Development of an analytical method to detect methyl salicylate in human plasma
Wintergreen is used as a common flavor in many moist smokeless tobacco (MST) products and is also widely used as a food flavorant as well as an over-the-counter analgesic to relieve muscle aches. MST products, including wintergreen flavored products, have been in the commercial market for decades. Unlike food, MST is not ingested and it is important to understand how much of the flavor is absorbed during use. Therefore, a method of detecting methyl salicylate (the main chemical component of wintergreen flavor) in human plasma is needed to estimate the absorption of this compound during use of wintergreen flavored MST. We report a novel high sensitivity LC-MS/MS method for the determination of methyl salicylate in human plasma. An aliquot (0.0750 mL) of human plasma containing the analyte and internal standard, d4-Methyl Salicylate is extracted using a protein precipitation with derivatization procedure. The extracted samples are analyzed by an HPLC equipped with an AB SCIEX API 4000™ triple quadrupole mass spectrometer using an ESI source. Positive ions are monitored in the multiple reaction monitoring (MRM) mode. Quantitation is determined using a weighted linear regression analysis (1/concentration2) of peak area ratios of the analyte and internal standard. The range of quantification is 1.75 -50.0 ng/mL. In a clinical study, peak plasma levels during a single 40-minute use of 2 g wintergreen flavored MST ranged from < LOQ - 4.62 ng/mL. Development of this method is an important step in being able to assess the absorption of methyl salicylate during wintergreen MST use. This method also has broader application and may be used to characterize methyl salicylate uptake from other consumer products as well.