AN ABSTRACT OF THE THESIS OF Boonthong Poocharoen for the degree of Doctor of Philosophy in Food Science and Technology presented on June 1, 1983 Title: Determination of Selected Secondary and Tertiary Amine Alkaloids in Barley Malt /7 Abstract approved: /7R. A. Scanlan The malt alkaloids N-methyltyramine, N,N-dimethyltyramine (hordenine), N-methyl-3-aminomethylindole and N,N-dimethyl-3-ami- nomethylindole (gramine) are known precursors for N-nitrosamines. Analytical procedures were developed and applied to extract and quantify these alkaloids from malt, green malt, malt roots, and raw barley. A ground malt sample under nitrogen atmosphere was extracted with 5 percent concentrated ammonium hydroxide in meth- anol at room temperature for 24 hours. After purification, the extracted alkaloids were quantitated by reverse phase high per- formance liquid chromatography using both phosphate buffer and ion-pairing solvent systems. The recoveries obtained for the al- kaloids listed were, respectively, 77.4, 92.0, 74.0 and 80.5 per- cent with detection limit of 0.2, 0.2, 0.1 and 0.1 pg/gm. Con- formation of identity of each alkaloid was accomplished by TLC, UV, and MS. Twenty-three samples of kilned malts, five samples of green malts, ten samples of kilned malt roots, five samples of green malt roots, and ten raw barley samples, from a total of 11 varie- ties of Hordeum vulgare were analyzed for the alkaloids of in- terest. N-Methyltyramine was found in every sample from all sources with a mean and range, respectively, of 26.8 (15.7-48.5), 21.4 (11.6-37.5), 1959.8 (1358.4-2798.4), 1532.5 (962.3-2091.1) and 4.7 yg/gm (0.4-17.9 yg/gm) on a dry weight basis. The means and ranges for hordenine were, respectively, 28.1 (8.9-42.4), 21.2 (9.6-35.5), 4066.4 (2741.2-5180.1), 3363.3 (1956.2-4744.3) and 0.7 yg/gm (0.5-1.0 yg/gm). Gramine was found in only two barley malt varieties examined where kilned and green malts con- tained, respectively, mean values and ranges of 7.4 (4.9-10.0) and 6.0 yg/gm (4.7-7.3 yg/gm). N-Methyl-3-aminomethylindole was not detected in any of the samples examined. The results of this investigation indicated that the variety of barley affected alkaloid production during malting. The re- sults also showed that the kilning step of the malting process in- creased the total amount of each alkaloid produced. Finally, room temperature nitrosation of five varieties of kilned and green malts, in which the contents of hordenine and gramine were known, was carried out with an excess of nitrite in acetic acid at pH 3.2 for 18 hours. The amounts of N-nitrosodi- methylamine (NDMA) obtained from this nitrosation were far greater than the expected yields for NDMA from the hordenine and gramine present in the malts. The results strongly suggest that the alka-' loids hordenine and gramine were not the only precursors for NDMA and that there must be additional precursors; dimethylamine was suggested as a possibility. Determination of Selected Secondary and Tertiary Amine Alkaloids in Barley Malt by Boonthong Poocharoen A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed June 1, 1983 Commencement June 1984 APPROVED: Professor of FoocJ/Science and Technology in charge of major Head of "Departmen^'o'ff Food Science and Technology Dean of Gradua/ui School J Date thesis is presented June 1, 1983 T^ed ^ Do'nna^el^rJen-Race ^r Boonthong Poocharoen ♦ DEDICATION ♦ The author wishes to express his deep appreci- ation to his family for their endless encouragement and understanding. This thesis is dedicated to his wife Duangduen3 his daughter Wccnnisa, and his son Phanupat. ACKNOWLEDGMENTS The author wishes to thank Dr. R. A. Scanlan, his major ad- visor, for directing this research and Dr. L. M. Libbey for his assistance with mass spectral analysis and help in writing this thesis. Thanks are expressed also to: Mr. B. K. Lukes, Project Chemist of Great Western Malting Company, and the Great Western Malting Company for providing and preparing all malt samples used in this study; Mr. J. F. Barbour of the Department of Food Science and Technology, O.S.U., for his help with the quantitation of NDMA using the Thermal Energy Analyzer; and Mr. D. A. Griffin, Department of Agricultural Chemistry, O.S.U., for his assistance with mass spectrometry. The author also wishes to extend his gratitude to Payap College, Chiang Mai, Thailand, the United Board for Christian Higher Education in Asia, New York, and the Myrtle L. Atkinson Foundation, California, for providing the opportunity and a scholar- ship to the author for studying in this country. This investigation was supported in part by Grant No. CA 25002, awarded by the National Cancer Institute, DHHS, and also a grant from the United States Brewers Association, Inc., Washington, D.C. TABLE OF CONTENTS Page I. INTRODUCTION 1 II. LITERATURE REVIEW 7 A. Introduction 7 B. N-Nitrosation of Secondary Amines 7 C. N-Nitrosation of Tertiary Amines 9 D. Gaseous Nitrosating Agents 14 E. Precursors of NDMA in Malt 15 F. Biosynthesis of Alkaloids in Barley during Germin- ation 26 -N-Methyltyramine and Hordenine Alkaloids .... 