A GC/MS method for the quantitation of N-nitrosoproline and N- acetyl-S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation Keary Cope 1 , Harold Seifried 2 , Rebecca Seifried 1,3 , John Milner 2,3 , Penny Kris-Etherton 2 , and Earl H. Harrison 1 1 Phytonutrients Laboratory, USDA Human Nutrition Research Center, Beltsville, MD 20705 2 Nutritional Sciences Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20892 3 Department of Nutritional Sciences Penn State University; University Park, PA 16802 Abstract Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl-S- allylcysteine from urine. The latter is a major metabolite of S-allyl cysteine which is abundant in garlic. Urine was acidified and the organic acids extracted by reversed phase extraction (RP-SPE) and use of a polymeric weak anion exchange (WAX-SPE) resin. NPRO was quantified by isotope dilution gas chromatography-mass spectrometry using 13 C 5 NPRO and N-nitrosopipecolic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46 ng of NPRO per mL urine was detected. The method is straightforward, reliable and can be performed with readily available GC/MS instruments. N-acetyl-S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng per mL of urine. The results suggest that 3 to 5 grams of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5 g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl-S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans. . Keywords biomarkers; allyl sulfur compounds; nitrosation; nutrition; cancer © 2009 Elsevier Inc. All rights reserved. Address correspondence to: Earl Harrison, Department of Human Nutrition, The Ohio State University, Columbus, OH 43210, 614-292-8189, Harrison.304@osu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Anal Biochem. Author manuscript; available in PMC 2010 November 15. Published in final edited form as: Anal Biochem. 2009 November 15; 394(2): 243–248. doi:10.1016/j.ab.2009.07.035. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript