Using CGMS as a forensic tool in discovering natural materials and creating fragrances

1. The Metabolite Map for Identifying Probabilities and Olfactory Analysis As A Supplement to GCMS Analysis Murray Hunter

2. 1. Olfactory Evaluation 2. Using CGMS in Forensic Fragrance Construction 3. Using CGMS in Identifying Essential oil constituents 4. Using CGMS in Quality Assurance of Flavours & fragrances

3. What are essential oils?

4. Essential Oil A volatile oil obtained from a wide variety of plant, scrub, and tree species and from various parts of the plant anatomy, such as the roots, rhizomes, wood bark, leaves, stems, fruit, flowers and seeds. Usually extracted by hydro or steam distillation, expression or effleurage - Hunter 1996

5. Concretes and Absolutes Volatiles and waxes extracted from plant material with hydrocarbon solvents (usually benzene and hexane) through washing and removal of the volatile solvent with distillation. A waxy aromatic substance remaining is called a concrete. The concrete is washed with alcohol to remove the volatile materials and ethanol removed through vacuum distillation to leave an absolute.

6. The Natural Aromatic Product Family

7. Olfactory Evaluation

8. ? The Olfactory System & Odour Classification

9. The Human Olfactory System Odour molecules pass through the olfactory organ

10. Olfactory epithelium Olfactory mucosa Mucus! High in nasal cavity Site of transduction Contains olfactory receptor neurons (ORN)

11. 11 Nose hair: Olfactory cilia ORN have cilia Cilia contain olfactory receptor proteins Similar to visual pigment Transduction Odorants bind to ORs Change shape of protein Ion flow across OR Electricity

12. Transmission Olfactory Receptors Olfactory Cells line the Olfactory Epithelium which is responsible for olfaction Each cell has cilia where receptor sites are located Replaced every month Axons of the olfactory receptors carry information to the olfactory bulb Olfactory bulb sends axons to several specific parts of the cortex with precise connections From the cortex, information is sent to other areas that control feeding & reproduction Olfactory Disorder Anosmia: the general lack of olfaction Specific Anosmia: the inability to smell a specific chemical

13. 13 How many receptor types are there? 1000 different kinds of olfactory receptors (OR) 10 million OR neurons 10,000 of each type of OR Each OR neuron has only one type of receptor 1000 neuronal chemical detectors Potential to differentiate between 5,000-10,000 different odours

14. Olfactory Bulbs An outcropping of the brain Its like a snail in your brain! Electrical responses in cilia passed through olfactory nerve to OB

15. The hedonic primacy of olfaction Sensory and emotional experience Not the same for vision/audition Seeing and feeling more distinct More intertwined in the chemical senses Why? Orbitofrontal cortex Plays multiple roles Critical for emotional experience Secondary sensory cortex for olfaction Also centre of creativity and imagination

16. The olfactory interpretation process from input to response

18. Classification of Odours Curtis & Williams (1994)

19. Floral Family Descriptions

20. Woody Family Descriptions Animalic Family Descriptions

21. Balsamic Family Descriptions Herbaceous Family Descriptions Agrestic Family Descriptions

22. Green Family Descriptions Minty Family Descriptions Coniferous Family Description

23. Marine Family Descriptions Aldehydic Family Descriptions Medicated Family Descriptions

24. Fruity Family Descriptions Sub-Class (Citrus)

25. Spicy Family Description Miscellaneous Notes

26. Odour Characteristics Additional Characteristics

27. Fragrance Description Workshop

30. Performance Test GC-MS A Fragrance Development Lab A trained human more accurate than a GC-MS

31. Sample of a Flavour Chart

33. Using CGMS in Forensic Fragrance Construction From This To This

36. Apples aren't the same Sweet, sour, green, red, sharp, fresh, wholesome, etc. This means different aromatic chemicals play a role in fragrance

38. Discovered in 1937, the First Publication on Raspberry Ketone appears in 1961

39. 1964 – Introduction of Furaneol® 1965 – First publications on isolation of Furaneol® from strawberry and pineapple

40. 1965-66 – α- & β-Sinensal isolated from orange oil

41. 1960’s – Nootkatone First isolated in 1962, it’s importance to citrus flavor was unknown until it’s isolation from grapefruit by Mcleod in 1964. In 1966, Coca-Cola introduced Fresca. By 1970, Nootkatone was GRAS and became a key ingredient in Fresca. Coca-Cola manufactured Nootkatone for many years and soon made it available to the flavor industry. (+)-Nootkatone strong grapefruit odor, bitter in taste (-)-Nootkatone weak woody (vetiver note); no grapefruit character; virtually no taste Threshold: 60,000 ppb 800 ppb

42. 1970’s-80’s – Other Furanones Sotolon (caramel furanone) & Maple furanone identified in cane sugar – powerful caramel maple notes Maple Furanone (Abhexone) Sotolon Threshold: 0.003 ppb 0.00005 ppb Sotolon – key flavorant of fenugreek, sake, sherry wine

43. 1968 – Pfizer patents Ethyl maltol Ethyl maltol (Veltol Plus®) was touted to be about 6X stronger than maltol and an important substitute for Coumarin. Well, at least it is stronger than maltol.

