Analytical determination of CML and CEL in oral use tobacco products by HPLC-MS/MS method

Abstract

Exogenous dietary advanced glycation endproducts (AGEs) have been suggested to increase health risk. Oral use tobacco products are used as substitute for cigarette smoking due to their “reduced risk” and no second-hand smoke. They may increase AGEs intake due to their oral form of use. However, research on AGEs in oral use tobacco products is limited. A HPLC-MS/MS method was developed to survey free and total Nε-Carboxymethyllysine (CML) and Nε-Carboxyethyllysine (CEL) levels in different oral use tobacco products in this work. HPLC conditions, such as HPLC column and mobile phase additive were optimized. With the optimized conditions, analytical method performance for three types of oral use tobacco matrix (i.e. CRP2, gum and dissolvable tobacco product) was characterized. CML and CEL showed higher recovery in all three sample matrix, and the recovery of CML and CEL was in the range of 88.7%-120%, 90.0%-110%, respectively. The developed method showed good intra-day and inter-day precision with RSDs for both free and total CML (or CEL) of less than 9.7%. Limit of detections (LODs) of CML and CEL were 0.36 and 0.25 ng/mL, respectively. Sample analysis for 21 oral use tobacco products was conducted with the developed method. As for oral use tobacco products, total CML and CEL was much more than those of their free state, indicating that CML and CEL exist mainly protein adducts. In addition, the content of CML was higher than that of CEL for both free and total state. Oral use tobacco products I (i.e. dry snuff, moist snuff, suns and chew) had the highest CML (i.e. 44.89–608.69 µg/g) and CEL (i.e. 15.98–77.70 µg/g) levels, compared with oral use tobacco products II (i.e. gum) with the least CML (i.e. 0.7–0.8 µg/g) and CEL (i.e. 0.14–0.18 µg/g) levels. It mainly depended on the composition and processing technology of oral use tobacco products. Both total CML and CEL showed linear relationship with lysine and total amino acid (i.e. R > 0.77). However, there was no linear relationship between total CML (or CEL) and total reducing carbohydrates (i.e. R < 0.5).