Limonene –Odour Profile and Oxidation to Hydroperoxide

15 October 2019Analytical Strategies 2019Susanne Kernkera@zhaw.ch

Overview

• Limonene - a versatile odorantPurity and odour profile

• Fragrance allergensLegislation, analytics

• Limonene hydroperoxidesAnalytical challenge and quatification

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Fragrance Limonene

• Monoterpene C10H16

• Naturally occurring as (R)-(+)-Limonene in e.g. orange peel, by-product of orange juice production

• (S)-(-)-limonene in e.g. mint oil• High octanol water partition coefficient: log Kow = 4.46 hydrophobic and diffusive

(R)-(+)-LimoneneOdour: orange, citrus

(S)-(-)-LimoneneOdour: terpentine-like

(R) (S)

Limonene often cited as a material possessing two enantiomers with different odours: (R)-(+)-Limonene "orange odour" (S)-(-)-Limonene "lemon odour" based on a erroneous publication

Purity of Limonene

• Pure limonene is "biological" biodegradable contains e.g. no hazardous air pollutants

• Highly refined, pure (R)-(+)-Limonene lower odour profile probably smell of limonene mostly attributed to aldehydes present in trace amounts

P. Kraft, K. Swift. Perspectives in flavour and fragrance research. Helvetica Chimica Acta: Zurich. 2005. DOI:10.1002/9783906390475

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CitronellalOdour: herbaceous-citrus, fresh

Enantiomeric Separation of Limonene

Chiral GC-olfactometry• Chiral GC columns based on cyclic oligosaccharides with different regioselective

derivatisation of hydroxy groups• Enantiomeric separation is not predictable try out rather than use one type of GC column for similar enantiomeric cases

Chiral GC column contain cyclodextrine phasese.g. Heptakis-(di-O-methyl-O-pentyl)-β-cyclodextrine

O

O

O

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7

Reference standards for both (R)-(+)-Limonene and (S)-(-)-Limonene needed

"GC-Sniff"• GC separation with a split to two detectors one detector e.g. FID second detector nose = "sniff" split ratio: 1:1 ~2 µg/µl concentration of sample

• Injection type:on-column mode or split/splitless

Olfactive Detection of Limonene

ORD Measurement

Optical rotatory dispersion (ORD)• Change in wavelength of circularly polarized light optically active chiral compounds differently absorb light coming from two directions

value given as [α]De.g. (R)-(+)-Limonene neat at 28°C at 589 nm: [α]D = 119 °(S)-(-)-Limonene neat at 28°C at 589 nm: [α]D = -103 °

• Comparison of measured values to literature values

F. Reinscheid, U.M. Reinscheid. Stereochemical analysis of (+)-limonene using theoretical and experimental NMR and chiroptical data. Journal of Molecular Structure 1106 (2016) 141-153.

M. de Mattos, R. B. Coelho, A. M. Sanseverino. Chemospecific Preparation of Both Enantiomers of α-Terpinyl Trifluoroacetate. Synthetic Communications 34 (2004) 541-545

Chirality and Odour Perception

• Olfactory receptors are proteins (i.e. chiral molecules) enantiomeric odour pairs should interact differently with receptors

• But: there are enantiomeric odour pairs with same smell no predictable relationship between fragrance structure and odour perception

• Newly assumption: odour enantiomer pairs interact differently with olfactory epithelium receptors but similar cortical response patterns result in similar behavioural responses structure-based "encoding" seems to be present in the periphery which is not necessarily translated into a central nervous electrophysiological or behaviouralresponse.

S. C. Poletti, A. Cavazzana, C. Guducu, M. Larsson, T. Hummel. Indistinguishable odour enantiomers: Differences between peripheral and central-nervous electrophysiological responses. Scientific Reports 7, (2017) 8978. DOI:10.1038/s41598-017-09594-3

Odour Threshold Measurements

• Olfaction is not an objective science large panel (min 15) and statistics to properly obtain odour thresholds usually pure reference material from synthetic chemists

• Concentration calculated from odorant’s vapour pressure and from dilution ratios assuming vapour saturation in sample generator dilute sample for good range measure vapour pressure check vapour saturation by

headspace analysis

Olfactometer

Odour Thresholds

• Individual values given as ml of diluted sample/min approximately Gaussian distribution 1 person is anosmic

• Odour threshold calculated (geom. mean) as ng/L air (25°C)• From this value and vapour pressure: 2-Isopropyl-4-methylpyridine: ca 200 µg/L

(25°C)

Odour Thresholds in Fragrances

• Odour threshold at different concentration levels compared to sensitivity of different analytical tools

N. Nibbe, H. Gygax, B. Maxeiner. Odour Measurement for ImprovedScent Performance in Consumer Goods. SOFW-Journal 141, 9 (2015).

