Trace Level Analysis of NNAL in Urine Using SupelMIP™ SPE – NNAL and LC-MS-MS

By: Olga Shimelis, Anna-Karin Wihlborg, Craig Aurand, An Trinh, Reporter US Volume 25.3

Olga Shimelis, Anna-Karin Wihlborg*, Craig Aurand, and An Trinh

*MIP Technologies AB, Lund, Sweden


Tobacco-specific nitrosamines (TSNAs) are created through the burning, curing, and fermentation of tobacco leaf. In 1989, the US Surgeon General provided a list of carcinogens found in tobacco products (1). Among that list were nine nitrosamines that can be found in chewing, smoking, and snuff tobacco. These TSNAs are considered to be highly carcinogenic and have been linked to tumors found in the lung, oral and esophageal cavity, cervix, and liver (1, 2). Because TSNAs are only found in tobacco products, their characterization is invaluable in the study of tobacco’s cancerous nature (3).

NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) is a TSNA found in tobacco smoke at significant amounts. Upon inhalation, NNK rapidly metabolizes into NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) (Figure 1). The extraction and quantitation of NNAL in urine is therefore a useful biomarker when assessing a subject’s exposure to tobacco smoke. NNAL is not only found in smokers but in non-smokers (via second-hand smoke) as well. Because NNAL is detected in urine at very low concentrations (<1 ng/mL), a highly specific and sensitive assay is required. Although such extraction and analysis protocols have been developed, many of them require extensive and timeconsuming (up to 2-3 days) sample preparation (4).

Figure 1. The Metabolism of NNK to NNAL

In an effort to develop a simple and highly sensitive assay for the extraction and LC-MS-MS analysis of NNAL in urine, Xia et al of the Center for Disease Control and Prevention, and MIP Technologies AB developed and validated a molecularly imprinted polymer phase and procedure specific for this application (5). In their study, they were able to achieve effective limits of detection of ~1.7 pg/mL. In this report, the utility of molecularly imprinted polymer technology is further demonstrated by comparing the SupelMIP SPE – NNAL procedure with a recently published procedure using a mixed-mode cation polymer SPE phase (6).

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Molecularly Imprinted Polymers

Molecularly imprinted polymers (MIPs) are a class of highly cross-linked polymer-based molecular recognition elements engineered to bind one target compound or a class of structurally related compounds with high selectivity. Selectivity is introduced during MIP synthesis in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities that are sterically and chemically complementary to the target analyte(s). As a result, multiple interactions (e.g., hydrogen bonding, ionic, Van der Waals, hydrophobic) can take place between the MIP cavity and analyte functional groups (Figure 2). The strong retention offered between an MIP phase and its target analyte(s) allows for the use of exhaustive wash procedures during solid phase extraction that results in superior sample cleanup prior to analysis.

Figure 2. Example of an MIP Binding Site

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The Extraction of NNAL from Urine

In this study, NNAL was extracted from human urine using SupelMIP SPE – NNAL prior to subsequent LC-MSMS analysis. The SupelMIP procedure was compared against a method adapted from a recently published study using mixed-mode cation polymer SPE sample prep approach (6). The protocols for both extraction methods are described in Table 1 .

Table 1. Description of SupelMIP SPE – NNAL and Mixed-Mode Cation-Exchange SPE Method for the Extraction of NNAL from Urine (53206-U)

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Improved Selectivity Using SupelMIP SPE – NNAL

NNAL was extracted from urine using both the SupelMIP (molecularly imprinted polymer) and mixed-mode cation-exchange procedures described in Table 1. Figure 3 is a representative ion-chromatogram (MRM 210.2/180.2) of a 1 ng/mL external standard injection of NNAL. The ionchromatograms (MRM transitions 210.2/180.2 and 210.2/93.2) for blank urine samples extracted from both procedures are detailed in Figure 4. Of the two MRM transitions, 210/93.2 was more intense; however, high level matrix impurities had co-eluted in the LC elution area of NNAL (0-2 min.) when extracting urine using the mixed-mode cation-exchange procedure. Such impurities can potentially result in signal-suppression of NNAL. Therefore, the MRM transition of 210.2/180.2 was used for quantitation. In contrast, the SupelMIP approach provided a much cleaner extract with a signal-to-noise ratio of at least 10 at the lowest spike concentration tested. This is further demonstrated by spiking NNAL into the SPE eluate of blank urine samples extracted with SupelMIP SPE – NNAL and comparing the response with NNAL standards diluted in LC-mobile phase. In Figure 5, we see that when overlapping the response of NNAL calibration curves diluted in both blank urine extracts (spiked post-extraction) and LC mobile phase, the curves overlapped almost perfectly, signifying almost no (less than 4%) signal suppression from co-extracted matrix impurities (data provided by MIP Technologies AB).