29 -Gramine and N-Methyl-3-aminomethylindole Alkaloids 34 I. Formation of Non-volatile N-Nitrosamines in Malt' and Beer 38 J. Analysis of Alkaloids in Raw Barley, Green Malt, Malt, and Malt Roots 42 -Isolation and Purification 43 -Separation and Identification 46 -Detection and Quantitation 50 -Confirmation 53 III. EXPERIMENTAL 56 A. Reagents and Equipment 56 B. Extraction Procedure for Isolation of Alkaloids from Malt 60 Part 1: Extraction of Alkaloids from Malt. ... 59 Part 2: Determination of Recovery 64 C. Separation and Quantitation of Alkaloids by HPLC. 67 D. Quantitative Determination of Alkaloids in Malt, Green Malt, Malt Roots and Raw Barley 68 Part 1: Survey of Alkaloids in Malted Barley . 68 Part 2: Determination of Alkaloids in Barley, Clean Malt and Malt Roots from Barleys that Were Grown under the Same Conditions 69 Part 3: Determination of Alkaloids Biosynthe- sized during the Malting Process .... 70 TABLE OF CONTENTS - CONTINUED Page E. Confirmation of Identity for Alkaloids Extracted from Malted Barley, Roots and Raw Barley 74 Part 1: High Performance Liquid Chromatog- raphy (HPLC) 74 Part 2: Thin Layer Chromatography (TLC). ... 75 Part 3: Ultraviolet Spectrometry (UV) 75 Part 4: Mass Spectrometry (MS) 75 F. Nitrosation of Alkaloids in Malted Barley under Laboratory Conditions 78 Part 1: Nitrosation of Freeze-dried Clean Malt Spiked with Standard Amines ... 79 Part 2: Nitrosation of Freeze-dried Clean Malts and Kilned Clean Malts 80 IV. RESULTS AND DISCUSSION 81 A. Extraction Procedure for Isolation of Alkaloids from Malt 81 Part 1: Extraction of Alkaloids from Malt. 82 Part 2: Determination of Recovery 87 B. Separation and Quantitation of Alkaloids by HPLG. 90 C. Quantitative Determination of Alkaloids in Malt, Green Malt, Malt Roots and Raw Barley 102 Part 1: Survey of Alkaloids in Malted Barley . 102 Part 2: Determination of Alkaloids in Barley, Clean Malt, and Malt Roots from Bar- leys that Were Grown under the Same Conditions 106 Part 3: Determination of Alkaloids Biosynthe- sized during the Malting Process . 110 D. Confirmation of Identity by HPLC, UV, MS and TLC for Alkaloids Extracted from Malted Barley, Malt Roots and Raw Barley 115 E. Nitrosation of Alkaloids in Malted Barley Under Laboratory Conditions 171 Part 1: Nitrosation of Freeze-dried Clean Malt Spiked with Standard Amines 172 Part 2: Nitrosation of Green (Freeze-dried) Clean Malts and Kilned Clean Malts . 172 TABLE OF CONTENTS - CONTINUED Page V. SUMMARY AND CONCLUSIONS 179 VI. BIBLIOGRAPHY 182 LIST OF FIGURES Figure Page 1 Proposed mechanisms for the nitrosative dealky- lation of tertiary amines 12 2 Proposed reaction products from the nitrosation of hordenine at elevated temperature 22 3 Proposed reaction products from the nitrosation of gramine 25 4 Unit operation in the malting process 28 5 Biosynthetic pathway for alkaloids in green malts 31 6 Proposed reaction products from the nitrosation of N-methyltyramine and N-methyl-3-aminomethylin- dole 40 7 Schematic procedure for extraction of alkaloids from malt 62 8 Schematic representation of malt sampling for the determination of alkaloids biosynthesized during malt processing 73 9 HPLC analysis using the phosphate buffer system for alkaloids from freeze-dried Morex malt ex- tract 92 10 HPLC analysis using the ion-pairing system for alkaloids from freeze-dried Morex malt extract 94 11 HPLC analysis using the phosphate buffer system for alkaloids from kilned Klages malt extract spiked with N-methytyramine 96 12 HPLC analysis using the ion-pairing system for alkaloids from kilned Klages malt extract spiked with N-methyltyramine 98 13 UV spectra of N-methyltyramine 117 14 Mass spectrum of N-methyltyramine collected as peak #2 from HPLC separation of Winter malt extract 120 LIST OF FIGURES - CONTINUED Figure Page 15 Mass spectrum of standard N-methyltyramine 122 16 Mass spectra of N-methyltyramine from malt 124 extract and standard 17 HPLC analysis using the phosphate buffer system for alkaloids from kilned Karla malt extract spiked with hordenine 126 18 HPLC analysis using the ion-pairing system for alkaloids from kilned Karla malt extract spiked with hordenine 128 19 UV spectra of hordenine 130 20 Mass spectrum of hordenine collected as peak #3 of HPLC separation using the ion-pairing system on Winter malt extract. 132 21 Mass spectrum of standard hordenine (free base form