Legal Situation

Fragrance and flavour legally different• Fragrance in EU: Substance information from ECHA (European Chemicals Agency)

Substances categorised by EINECS (EC) Numberse.g. EC No. 232-433-8 for "Orange, sweet, ext." being a mixture including Limoneneor EC no. 227-813-5 for (R)-p-mentha-1,8-diene i.e. (R)-(+)-Limonene

• Flavour: EU Lists of Flavourings; Generally Recognized As Safe (GRAS) by FDA (United States Food and Drug Administration)

European Commission, European Union, 1995-2019. Scientific Committee on Consumer Safety (SCCS) –OPINION on Fragrance allergens in cosmetic products. 2012.https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_102.pdf

Registration in the EU?According to ECHA (European Chemicals Agency): Definition of substance

Mono-constituent / Multi-constituentsubstances

• Impurities ≥1% have to be identified: • Analytics with GC-MS, NMR, IR, UV/Vis

UVCB

• Substances ≥10% in mixture have to be identified

• Manufacturing process can be described

ECHA European Chemicals Agency. Substance Identification. Retrieved fromhttps://echa.europa.eu/de/support/substance-identification/what-is-a-substance (7.9.2019).

ECHA Limonene Registration

ECHA European Chemicals Agency. Registered Substances. Derieved from https://echa.europa.eu/de/information-on-chemicals/registered-substances (14.10.2019).

EC no.: 227-813-5

Fragrance Allergens

• In year 2003: European Commission introduced list of 26 allergenic substances (including Limonene) Fragrance chemicals most frequently reported & well-recognised consumer allergens (List A) Fragrance chemicals less frequently reported & less documented as consumer allergens (List B, contains Limonene)

• Current regulatory proposal: List of up to 90 fragrance allergens Declaration in products at 0.01% level

• Three allergens (HICC, atranol and chloroatranol) considered not safe

European Commission, Health and Food Safety “SCCS final opinion on Fragrance allergens in cosmetic products now available.” Retrieved from https://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_102.pdf(15.10.2019).

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Analytical Technique for Fragrance Allergens

• List of 26 allergenic substances contains 24 volatile substances GC method of choice

• 2 dimensional GC for better separation:GCxGC-MS

OOH

OO OH

OOH

cis/ trans-Limonene-1-OOH

EC3 = 0.33%

cis/ trans-Limonene-2-OOH

EC3 = 0.83%

Limonene

cis/ trans-Limonene-epoxide

EC3 ~ 25%

cis/ trans-Carveol

CarvoneEC3 = 13/10.7%

Chemical Transformation of LimoneneHydroperoxide of limonene (and other terpenes) are moderate to strong sensitizers (LLNA EC3 0.5 – 2%)

Hydroperoxide: Primary oxidation products

Main suspects for skin allergen

Kern, S.; Granier, T.; Dkhil, H.; Haupt, T.; Ellis, G.; Natsch, A. Stability of limonene and monitoring of a hydroperoxide in fragranced products. Flavour and Fragrance Journal. 2014, 29, 277-286.

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Limonene from Production to Product

Extraction from natural source

Dilution in finalproduct

Storage as oil

Storage offragrance oilFragrance

compounding

Repeated application to skin, topical

Storage of final product: sealed

Storage of final product: Opened, partly used

Where in this value chain would you expect hydroperoxide formation to be most likely?

Only analytics can tell ....

Chemical production

Conventional Analysis of Hydroperoxides

• Peroxide values via iodometric titration, e.g. from citrus oils representative for total molar equivalent of all oxidizing species in a sample

• No information about chemical structure of oxidizing species• Difficult in mixtures• Time consuming sample preparation and high sample amount needed

Two Redox reactions and measurement of colour change with starch added as an indicator

Norveel Semb, T. Analytical methods for determination of the oxidative status in oils. Master Thesis Norwegian University of Science and Technology Trondheim. 2012.

