Matthew Feldhammer (Presenter)
Emory University
Authorship: Matthew Feldhammer (1), Dan Menasco (2), Willa Zhang (1) and James C. Ritchie (1)
(1) Emory University, Department of Pathology and Laboratory Medicine, (2) Biotage Inc.
| Short Abstract The need to provide accurate and quantifiable data in the detection of patient adherence to smoking cessation programs necessitates the utilization of high-resolution instrumentation. Reliance on immunoassay-based approaches can lack the sensitivity and specificity to provide a complete clinical picture in the context of various nicotine replacements and smoke cessation therapies. In the healthcare setting, the ability to provide clinicians with this information can have a significant impact on organ allocation and transplantability in addition to informing surgical eligibility. Anabasine, an alkaloid present in tobacco plants, is often utilized as a primary marker of active smoking. Surprisingly, during our validation of a new LC-MSMS assay, we determined that this utilization resulted in a very high false negative rate when compared to patients’ self-reported smoking status. |
Long Abstract
Introduction:
Tobacco use is one of the single largest contributors to preventable diseases in the world today. Both long and short-term health risks are associated with tobacco use including increased incidences for various cancers, coronary artery disease, and atherosclerosis. In addition tobacco exposure is strongly correlated with increased postoperative risks for infections as well as negative outcomes in both transplant donors as well as recipients [1, 2]. Nicotine screening has become a routine part of the clinical workup for pre-transplant eligibility as well as many elective surgical procedures. Routine screening protocols often rely on EMIT based assays, which lack the capability to distinguish nicotine which is has a very short half life from some of it’s longer lived metabolites such as trans-3’-hydroxycotinine. Additionally these screens also fail to distinguish between active tobacco use and nicotine replacement therapy (NRT) leaving some patients with false positive results (positive nicotine due to patches or gum but no anabasine) potentially making them ineligible for organ transplantation and various surgeries. The clinical utilization of LC-MS/MS technology can bypass this limitation by simultaneously providing quantitative analyses of nicotine as well as several of the metabolic products including those purported to distinguish patients using NRTs. One of several reasons limiting the widespread adoption of the use of clinical mass spectrometry has been the extended amount of time required for sample by highly skilled technologists. In an effort to overcome this hurdle several manufacturers have begun implementing automated pipetting and sample extraction stations to reduce the workload and improve sample turn-around time. We therefore set out to validate an LC-MS/MS method for the quantification of nicotine, its metabolites: cotinine, trans-3’-hydroxycotinine, and the tobacco plant alkaloid anabsine in patient urine. All four metabolites will be examined under routine clinical screening applications using the Biotage® Extrahera™ liquid handling station to evaluate the utility of sample prep automation.
Methods:
Patient urine samples were obtained from our orthopedics and transplant centers. Samples analyzed by automation were purchased from UTAK. Interfering matrix components were removed using supported liquid extraction (SLE) columns either by vacuum manifold or on the Extrahera (Biotage®) automation station. The extracted analytes (nicotine, cotinine, trans-3’-hydroxycotinine and anabasine) were chromatographically resolved over 7 minutes on a Waters 50 x 2.1, 1.7 μm BEH-C18 column prior to injection and analysis by tandem mass spectrometry (Waters Corp.). Linear responses were evaluated across the typical range reported for routine tobacco users. Patients smoking status was self reported to Emory clinicians and obtained by analyses of the electronic medical record. Patients were categorized as either active smokers (nicotine > 30 ng/mL; cotinine > 50 ng/mL; trans-3’-hydroxycotinine > 120 ng/mL; anabasine > 3 ng/mL) or non-smokers if they did not fulfill the above criteria. [3].
Results:
The quantification limits of the method were 1-5 ng/mL and the limits of detection ranged from 0.6-5 ng/mL. The linear range for all analytes was confirmed over 5-5000 ng/mL. Interday and intraday precisions for all analytes had a C.V. of <15% at the low end of the quantification limit. Method comparison studies were performed with either GC-MS (within institution method) or by LC-MS/MS (ARUP) correlation coefficients were determined to be between (0.95-0.99). Under the auspice of automation, recoveries were determined to be 107, 95, 53, and 107 percent for nicotine, anabasine, trans-3’-hydroxycotinine, and cotinine, respectively. Moreover, sample processing times (completed in batches of 24) decreased by 50% using the Extrahera™ automation station when compared to manual extractions. Most surprisingly we determined that 64% of our patients that self declared as active smokers and tested positive for nicotine had anabasine levels below <3 ng/ml.
Conclusions.
Nicotine, cotinine, trans-3’-hydroxycotinine and anabasine can be simultaneously accurately quantified in human urine by LC-MS/MS to distinguish between smokers and non-smokers. Moreover, workflow efficacy improved significantly when automation was integrated into the pipeline. Interestingly the clinical utility of anabasine as a means of detecting active tobacco use is highly questionable, as up to 64% of admitted smokers had no detectable levels of the alkaloid.
References & Acknowledgements:
References
1. Corbett, C., M.J. Armstrong, and J. Neuberger, Tobacco smoking and solid organ transplantation. Transplantation, 2012. 94(10): p. 979-87.
2. Sorensen, L.T., et al., Risk factors for tissue and wound complications in gastrointestinal surgery. Ann Surg, 2005. 241(4): p. 654-8.
3. Moyer, T.P., et al., Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles. Clin Chem, 2002. 48(9): p. 1460-71.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |