Nicotine & Tobacco Research, Volume 16, Number 11 (November 2014) 1533
Letter
Response to Detection Limit and Statistical Results Lisanne Brown PhD1, Nikki L. Rider PhD, ScD2, David K. Lirette PhD3, James H. Diaz PhD4 1Evaluation and Research, Louisiana Public Health Institute, New Orleans, LA; 2Research and Evaluation, National Network of Public Health Institutes, New Orleans, LA; 3Research and Evaluation, Sagenex Labs, Slidell, LA; 4School of Public Health and School of Medicine–New Orleans, Louisiana State University Health Science Center, New Orleans, LA
Corresponding Author: Lisanne Brown, PhD, Evaluation and Research, Louisiana Public Health Institute, 1515 Poydras Street, Suite 1200, New Orleans, LA 70112, USA. Telephone: 504-301-9818; Fax: 504-301-9820; E-mail: [email protected] Received June 24, 2014; accepted June 27, 2014
have not recently smoked. Findings from the analysis of the saliva nicotine levels confirmed the findings based on saliva cotinine. Further, we selected the method because assessment of the saliva cotinine levels combined with nicotine levels and the subject’s attestation of smoking status were sufficient to assess smoker/nonsmoker status and to measure pre–post shift exposure levels at the highest level of accuracy (specificity). Changing the LOD most likely would not reliably differentiate the two classes and the combined use of cotinine and nicotine levels did. If using the LOD is as great a confounder as suggested, there are several additional ways that the data could be analyzed to confirm the findings. We appreciate Dr. Kwanda’s insights into different approaches to the analysis and are considering how reexamining the data could provide evidence to support the selection of laboratory and methodological approaches, such as the use of both nicotine and cotinine levels, to strengthen future studies.
Declaration of Interests None declared.
References 1. Matsumoto A, Ino T, Ohta M, et al. Enzyme-linked immunosorbent assay of nicotine metabolites. Environmental Health and Preventive Medicine. 2010;15(4):211–216. doi:10.1007/s12199-009-0129-2 2. Wielkoszyński T, Tyrpień K, Szumska M. The enzymelinked immunosorbent assay (ELISA) method for nicotine metabolites determination in biological fluids. Journal of Pharmaceutical and Biomedical Analysis. 2009;49(5):1256–1260. doi:10.1016/j.jpba.2008.12.026. 3. Miller E, Norris HR, Rollins DE, et al. Identification and quantification of nicotine biomarkers in human oral fluid from individuals receiving low-dose transdermal nicotine: a preliminary study. Journal of Analytical Toxicology. 2010;34(7):357–366.
doi:10.1093/ntr/ntu130 © The Author 2014. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: [email protected].
1533
Downloaded from http://ntr.oxfordjournals.org/ at Rensselaer Polytechnic Institute on April 8, 2015
Dr. Kwanda from the Department of Hygiene and Public Health, Nippon Medical School, expressed concern about the use of “limit of detection (LOD) values as real values” in our statistical analysis. While Dr. Kwanda concurs with our selection of the nonparametric statistical tests used, his main objection is with including the LOD in the analysis, and he also objected to the use of exposure as a predictor in a model of postshift cotinine with the inclusion of controls. He suggests the estimate of exposure effect be used with caution as it is “likely biased because of the inclusion of LOD values.” Dr. Kwanda also suggests that we should have used more sensitive instrumentation. The liquid chromatography–mass spectrometry (LC/MS) methodology was used because of its “gold standard” level of specificity, and it was the equipment available within our given budget. The very narrow range from LOD to level of quantitation (LOQ), 0.5 to 1.0 ng/ml, was not considered a hindrance at the time. Matsumoto et al. (1) describe the characteristics of multiple enzyme-linked immunosorbent assay (ELISA) methods, and it is noted how the specificity of these assays varies widely, and sensitivity is comparable to our method. Wielkoszyński, Tyrpień, and Szumska (2) also describe an ELISA technique, but comment that the method is less specific to their reference method (thin-layer chromatography). Miller et al. (3) mention the differences in nicotine metabolism due to genetic polymorphisms and the resultant differences in detected nicotine metabolites. Our LC/MS method, because of its specificity, measures the most studied metabolite cotinine and unmetabolized nicotine, unlike ELISA that is variably sensitive to multiple nicotine metabolites. The LOD/LOQ in most of the ELISA methods mentioned are not orders of magnitude different from our method and not in these researchers opinion enough to justify the use of a less specific method. While the results were not presented, as mentioned above, saliva nicotine levels were also measured. Based on the kinetics of nicotine and cotinine, examination of the nicotine levels allowed the study team to differentiate nontobacco users from users that have recently smoked and from chronic users that