Analytical Challenges with Hydroperoxides

OOH O

OH

cis/ trans-Limonene-2-OOH

cis/ trans-Carveol

LC-MS:unspecific ionC10H14O

PPH3reduction

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Selective detection by GC-MS in complex matrix

• Hydroperoxides known to be not stable (e.g. GC injector)• Heavy matrix from perfumes and cremes/deodorants expected,

sample purification difficult due to instability of hydroperoxides and chemical similarity to other fragrance constituents

• Sensitivity down to low ppm levels needed• Selectivity for these specific limonene hydroperoxides needed• No internal standards available and reference standards had to be made first

Try out with high resolution LC-MS

Two diastereomers

Full MS of trans-Limonene-2-OOH

m/z 151.1117

Extracted ion chromatogram

LC-MS vs GC-MS Method for Hydroperoxides

OOH O

OH

cis/ trans-Limonene-2-OOH

cis/ trans-Carveol

LC-MS:unspecific ionC10H14O

PPH3reduction

H

Selective detection by GC-MS in complex matrix

Matrix effect in LC-MS:

Quantification with GC-MS of Carveol:

GC-MS Method for Reduced Hydroperoxides

• GC-MS: split injection for less artefacts, long method (>1 hour analysis time) for better separation good selectivity, less false positives

• Fragrance D with 200ppm spiking of hydroperoxide mix and triphenylphosphineRT: 5.00 - 83.00

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80Time (min)

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60.7724.04 45.9347.80 81.58

54.39 79.1245.0636.52 38.59 60.34

55.728.43 31.78 49.4644.7639.6033.375.72 59.2513.44 74.5418.79 30.42

NL:3.05E8m/z= 35.0-350.0 - m/z= 39.5-40.5+43.5-44.5 MS K96634B

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• Interlaboratory ring trial: Accurate detection and quantification with GC-MS reduction method by five labs

Method Validation in Real Products – Fine Fragrances

EdT: Eau de ToiletteEdP: Eau de Parfum

Grey squares: Spiked levels of hydroperoxides in different perfumesBlack diamonds: Found levels of corresponding carveol in these perfumes

Analytical Methods Summary

• Sensitivity: LOD (limit of detection) of all analytes for both GC-MS and LC-MS around 0.5 ppm in pure reference standards, ≥5 ppm in matrix samples

• Robustness (risk of false negatives/positives):GC-MS method more robust additional confirmation by LC-MS for hydroperoxides of linalool matrix effects can be big for LC-MS LC-HRMS versatile if other analytes wanted to be tracked (e.g.

hemiacetals) limitation of GC-MS when coelution

• Studies done with limonene, linalool and other similar compoundsAnalytical effort (within almost 5 years):GC-MS and LC-MS: each >2500 samples analyzed

• Application to other cosmetic products such as cremes, deodorants, lotions…

Comparison of Analytical Methods

• Within IFRA analytical taskforce: detection of fragrance hydroperoxides with six different analytical methods

• Blinded samples being spiked orange oil with 500 and 3000 ppm hydroperoxides.

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PPh 3

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Extraction and Recovery

n.d. = not detected

Limonene-2-OOH(analyzed as trans-Carveol)

Linalool-6-OOH (analyzed as 6-OH-linalool)

Woolwax Alcohol Cream 107 113

Anti aging cream 96 86

Bodylotion 94 81

Face Lotion 88 128

Roll on Deodorant 84 111

All natural deodorant 88 128

All natural deodorant - not spiked n.d. 25

• Quantification of hydroperoxides (µg/g) in different consumer products with Extrelut® NT extraction followed by the GC-MS reduction method

• All products spiked with 100 µg / g with hydroperoxides In parallel, non-spiked samples tested

Exposure to Hydroperoxides

• Analytical and literature data calculated as dose-per area, here shown for Linalool-hydroperoxides Levels in products 3 – 4 orders lower as compared to animal tests and patch tests !

Dose of hydroperoxide in test preparation Dose per area

LLNA a Dose inducing sensitisation (EC3) 16‘000 µg/g (1.6%) 400 µg/cm2

Patch test 2% oxidized linalool (0.83% response) 3’800 µg/g (0.38%) 152 µg/cm2

Patch test 6% oxidized linalool, diagnostic level 10’000 µg/g (1%) 456 µg/cm2

Patch test 11% oxidized linalool(7.2% response) 20’900 µg/g (2.09%) 836 µg/cm2

Analytical data fine fragrance: median 14 µg/g (0.0014%) 0.031 µg/cm2

Analytical data fine fragrance: (Max. value of n=39) 132 µg/g (0.0132%) 0.29 µg/cm2

Current Status

• Validated methods to detect hydroperoxides• Natural ingredients can be a source of hydroperoxides added to products Low levels if working under IFRA rules, QC of ingredients includes peroxide titration

• Synthetic linalool has very low hydroperoxides and is stabilized by Tocopherol Even with forced oxidation extremely stable (data not shown)

• No evidence for oxidation taking place in formulated products No evidence even in repeatedly opened products after 9 months 45°C

• Aged consumer samples have levels far below the levels used in toxassays and in dermatological clinics 3 – 4 orders of magnitude!

• No evidence for oxidation on the skin

• Alternative source? Aromatherapy?• Or: False-positive tests in dermatological clinics?

Thank you