Trace analysis of tobacco-specific nitrosamines

By: Nicole Amann, Analytix Volume 8 Issue 2

New standards complement SupelMIP™SPE phases for highly sensitive LC-MS analysis of TSNAs in urine

Nicole Amann, Product Manager Analytical Standards

There is a perpetual need for sample preparation devices that provide high analyte selectivity and recovery, but do so simply and economically. The need is especially strong when dealing with low-level or trace detection in complex matrixes. Mass spectrometric detection can provide the high sensitivity and specificity, but its effectiveness is dampened by interferences from the sample matrix that suppress ionisation.

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SupelMIP – Highly selective SPE phases

Recently, we addressed this dilemma by introducing SupelMIP solid phase extraction (SPE) products to the analytical market. “MIP” stands for molecularly imprinted polymers. They comprise highly cross-linked polymers that are engineered to extract a single analyte of interest or a class of structurally related analytes with an extremely high degree of selectivity. This is possible because selectivity is introduced during MIP synthesis in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities or imprints that are sterically and chemically complementary to the analyte(s) of interest. Figure 1 shows a schematic of the highly selective SupelMIP binding site and representative molecular interactions.

Figure 1. Schematic of SupelMIP SPE Binding Site

Lower detection limits

SupelMIP phases offer multiple points of interaction with only the analytes of interest. Consequently, harsher wash conditions can be used that provide cleaner extracts, lower background and better sensitivity than possible with conventional SPE phases.

Save SPE method development time

There is no need to conduct time-consuming method development. We have developed both the SupelMIP phase and the specific extraction procedure to follow to guarantee the sensitivity, precision and accuracy required from the assay.

Reduce ion suppression

LC-MS is an invaluable tool for trace analysis. Because the SupelMIP phases are extremely selective only for the target imprinted analytes, matrix interference and resulting ion suppression are not problematic. Even in complex matrixes, such as biological fluids, rapid analysis and high sensitivity are possible with SupelMIP.

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Featured analysis: Tobacco-specific nitrosamines

One application area that has benefited from the high selectivity of SupelMIP phases is the analysis of tobacco-specific nitrosamines (TSNAs). Two recent Supelco Reporter articles and a Technical Bulletin described this application in detail 1-3. TSNAs are formed through the burning, curing and fermentation of tobacco leaves. They are considered highly carcinogenic. Five TSNAs are of particular interest: N‘-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB), N-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1- butanone (NNK). Total urinary NNAL is a biomarker for monitoring exposure to TSNAs in smokers and in nonsmokers who have been exposed to second-hand smoke.

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SupelMIP SPE phases for NNAL and other TSNAs

Because NNAL is detected in urine at very low concentrations, as low as pg/mL, a highly specific and sensitive assay is called for. Although TSNA extraction and analysis protocols already exist, they require complex and time consuming sample preparation that takes on the order of days to complete. We eliminated this problem by introducing SupelMIP SPE phases for NNAL and four other TSNAs. By following the clear and simple protocol included with the SupelMIP tubes, higher sample throughput, lower limits of quantitation and greater reproducibility over conventional SPE methods can be achieved, even in the presence of high levels of nicotine. The chromatogram in Figure 2 shows the LC-MS analysis of NNAL in urine following extraction by SupelMIP NNAL. The high extraction selectivity of SupelMIP NNAL gave low matrix interferences, and permitted a highly sensitive and rapid analysis. With traditional SPE methods, because of their lack of selectivity, non-extracted matrix components reduce sensitivity and make quantification of early-eluting peaks unreliable if not impossible.

Figure 2.Ion chromatogram of NNAL standard (581307-U)

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A perfect complement: TSNA

The importance of high-quality analytical standards for reliable and quantitative analysis cannot be overstated. To complement the SupelMIP NNAL and TSNAs SPE tubes, we have developed standards of all five TSNAs. Additionally, we provide deuterated NNAL and NNK to enable low detection limits for trace analysis. For more information on TSNA analysis and SupelMIP in general, including the comprehensive SupelMIP brochure, please use the enclosed reply card, contact our technical service or visit our dedicated website:

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  1. Shimelis, O.; Whilborg, A.; Aurand, C.; Trinh, A. “Trace Level Analysis of NNAL in Urine Using SupelMIP SPE–NNAL and LC-MS-MS.” Supelco Reporter (International), 2007, 28, 7-10.
  2. Boyd, B.; Lundberg, D.; Kronauer, S.; Wihlborg, A.; Trinh, A. “The Extraction of TSNAs using Molecularly Imprinted Polymer SPE.” Supelco Reporter (US), 2007, 25.5, 12-14.
  3. Boyd, B.; Lundberg, D.; Kronauer, S.; Wihlborg, A. “Tobacco-Specific Nitrosamines: Efficient Extraction of Toxic Compounds from Complex Matrices using Molecularly Imprinted Polymers.” Supelco Technical Report (T407117).


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