Letter
Response to Detection Limit and Statistical Results Lisanne Brown PhD1, Nikki L. Rider PhD, ScD2, David K. Lirette PhD3, James H. Diaz PhD4 1Evaluation and Research, Louisiana Public Health Institute, New Orleans, LA; 2Research and Evaluation, National Network of Public Health Institutes, New Orleans, LA; 3Research and Evaluation, Sagenex Labs, Slidell, LA; 4School of Public Health and School of Medicine–New Orleans, Louisiana State University Health Science Center, New Orleans, LA
Corresponding Author: Lisanne Brown, PhD, Evaluation and Research, Louisiana Public Health Institute, 1515 Poydras Street, Suite 1200, New Orleans, LA 70112, USA. Telephone: 504-301-9818; Fax: 504-301-9820; E-mail: [email protected] Received June 24, 2014; accepted June 27, 2014
have not recently smoked. Findings from the analysis of the saliva nicotine levels confirmed the findings based on saliva cotinine. Further, we selected the method because assessment of the saliva cotinine levels combined with nicotine levels and the subject’s attestation of smoking status were sufficient to assess smoker/nonsmoker status and to measure pre–post shift exposure levels at the highest level of accuracy (specificity). Changing the LOD most likely would not reliably differentiate the two classes and the combined use of cotinine and nicotine levels did. If using the LOD is as great a confounder as suggested, there are several additional ways that the data could be analyzed to confirm the findings. We appreciate Dr. Kwanda’s insights into different approaches to the analysis and are considering how reexamining the data could provide evidence to support the selection of laboratory and methodological approaches, such as the use of both nicotine and cotinine levels, to strengthen future studies.
Declaration of Interests None declared.
References 1. Matsumoto A, Ino T, Ohta M, et al. Enzyme-linked immunosorbent assay of nicotine metabolites. Environmental Health and Preventive Medicine. 2010;15(4):211–216. doi:10.1007/s12199-009-0129-2 2. Wielkoszyński T, Tyrpień K, Szumska M. The enzymelinked immunosorbent assay (ELISA) method for nicotine metabolites determination in biological fluids. Journal of Pharmaceutical and Biomedical Analysis. 2009;49(5):1256–1260. doi:10.1016/j.jpba.2008.12.026. 3. Miller E, Norris HR, Rollins DE, et al. Identification and quantification of nicotine biomarkers in human oral fluid from individuals receiving low-dose transdermal nicotine: a preliminary study. Journal of Analytical Toxicology. 2010;34(7):357–366.
doi:10.1093/ntr/ntu130 © The Author 2014. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: [email protected].
1533
Downloaded from http://ntr.oxfordjournals.org/ at Rensselaer Polytechnic Institute on April 8, 2015
Dr. Kwanda from the Department of Hygiene and Public Health, Nippon Medical School, expressed concern about the use of “limit of detection (LOD) values as real values” in our statistical analysis. While Dr. Kwanda concurs with our selection of the nonparametric statistical tests used, his main objection is with including the LOD in the analysis, and he also objected to the use of exposure as a predictor in a model of postshift cotinine with the inclusion of controls. He suggests the estimate of exposure effect be used with caution as it is “likely biased because of the inclusion of LOD values.” Dr. Kwanda also suggests that we should have used more sensitive instrumentation. The liquid chromatography–mass spectrometry (LC/MS) methodology was used because of its “gold standard” level of specificity, and it was the equipment available within our given budget. The very narrow range from LOD to level of quantitation (LOQ), 0.5 to 1.0 ng/ml, was not considered a hindrance at the time. Matsumoto et al. (1) describe the characteristics of multiple enzyme-linked immunosorbent assay (ELISA) methods, and it is noted how the specificity of these assays varies widely, and sensitivity is comparable to our method. Wielkoszyński, Tyrpień, and Szumska (2) also describe an ELISA technique, but comment that the method is less specific to their reference method (thin-layer chromatography). Miller et al. (3) mention the differences in nicotine metabolism due to genetic polymorphisms and the resultant differences in detected nicotine metabolites. Our LC/MS method, because of its specificity, measures the most studied metabolite cotinine and unmetabolized nicotine, unlike ELISA that is variably sensitive to multiple nicotine metabolites. The LOD/LOQ in most of the ELISA methods mentioned are not orders of magnitude different from our method and not in these researchers opinion enough to justify the use of a less specific method. While the results were not presented, as mentioned above, saliva nicotine levels were also measured. Based on the kinetics of nicotine and cotinine, examination of the nicotine levels allowed the study team to differentiate nontobacco users from users that have recently smoked and from chronic users that
