwell, interscan, and polarity switch times chosen? This information is lacking in the Materials and Methods section. (4) It is not clear how the MS performance was checked. The authors indicate that they checked performance of the equipment before starting analysis but only refer to the HPLC pumps and retention times. This paper’s conclusions are focusing on the mass spectrometric detection and therefore we would expect that the performance check would have been focused on the MS performance before starting analysis. Which criteria are used and how is a conclusion drawn whether an uncontrolled factor is caused by the method applied or by the system used? (5) It would have been more balanced if the authors would have pointed at the fact that the mouse bioassay was never formally validated in an interlaboratory validation study. In addition, they should have more elaborated on the fact that in the relevant opinion of the European Food Safety Authority (EFSA), a large list of drawbacks of the mouse bioassay is given.5 (6) Throughout the paper several citations are made to the work of Gerssen et al. These were not correctly done. For example, it is mentioned that Gerssen et al. published an interlaboratory study but the cited paper is only referring to an in-house validation study.6 Gerssen is coauthor of an accepted paper in Food Additives and Contaminants, “Quantitative determination of marine lipophilic toxins in mussels, oysters and cockles using liquid chromatography mass spectrometry. Interlaboratory validation study”, which focuses on an interlaboratory study of the method described in the in-house validation paper.7 Furthermore, the authors are referring to papers that apply simultaneously positive and negative ionization.6,8 The method described by Gerssen et al. is using alkaline chromatographic conditions. Unlike chromatography under acidic conditions, Published: October 18, 2011 476
dx.doi.org/10.1021/ac2022663 | Anal. Chem. 2012, 84, 476–477
Analytical Chemistry
COMMENT
under alkaline conditions there is a separation of toxins preferably analyzed in negative (OA group and yessotoxin group toxins) and positive (azaspiracid group toxins and pectenotoxin-2) ionization mode is occurring.9 Therefore, this method does not run positive and negative ionization simultaneously. (7) Reference to 2002/657/EC is not appropriate. This document is not for the purpose of interlaboratory validation studies and can only be applied to intralaboratory validation studies.10 We conclude that the uncontrolled factors identified by Otero and co-workers1 are not valid and that the concerns of the authors are not convincing. The experiments and observations described in this paper do not justify the questioning of the convenience or the reliability of having MS/MS as a reference method for protecting consumers.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +31-317-480256. Fax: +31-317-417717. E-mail: Arjen.
[email protected].
’ REFERENCES (1) Otero, P.; Alfonso, A.; Alfonso, C.; Rodríguez, P.; Vieytes, M. R.; Botana, L. M. Anal. Chem. 2011, 83, 5903–5911. (2) Commission Regulation 15/2011, Off. J. Eur. Union, 2011, L6, 3 6. (3) EU Harmonised SOP LIPO LCMSM, version 4; http://www. aesan.msps.es/en/CRLMB/web/procedimientos_crlmb/crlmb_ standard_operating_procedures.shtml, accessed July 22, 2011. (4) These, A.; Klemm, C.; Nausch, I.; Uhlig, S. Anal. Bioanal. Chem. 2011, 399, 1245–1256. (5) EFSA Contaminants Panel., EFSA J. 2008, 589, 1-62. (6) Gerssen, A.; van Olst, E. H.; Mulder, P. P.; de Boer, J. Anal. Bioanal. Chem. 2010, 397, 3079–88. (7) van den Top, H. J.; Gerssen, A.; McCarron, P.; van Egmond, H. P. Food Addit. Contam. 2011, 28, 1745–1757. (8) Fux, E.; McMillan, D.; Bire, R.; Hess, P. J. Chromatogr., A 2007, 1157, 273–280. (9) Gerssen, A.; Mulder, P. P. J.; McElhinney, M. A.; De Boer, J. J. Chromatogr., A 2009, 1216, 1421–1430. (10) Commission Regulation 657/2002, Off. J. Eur. Commun, 2002, L221, 8 36.
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dx.doi.org/10.1021/ac2022663 |Anal. Chem. 2012, 84, 476–477
