A Collaborative Study for the Determination of Tobacco Specific Nitrosamines in Tobacco

The manuscript presents results from a collaborative study by 15 different laboratories using two different methods to determine tobacco specific nitrosamines (TSNAs) in tobacco and was performed under the auspices of the Tobacco Science Research Conference Analytical Methods Committee (TSRC-AMC). Although it is apparent that some of the laboratories failed to follow the provided protocols, both methods proved robust for determining TSNAs in a variety of different tobacco types. Twelve laboratories extracted the tobacco sample using an alkaline-methylene chloride extraction (Method 1) and nine used a buffer to extract the tobacco sample (Method 2). Six laboratories performed both methods. All participants used gas chromatography (GC) to separate the TSNAs and chemiluminescence detection. Method 1 used N-hexyl-N-nitroso-1-hexanamine (NDHA) as a surrogate (added prior to extraction) internal standard for quantitation. Method 2 used N-nitrosoguvacoline (NG) as the surrogate internal standard, NDHA as a chromatographic (added after extraction, prior to analysis) internal standard and external standard quantitation. After demonstrating that the average accuracy of both methods was at least about 92% through recovery studies, eight different tobacco types were analyzed in triplicate by each method. Means, reproducibility (precision between laboratories) and repeatability (precision within a laboratory) of results were determined for each method. After statistical analyses, it was established that both methods were capable of analyzing a variety of tobacco types and repeatability between methods was not significantly different. The limit of detection (LOD) and limit of quantitation (LOQ) were lower for Method 2 as compared to Method 1 when using the surrogate internal standard. Reproducibility variation, analyzed as the coefficient of variation, was 6% lower for Method 2 vs. Method 1 for N-nitrosonornicotine (NNN) and directionally 12% lower for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Method 2 using NDHA as the chromatographic internal standard has been recommended by the TSRC-AMC for use as a reference method. However, Method 1 using NDHA as surrogate internal standard is favored by four of the study participants because of lower chemical and material costs and higher sample throughput.