44. 1975 - Introduction of α-Damascone and β-Damascone 1982 – Introduction of Damascenone 1970 – Discovery Damascenone & β-Damascone

45. 1974 – Thiomenthones identified in Buchu oil Key component for black currant and the “fuzzy” peach skin note

48. Coumarinic p-Menthane Lactones Dihydromintlactone (1995) Wine lactone (1996) Mintlactone (1983) GRAS No. -- 3764 4032 Odor: Vinous, lactonic Minty, coumarinic, Coumarinic, hay, (coconut) tenacious lactonic, tonka Flavor: Lactonic, sweet, herbal Coumarinic, fatty, Coumarinic, herbal lactonic, coconut Threshold*: 0.0000016ppb 0.00012 ppb 0.000039 ppb *In air Ref: Frerot, et. al., FlavourFragr. J., 2002; 17: 218–226; Gaudin, Tetrahedron, 2000, 56: 4769-4776

49. 1962 – First publications appear on Methyl Jasmonate & Methyl Dihydrojasmonate 1R,2R-(-)-Z-methyl jasmonate 1S,2S-(+)-Z-methyl jasmonate 1R,2S-(+)-Z-methyl epijasmonate 1S,2R-(-)-Z-methyl epijasmonate Methyl Jasmonate – odorant, insect pheromone, plant growth regulator

50. Advances in Flavoring Materials Chirality & Odor - Methyl Jasmonate & Methyl Dihydrojasmonate ConfigurationOdor DescriptionOdor Threshold (in PPB) Methyl jasmonates 1R,2R-(-)- Weak odor >70* 1S,2S-(+)- Odorless Odorless 1S,2R-(-)-epi- Odorless Odorless 1R,2S-(+)-epi- Strong odor; floral, true jasmin-like 3* Ref: T.E. Acree et. al., J.Agric.Food.Chem. 1985 Methyl dihydrojasmonates 1R,2R-(-)- Floral, sweet, jasminelike 240** 1S,2S-(+)- Floral, fatty, cis-jasmone, hay character, 15,360** tea note, slightly lemon peellike (weak) 1S,2R-(-)-epi- Herbal, fatty, tea-like, tobacco, 12,500** β-damascone, cis-jasmone 1R,2S-(+)-epi- Intensely floral, jasmine-like, bright, cis- 15** jasmone, slightly fatty, woody, β-ionone-like, extremely long lasting Ref: Werkhoff, P., et. al., Food Reviews International, 2002 * detection threshold **recognition threshold

51. Advances in Flavoring Materials 2001 – Takasago’s Professor RyojiNoyori wins the 2001 Nobel Prize for Chemistry

54. Individual Aromatic Chemicals Probable Constituents of an essential oil

57. Chypre

58. FougereCitrus Floral Aldehydic Spicy

69. Using CGMS in Identifying Essential oil constituents

74. Ylang Ylang (Cananga odorata) benzyl acetate (ca. 25 %), p-cresyl methyl ether (ca. 20 %), methyl benzoate (ca. 5 %), methyl salicylate, cinnamyl acetate, (-)-linalool (ca. 15 %), geranyl acetate (ca. 10 %), farnesyl acetate (ca. 3 %), as well as a number of other sesquiterpenes and their oxygenated derivatives, e.g. muurolol T (ca. 2 %) Extraction Method: Steam distillation of the flowers Origin: Indonesia, Madagascar

75. Patchouli Oil (Pogostemon cablin) There are no synthetic equivalents of the patchouli scent. Main Constituents: (-)-patchoulol (30-40 %). However, it is maintained that norpatchoulenol, present in only 0.3-0.4 %, is playing a principal part in the overall odour picture. (-)-patchoulol andnorpatchoulenol Obtained by steam distillation under pressure or CO2- extraction of the dried leaves Patchouli is mostly grown in Indonesia

76. Pandanus Oil (Pandanus odoratissimus) phenethyl methyl etherpandanol phenethyl methyl ether ( pandanol) (38 %), together with terpinen-4-ol (19 %), alpha-terpineol (8 %) and phenethyl alcohol (7 %) [79]. Phenethyl alcohol and its derivatives are common odorants in flowers The flowers are hydro-distilled to yield a 'kewda attar Origin: native of South East Asia and is much cultivated on the Indian East Coast

77. Backhousia citriodora (Lemon Myrtle) High Investment to Expand Industry in Australia Strong Demand as an Ingredient for tea Good Crop to Grow in Most Parts of Thailand Citral (geranial 51.43 % and neral 42.12 % )

88. Different Major Chemical and Olfactory Profiles of Five Basil Oils

93. Harvest Timing Critical for Some Crops (Mentha piperata)

94. 1959 – Bain & Webb – Turpentine into Fragrance & Flavor beta-Pinene Linalool Linalyl Acetate Menthol Myrcene Geraniol Citronellol Citronellal

95. 1959 – Roche Process for Linalool & Citral via Acetylene

96. Points to Remember The highest probability is not necessarily the correct one Check back to see if that chemical could physically be present You will have to add new materials to your database (Both NIST & Wiley databases) Is it an artifact? Confirm your analysis with other supportive data

98. Using CGMS in Quality Assurance of Flavours & fragrances

99. QC lab. in the days before the concept of ‘Health & Safety at Work’ – note lighted cigarettes dangling from lips of staff!

101. Gas Chromatography – Mass Spectrometry Detective work GC-MS analysis Looking at metabolite paths Olfactory analysis of living plants

103. Helps elucidate many of the C10H16 group terpene structures present in essential oils utilizing common reagents such as hydrogen chloride and hydrogen bromide. In 1909 he published the results of his extensive studies in the book Terpene und Campher, a volume of 600 pages dedicated to his pupils.beta-Pinene alpha-Pinene Camphene Camphor

106. Elucidated the structures of the naturally occurring musk perfumes, civetone and muscone thus replacing scents prized since antiquity – but only available from endangered species. Civetone Muscone Musk Deer Moschusmoschiferus L. Civet Cat Viverracivetta

107. Have fun with your new toy Thank You