Figure 3. Ion-Chromatogram (MRM 210.2/180.2) of 1 ng/mL NNAL standard (581307-U)

Figure 4. Blank and Spiked NNAL Urine Samples Extracted with SupelMIP SPE – NNAL and Mixed-Mode Cation Exchange Polymer SPE

Figure 5. Response Comparison of NNAL Calibration Curve Generated from SupelMIP SPE – NNAL Urine Extract (post-SPE spike) vs. External Standards

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Improved Recovery Using SupelMIP NNAL

From Table 2, we see that at the 50-60 pg/mL spike level, absolute recovery using the SupelMIP protocol was higher than Mixed-Mode Cation-Exchange procedure. Note that MIP Technologies AB, the manufacturers of SupelMIP SPE – NNAL, recommends quantitating relative recovery against NNAL-d3 internal standards that are spiked into urine samples prior to SupelMIP extraction. Under such conditions analysts can readily achieve relative recovery values > 90% and limits of detection and limits of quantitation values of 5 pg/mL and 13 pg/mL, respectively (data not shown).

Table 2. Absolute Recovery of SupelMIP SPE – NNAL vs. Mixed-Mode Cation Exchange Polymer SPE

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In this report, we demonstrated the extraction of NNAL from urine using SupelMIP SPE – NNAL method against a published mixed-mode cation exchange polymer phase. Because selectivity is introduced during the development of the MIP itself, it allows for a binding site that is sterically and chemically complementary to the target analyte(s). The multiple interactions that take place between the imprint binding site and analyte(s) of interest offer strong interactions enabling the use of exhaustive wash conditions during the SPE process to provide cleaner extracts prior to analysis. The SupelMIP approach offered improved selectivity over the mixed-mode approach. This was particularly evident when analyzing blank urine sample extracts at MRM transitions 210.2/93.2. In addition, absolute recovery was also higher for the SupelMIP method relative to the mixed-mode procedure. Higher recovery is essential when analyzing NNAL at trace levels.

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  1. U.S. Department of Health and Human Services, “Reducing the Health Consequences of Smoking: 25 Years of Progress. A report of the Surgeon General, 1989”
  2. “Identification of Tobacco-specific carcinogen in the cervical mucus of smokers and non-smokers”. B. Prokopczyk, J.E. Cox, D. Hoffmann, S. E Waggoner, Journal of the National Cancer Institute, Vol. 89, No. 12, June 18, 1997
  3. “Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-specific carcinogen in the cervical mucus of smokers and non-smokers”. M. Meger, I. Meger-Kossien, K. Riedel; G. Scherer; Biomarkers, Volume 5, Issue 1 January 2000, pages 33 – 45
  4. “Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke”. S.S. Hecht and D. Hoffman, Commentary, American Health Foundation, pages 875-884.
  5. “Analysis of the Tobacco-Specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)- 1-butanol in Urine by Extraction on a Molecularly Imprinted Polymer Column and Liquid Chromatography/Atmospheric Pressure Ionization Tandem Mass Spectrometry”, Xia Y, McGuffey JE, Bhattacharyya S, Sellergren B, Yilmaz E, Wang L, and Bernert JT, Anal. Chem. 77 (2005) 7639-7645
  6. “Liquid chromatographic/tandem mass spectrometric method for the determination of the tobacco-specific nitrosamine metabolite NNAL in smokers’ urine”, G.D.Byrd, M.W.Ogden in J. Mass Spectrom. 2003 (38) 98-107

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